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BIOLOGIA (PAKISTAN) ISSN 0006-3096

BIOLOGIA

(PAKISTAN) Vol. 56, No.1&2, 2010

Editor-in-Chief

AZIZULLAH

Editors

PAKISTAN FOREIGN

Nusrat Jahan Jonathan Palmer (New Zealand) Ghazala Yasmeen Athar Tariq (U.S.A.) Ikram-ul-Haq C.J. Secombes (U.K.) M. R. Mirza B. Faye (France) A.U.Khan Salih Dogan (Turkey) Zaheer-ud-din Khan Wolfgang Von Engelhardt M.Sharif Mughal (Germany) Qi Bin Zhang (China)

BIOLOGICAL SOCIETY OF PAKISTAN Biological Laboratories, GC University, Lahore, Pakistan


PRESIDENT

M. Anwar Malik Department of Zoology,

GC University, Lahore, Pakistan

VICE PRESIDENTS

Zaheer-ud-din Khan Department of Botany,


Anjum Perveen Department of Botany,

University of Karachi, Karachi, Pakistan

Rehana Asghar

Department of Biology

Mirpur, University, AJK

Nusrat Jahan

Department of Zoology


Syed Akram Shah Department of Zoology,

Peshawar University, Peshawar, Pakistan

Asmatulla Kakar Department of Zoology,

University of Balochistan, Quetta, Pakistan

GENERAL SECRETARY

Ikram-ul-Haq, SI Institute of Industrial Biotechnology,


JOINT SECRETARY

Muhammad Afzal Agricultural University, Faisalabad

Pakistan

MANAGING EDITORS Abdul Qayyum Khan Sulehria

Department of Zoology

GC University, Lahore. Pakistan.

Safdar Ali Mirza

Department of Botany


EDITOR-IN-CHIEF

Azizullah Department of Zoology,


ADVISORY BOARD Tasneem Farasat (LCU for Women, Lahore) M. Fiaz Qamar (GC University, Lahore) Altaf Dasti (B. Z. U., Multan) Wazir Ali Baloch (University of Sindh, Jamshoro) Muhammad Ayub (DG, Fisheries Punjab) Sana Ullah Khan Khattak (University of Peshawar, Peshawar) Zahid Hussain Malik (University of AJK, Muzaffarabad) Tahira Aziz Mughal (LCU for Women, Lahore) Atta Muhammad (Univ. of Balochistan, Quetta) Aliya Rehman (karachi University) Moin-ud-Din Ahmad (Urdu Uni., of Sci. Tech., Karachi)

Pei Sheng-Ji (China) Kazuo N. Watanabe (Japan) Jin Zou (U. K.) Mary Tatnar (U. K.) William Bill Radke (U. S. A.) David B. Wilson (U. S. A.) Lee A. Meserve (U. S. A.) Fabrizio Rueca (Italy) Silvana Diverio (Italy) Giorgia Della Rocca (Italy) R. Pabst (Germany)

Vol. 56, No.1&2, 2010 PK ISSN 0006-3096

BIOLOGIA (PAKISTAN) CONTENTS

Chatta, M. A. and Ayub, M., Growth Performance of Hatchery Reared Golden

Mahseer (tor macroplepis) at Sialkot, Pakistan. 1

Jahan, N. and Mumtaz, N., Evaluation of resistance against Deltamethrin in Aedes mosquitoes from Lahore, Pakistan.

9

Ajaib, M. and Khan, Z., Diospyros Malabarica (desr.) Kostel. of family Eebenaceae and Ochna Serrulata (Hochst.) Walp. Of family ochnaceae: addition to the flora of pakistan

17

Qamar M. F., Zafar, M., Anjum A. A., Najeeb, M. I. and Maqbool, A., Effect of

Livol (herbal polysaccharide) as an Immunomodulator in Commercial Broilers 23

Sulehria, A. Q. K., Younus, I. and Hussain, A., Effect of Artificial Diets on the Growth and Survival of Rotifers

31

Raees, K. Asmatullah and Ahmad, K. R., Pregnancy and Fetal Correlations of Cypermethrin in Mice (Mus musculus)

39

Ali, S. and Iqbal, J., Facile Regeneration Through Adventive/Somatic Embryogenesis From In Vitro Cultured Immature Leaf Segments Of Elite Varieties Of Sugarcane (Saccharum Officinarum L)

55

Mustafa, Y. S., Sulehria, A. Q. K. Muneer, M. A. and Munir R., Effect of Water Restriction on the Lymphoid Organs and Production of Broilers

63

Mohsin, M. Sulehria, A. Q. K. Yousaf, I., Ejaz, M., Jameel, M. Y. and Hussain, A., Comparison of Spider Guilds Found in Various Oilseed Crops of Pakistan

69

Raza, C., Bilal, A. and Jahan, N., CEvaluation of Biodegradation Potential of Bacteria in Crude Oil Contaminated Soil

77

Javed, W., Khan, U. and Hayyat, M. U., Evaluation of Industrial Waste Water Quality by Monitoring the Effect on Growth Attributes of Pistia stratiotes L. and Eichornia crassipes Solm.

87

Ahmad, K. R., Asmatullah, Khan, S. Y., Raees, K., Abbas, T. and Ahmad, S. M., Alterations In Mammogenesis, Gestational Length And Nurturing Behaviors In Mice Following Co-Gestational Chlorpyrifos Exposure In Mice

95

Khalique, A., Khan, N., Mughal, M. S. and Anjum, K. M., An Overview on Nutrition and Feeding of Prawn (Penaeus Japonicus)

107

Anjum, A. A., Qamar, M. F., Ali, M. A., Usman, M. and Khan, M. T., Bacteriology of Mastitic Milk of Cattle and In vitro Antibiogram of the Pathogens Isolated, in

District Hafizabad, Pakistan

117

Anjum, K. M., Yaqub, A., Mumtaz, N., Sayyed, U., Asad, M. and Khan, S. A.,

Effect of Acute Fluoride Intoxication on some Hematological Changes in Chicken (Gallus domesticus).

123

Khan, Z. and Ajaib, M., Clerodendrum Splendens g. Don of Family

Verbenaceae: An Addition to the Flora of Pakistan 129

Ajaib, M. and Khan, Z., Melochia Corchorifolia l. of Family Sterculiaceae: An Addition to the Flora of Pakistan

133

Khatoon, A, and Pirzada, Z. A., Bacteriological quality of Bottled water brands in Karachi, Pakistan

137


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BIOLOGIA (PAKISTAN) 2010, 56 (1&2), 1-8 PK ISSN 0006-3096

Growth performance of hatchery reared golden mahseer

(Tor macrolepis) at Sialkot, Pakistan.

ANSER MAHMOOD CHATTA & MUHAMMAD AYUB

Fisheries Research & Training Institute, Department of Fisheries Punjab, Lahore.

ABSTRACT

The weight-length and condition factor parameters of hatchery reared Golden Mahseer, Tor macrolepis were analyzed. Log transformed regressions were used to test the growth performance. It was observed that growth in weight was almost proportional to the cube of its length (isometric): log W = -5.045 + 3.143 log TL. The value of the slope b = 3.143 coincides with the slope b = 3.0 for an ideal symmetrical fish. The condition factor (K) shows statistically significant relationship with both length and weight i.e. as fish grows K increases with increasing length or weight.

Key words: Growth, Tor macrolepis, weight-length, condition factor

INTRODUCTION

Golden Mahseer, the king of Himalayan fishes is a renowned, high valued game and food fish of South Asian countries including Pakistan, distributed in most Trans-Himalayan countries ranging from Afghanistan to Myanmar (McDonald, 1948; Day, 1958; Desai, 1994). Mahseer inhibits torrential waters and mid-hill lakes (Shrestha, 1981; 1991). It is known as pride of Anglers (Mirza & Bhatti, 1996). There are two species of mahseer known to occure in Indus River System in Pakistan, the Zobi Mahseer Naziritore zhobensis and Golden Mahseer Tor macrolepis (Mirza, 2004). Natural population of Mahseer is declining in all parts of its geographic distribution (Bista et al., 2006). This fish is now recognized as a critically endangered species in many countries. The major possible factors for the depletion of Mahseer stocks are degradation of environmental conditions of water bodies, indiscriminate fishing, industrial pollution, use of explosives, poisons and electro fishing, introduction of exotic fish species, and low fecundity rate (Das & Joshi, 1994; Rehman et al., 2005). Growth of Mahseer in captivity is less than in nature (Shrestha, 1997). Fisheries Department Punjab successfully did artificial breeding of Mahseer a few years back. Now further research is being undertaken to develop culture techniques to introduce this fish species in commercial aquaculture system.

Present study deals with the weight-length and condition factor relationship of Mahseer. This study has applied value in fish biology (Salam et al., 2005). The weight-length relationship provides a parameter to calculate an index commonly used by fisheries biologists to compare the “condition factor” or “well being” of a fish (Bagenal & Tesch, 1978).

Several studies on length-weight relationship have been carried out in other parts of the world (LeCren, 1951; Jhingran, 1952 & 1968; Chakrbborty & Singh, 1963; Saigal, 1964; Lal & Dwivedi, 1965; Khan, 1972; Willis, 1988; Wootton, 1998). This topic has also received attention in Pakistan (Javaid &

A. M. CHATTA & M. AYUB BIOLOGIA (PAKISTAN) 2

Akram, 1972; Salam & Janjua, 1991; Chatta & Salam, 1993; Salam et al., 1994; Naeem, et. al., 2000; Salam et al., 2005).

MATERIAL AND METHODS

One hundred seventy (170) freshwater hatchery reared Golden Mahseer Tor macrolepis were sampled during April to August at Fish Nursery Kotly Araian District Sialkot, Punjab, Pakistan. Their size ranged from 3.70-22.0 cm in length and 0.33-105.00 g in body weight. Fish were caught by using cast net early morning before feeding, selected at random and transported live to Lab. Total length measured form the tips of the maxilla to the last caudal fin ray fitted with a millimeter scale to nearest 0.1 cm by using Perspex measuring tray and weight to 0.01g with electronic digital balance (Salam et al., 1993; Naeem & Salam, 2004). Condition factor (K) was calculated using formula K = 100 x W/L

3 following

Weatherley & Gill, 1987. Statistical Analysis including regression analysis and correlation was done using statistical program, SAS.

RESULTS

The relationship between wet body weight (W) and total length (TL) is exponential (Fig.1) having the general from; Y = aX

b or W= aL

b. Whereas Y is

independent and X is dependent variable, a is intercept and b is power. When the data is transformed into logarithmic from, a linear relationship is obtained (Fig.2) with high correlation coefficient (r = 0.998; P<0.001) having the general formula; Log10 W = Log10 a + b Log10 TL

The values of these constants and other regression parameters are given in the Table-I. The regression coefficient of b = 3.143 when compared to b = 3.0 (an isometric slope) was found to be similar. It is therefore concluded that growth in weight of Golden Mahseer is directly proportional (isometric) to increasing length.

Condition factor, “K” when computed against total length and body weight using formula K = 100 W/L

3, it shows statistically significant relationship

(Fig. 3 & 4) both with length and weight i.e. as fish grows K increases with increasing length or weight. The values of regression parameters of condition factor (K) on log total length (TL) and log wet body weight are given in the Table-II. Table 1: Regression parameters of log wet body weight (W) on log total

length (TL) for Golden Mahseer, Tor macrolepis.

Regression equation

N

Size range(cm)

Correlation coefficient

(r)

S.E. (b)

T-value When b=3

Log W = log -5.045+3.143 log TL 170 3.70-22.00 0.998*** 0.117 0.74 n.s.

n.s. = P>0.05 *** = P<0.001

Vol. 56 (1&2) Growth Performance of Golden Mahseer 3

Table 2: The regression parameters of condition factor (K) on log total length (TL) and log wet body weight for Golden Mahseer Tor macrolepis.

Regression equation

N

Size (cm)

Correlation coefficient (r)

S.E. (b)

K = 0.765+0.0117 TL K = 0.830+0.0025 W

170 170

3.70-22.00 3.70-22.00

0.569*** 0.553***

0.106 0.108

n.s. = P>0.05 *** = P<0.001

Table 3: Weight-Length Relationship for Different Fish Species from

Different Locations

Fish Species Slope (b) Source

Catfish

Rita rita Immature 2.84 Lal & Dwivedi, 1965

Mature 2.42 -do-

Mystus vitatus 3.14 Javaid & Akram, 1972

Wallago attu 3.34 Salam et al., 1993

Clarius batrachus 3.33 Sinha, 1975

Onchorhyncus mykis 2.98 Salam et al., 1994

Oreochromis nitoticus 3.10 Naeem & Salam, 2004

Carps

Labeo rohita Immature 3.01 Jhingran, 1952

Ripe female 3.38 Khan, 1972

Farmed 3.99 2.98 3.07

Chatta & Salam, 1993

Cirrhinus mrigala 3.01 Salam & Khaliq, 1992

Catla catla Juvenile 3.39 Jhingran, 1968

Male 3.28

Female 3.19

Crassius auratus Farmed 3.03 Salam & Davies, 1992

Puntius chola 2.80 Salam et al., 2005

Hypophtalmichthys molitrix

Farmed 2.89 2.92 3.37

Salam et al., 1993

Ctenopharyngodon idella 3.09 Salam et al., 1993

Cyprinus carpio 3.17 Salam & Naeem, 2004

Tor macrolephis (Golden Mahseer)

3.143 Present study


DISCUSSION

In the present study with Golden Mahseer, the value of b = 3.143 shows that the fish is growing isometrically with increasing length maintaining constant proportions throughout its growth assuming that the specific gravity of the fish does not change.

The literature survey on weight-length relationship shows that the weight of a fish is considered as a function of its length (Weatherly & Gill, 1987; Wootton, 1990). If the fish maintains its shape, body proportions and specific gravity during growth, the value of its length exponent b would be near or equal to 3.0 expressing isometric growth pattern. A value less then 3.0 is interpreted as the fish becoming lighter in weight (negative allometery) and greater than 3.0 as getting heavier (positive allometery) for a particular length with increasing size (Wootton, 1990). The present study with Golden Mahseer generated the value of b = 3.143 which is not significantly different from b value 3.0 and thus the cube law appears to be followed in this population during growth and therefore conforms the cube law.

The cube law may be held in some species as in the present study (Salam and Janjua, 1991; Salam & Davies, 1992; Salam & khaliq, 1992; Salam et al., 2005). These results show that Golden Mahseer being indigenous species is not only growing comfortably in our hilly areas but can also grow in pond culture system of Punjab, Pakistan, while many species have been found violating the cube law (Javaid & Akram, 1972; Salam & Mahmood, 1993 Salam et al., 1994).

Condition factor (K) shows increasing trend with increasing length or weight. In the present study, slope of weight-length relationship i.e. b = 3.143 although not significant shows slightly increasing trend in weight and resultantly increasing K value with the increase in weight and length.

Keeping in view the multiple factors affecting growth, the interpretation is a complicated matter unless more studies are conducted on weight-length relationship of Golden Mahseer under different ecosystems, sex, feeding, maturity and seasonal variations to understand the mechanism of growth. Present study may act as first step to generate interest in this kind of research on this important game and food fish of Pakistan.


Fig. 1

Fig. 3

Fig. 2

Fig. 4

Fig., 1: The relationship between wet body weight (g) and total length (cm) in Golden Mahseer Tor macrolepis. Each point represents a single fish.

Fig., 2: The relationship between log wet body weight (g) and log total length (cm) in Golden Mahseer Tor macrolepis. Each point represents a single fish.

Fig., 3: The relationship between total length (cm) and condition factor in Golden Mahseer Tor macrolepis.

Fig., 4: The relationship between total weight (g) and condition factor in Golden Mahseer Tor macrolepis.

0

50

100

150

200

250

0 5 10 15 20 25

Total Length

Bo

dy W

eig

ht

-1

-0.5

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5

Log T Length

Lo

g B

od

y W

eig

ht

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-1 -0.5 0 0.5 1 1.5 2 2.5

Log Body Weight

Co

nd

itio

n F

acto

r

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.5 1 1.5

LogTotal Length

Co

nd

itio

n F

acto

r


REFERENCES Bagenal, T. & Tesch, F.W., 1978. Age and growth. In: Method for Assessment of

Fish Production I Fresh Water. (T. Bagenal). IBP Handbook, Blackwell Scientific Press, Oxford.

Bista, J.D., Suresh, K.W. & Arun, P.B., 2006. Maturity stage and spawning performance of Mahseer Tor putitora in ponds of Mid Hills Nepal (Abstract) International Symposium on the Mahseer Kuala Lsimpur, Malaysia (March 29-30): 11.

Chakrborty, R. D. & Singh, S.B., 1963. Observation on some aspects of fishery and biology of mrigal Cirrhina mrigala from Allahabad. Ind. J. Fish., 10: 209-230.

Chatta, A.M. & Salam, A., 1993. Growth performance of cultured rohu, Labeo rohita (Ham.) Under semi-intensive farming; An ecological approach. Proc. Pak. Congr. Zool. vol., 13: 473-482.

Das, P. & Joshi, K.D., 1994. Mahseer conservation – present and future, P. Nautiyal (com. & ed.) Mahseer the Game Fish., D3-D9.

Day, F., 1958. The fishes of India: Being a natural history of the fishes known to inhibit the seas and fresh waters of India, Burma and Ceylon. Vol (text), London. William Dawson and Sons Ltd. London., 564.

Desai, V.R., 1994. Ecostatus of mahseer in river Narmada (Madhya Pradesh). In Mahseer the game fish natural history, status and conservation practices in India and Nepal, ed (P. Nautiyal), Rachna, Garhwal UP, India.

Javaid, M.Y. & M. Akram, 1972. The length-weight relationship and condition factor of seven fresh water fishes of Pakistan. Bull. Dep. Zool. Univ. Punjab, Lahore. Article., 6:1-27.

Jhingran, V.G., 1952. General length-weight relationship of three major carps of India. Proc. Natl. Inst. Sci. India., 18: 449-460.

Jhingran, V.G., 1968. Synopsis of biological data on Catla catla (Ham. 1822). FAO Fish synopsis., 32 Rev. I.

Khan, R.A., 1972. Studies on biology of some important major carps. Thesis, Department of Zoology, Aligarah Muslim University, Aligarah.

Lal, M.S. & Dwivedi, A.S., 1965. Studies on the biology of the fishery of some fresh-water fishes of U. P., length-weigth relationship of Rita rita. Ichthyologica., 4: 21-26.

LeCren, E.D., 1951. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch Perca fluviatilis. J. Anim. Ecol., 20: 201-219.

McDonald, A. St. J., 1948. Circumventing the mahseer and other sporting fish in India and Burma. The Bombay Nat. Hist. Soc. 14(6): 306.

Mirza, M.R., 2004. Statusof Golden Mahseer (Pisces: Cyprinidae) of the Indus River System. Rec. Zool. Surv. Pakistan., 15: 42-44.

Mirza M.R. & Bhatti M.N., 1996. Systematics and Biology of the Golden Mahseer of the Indus River system. Biologia (Pakistan)., 42(1 & 2): 31-35.

Naeem, M., Salam, A. & Bhatti, M.N., 2000. Morphometric studies of cold water fish Rainbow trout Oncorhynchus mykiss in relation to body size. Pak. J. Zool., 1: 55-62.


Naeem, M. & Salam, A., 2004. Morphometric studies on Oreochromis nilotica (male) in relation to body size from Islamabad, Pakistan. Punjab Univ. J. Zool., 19: 73-81.

Rahman, M.A., Mazid, M.A., Rahman, M.R., Khan, M.N., Hossain, M.A. & Hussain, M.G., 2005. Effect of stocking density on survival and growth of critically endangered mahseer, Tor putitora (Hamilton) in nursery ponds. Aquaculture., 249: 275-284.

Saigal, B. N., 1964. Fishery and biology of cat fish in Ganga River system. Ind. J. Fish., 11: 1-144.

Salam, A. & Janjua, M.Y., 1991. Morphometric studies in relation to body size of farmed Rohu, Labeo rohita: A culturable major carp from Multam. J. Res. (Sci.) B. Z. University Multam (Pakistan)., 3: 59-63

Salam, A. & Davies, P.M.C., 1992. Weight-length relationship of a fresh water cold fish Carassius auratus L. Pakistan. J. Res. (Sci.) B. Z. University, Multam (Pakistan), 4: 15-18.

Salam, A. & Khaliq, R., 1992. Weight-length and condition factor relationship of a farmed mrigala, Cirrhinus mrigala. Pak. J. Zool., 24 (4): 329-33.

Salam, A. & Mahmood, J.A., 1993. Weight-length and condition factor relationship of a freshwater under yearling wild, Catla catla. Pak. J. Zool., 25 (2): 127-130.

Salam, A., Ali, M., Chatta, A.M., Akhtar, Q.A. & Zaman, Q., 1993. Growth performance of exotic Silver carp Hypophthalmichthys molitrix (VAL.) under semi-intensive farming; An ecological approach. Acta Sci., 3 (1&2): 89-100.

Salam, A., Bhatti, M.N., Chatta, A.M., Akhtar, Q.A. & Qureshi, T.S., 1994. Weight-length and condition factor relationship of Chinese Grass carp, Ctenopharyngodon idella, from Shah Mahmood Fish Farm, Multan, Pakistan. Proc. Sem. Aqua. Dev. Pakistan., 39-46.

Salam, A., Chatta A.M., Zaman, Q., Akhtar, Q.A. & Khan, S., 1994. Weight-length and condition factor relationship of a fresh water hatcher reared Rainbow Trout, Oncorhynchus Mykiss, From, Swat, Pakistan. Acta Sci., 4 (1&2): 67-72.

Salam, A. & Naeem, M., 2004. Some morphometric studies of common carp Cyprinus carpio in relation to body size, from Islamabad, Pakistan. Sindh Univ. Res. J. (Sci. Ser.)., 36: 21-48.

Salam, A., Naeem, M. & Shehnaz, K., 2005. Weight-length and condition factor relationship of fresh water wild Puntius chola from Islamabad, Pakistan. Pak. J. Bio. Sci. 8 (8): 1112-1114.

Shrestha, T.K., 1981. Fishing with open Wale Catch Boat in Narayani River System. J. Nat. Hist. Mus. 5 (1-4): 99-104.

Shrestha, T.K., 1991. Cold water Fish and Fisheries of Nepal. Fish and Fisheries at higher altitudes, Asia. FAO. Fisheries Technical Report., 385.

Shrestha, T.K., 1997. Prospects of propagation the Mahseer in Phewa Lake of the Pokhara Vally, The Mahseer, 70-71.

Sinha, A.L., 1975. Length-weight relationship of a fresh water cat fish Clarias batrachus (Lin.) Indian J. Zootomy., 14: 97-102.

Weatherley, A.H. and H.S. Gill, 1987. The biology of fish growth. Academic Press, London.


Willis, D.W., 1988. Proposed standard length-weight equation for Northern pike. N.Am. J. Fish. Manage., 9: 203-208.

Wootton, R.J., 1990. Ecology of Teleost Fishes; Chapman and Hall, London. Wootton, R.J., 1998. Ecology of Teleost Fishes; 2

nd Dordrecht: Kluwer.


Evaluation of resistance against deltamethrin in Aedes

mosquitoes from Lahore, Pakistan.

NUSRAT JAHAN & NOREEN MUMTAZ

Department of Zoology, GC University Lahore, Pakistan.

ABSTRACT

In the current study, resistance status of Aedes aegypti was evaluated against pyrethroid insecticide. The resistance in field collected population against Deltamethrin 2.5% EC was compared with susceptible (laboratory reared) population. Field population was collected from two localities of Lahore i.e. Government College University and Government Islamia College for Women Cooper Road, Lahore during July- September, 2010. CDC Bottle Bioassays were carried out on adult female mosquitoes (susceptible) in order to determine the diagnostic dose. A range of concentrations (10, 5, 2.5 and 1.25 µg/ml) of deltamethrin was used for 60 minutes exposure. Diagnostic dose 1.25 ug/ml was found in post 30 minutes exposure. The same concentration caused 100% mortality of field collected populations from GCU and Govt. Islamia College for Women in 30 and 40 minutes exposure respectively. Resistance level was expressed as resistance ratio (RR) of lethal time for 50% death determined in field collected and susceptible strain. Results of bioassays indicated that Ae. aegypti field collected population from Government Islamia College for Women Cooper Road, Lahore was resistant to Deltamethrin at RRLT50 =1.3 and RRLT95=1.37 as compared to laboratory reared susceptible population. However, field population of Ae. aegypti from Government College University, Lahore was found susceptible at RRLT50=1.03 and RRLT95=1.0. Key words: Aedes aegypti, resistance, Deltamethrin, CDC Bioassay.

INTRODUCTION

Many pathogenic diseases spread through different species of mosquitoes due to their hematophagus nature. Among these diseases Dengue fever (DF) and its complications Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) have posed serious threats since last few decades. The vector of Dengue belongs to genus “Aedes” (Subfamily: Culicinae). Two important species Aedes aegypti and Aedes albopictus, are involved in the spread of DF/ DHF in North and South America, Africa, Europe (Gratz, 1967) and South East Asian Region (SEAR) (Tewari et al., 2004) including Pakistan..

Aedes aegypti is a black colored mosquito and has silvery white scales in the form of lyre shaped pattern on the thorax region. These mosquitoes primarily breed in artificial containers like earthen jars, plastic and metal drums, used tyres, potted plants and main-hole covers etc (Chareonviriyaphap et al., 2003). Dengue is caused by “Flaviviruses” having four distinct serotypes “DEN-1, DEN-2, DEN-3 and DEN-4” (Westaway & Blok, 1997). WHO (2008) has reported dengue as re-emerging disease in SEAR and considered as it more fatal vector borne disease spreading rapidly all over the world.

There is no vaccine or proper treatment for dengue, therefore, vector control is the only option to control the disease. Chemical insecticides are

N. JAHAN & N. MUMTAZ BIOLOGIA (PAKISTAN) 10

primarily being used to control mosquito vectors. There are four classes of insecticides used for the control of mosquitoes; (1) organochlorine, (2) organophosphate, (3) carbamates and (4) pyrethroids. Pyrethroids have been successful in eradication of mosquitoes in last few decades (Nauen, 2007). Globally vector control measures are facing challenges due to the development of resistance in vector population against wide variety of insecticides (Chandre et al., 1999). Resistance in insects against commonly used insecticides was reported by many authors (Brengues et al., 2003; Liu et al., 2006; Nauen, 2007). It has become necessary to develop effective resistance management strategies due to increase in vector resistance and highly evolved resistance mechanisms (McCaffery & Nauen, 2006). Surveillance to the susceptibility status of vector is a necessary step to evaluate resistance in nature against particular insecticide (Brogdon & McAllister, 1998a). The knowledge of resistance status of Dengue vector may provide an effective data for the development of integrated vector control strategies. The main objectives of the present study were; to evaluate the resistance status of Aedes mosquitoes against Deltamethrin commonly used insecticide in Lahore, Pakistan and to find the level of resistance in two selected localities which will help in proper management of vector population control particular in areas of Lahore.

MATERIALS AND METHODS

Mosquitoes Rearing and Maintenance

Aedes aegypti were reared according to standardized conditions in the laboratory and maintained in GCU insectary since last 05 years. In the present study this colony was used as susceptible strain for the evaluation of resistance. Field Collection

The status of resistance was evaluated in the field collected Aedes aegypti against Deltamethrin (2.5% EC) in two different localities (Govt. College University (GCU) and Govt. Islamia College for Women (GICW), Cooper Road) of Lahore from July –September, 2010. Mosquito larvae were collected weekly from July 2010 to 2

nd week of September 2010. Larvae were identified on the

basis of short and thick siphon along with the black hooks at the thorax region. Moreover, Aedes aegypti had biforked comb rows on the last abdominal segment

as compared to needle like comb rows of Aedes albopictus (Rueda, 2004). Insecticide used for CDC Bottle Bioassays

Deltamethrin 2.5% EC (emulsifiable concentrate) was kindly provided by Health Directorate Cooper Road Lahore and was used to evaluate the susceptible and resistance status in laboratory reared and field collected adults Aedes aegypti females. Deltamethrin contained 2.5 gm active ingredient by weight in 100 ml of water by volume (25000 µg/ ml). Evaluation of resistance was carried out using CDC bottle bioassays (Brogdon & McAllister, 1998b). The purpose of CDC bioassay was to measure the rate of mortality of a population at a given dose of insecticide (usually the diagnostic dose). Preliminary tests were performed to determine the diagnostic dose (Benedict, 2007).

Vol. 56 (1&2) Resistance against Deltamethrin in Aedes mosquitoes 11

Experimental protocol Preliminary test for diagnostic dose

A diagnostic dose is an amount of insecticide needed to kill 100% susceptible population within specific time (30 min-1 hr). A range of insecticide concentrations (10, 5, 2.5, 1.25 µg/ml) was used to find the diagnostic dose for the susceptible population (laboratory reared Ae. aegypti). Each concentration was dissolved in 1 ml technical grade acetone with 3 replicates in 250 ml reagent bottles. Each concentration was properly labeled on the bottle and lid. Three untreated bottles containing 1 ml of acetone only acted as control. Performance of Bottle Bioassays

Bottles were prepared the day before used for testing. Each concentration (1ml) was transferred with the help of sterile disposable syringe to the relevant labeled bottle. Even coating was done on all surfaces with circular movement and upright / linear position. When the liquid disappeared lids were taken off and bottles were left on an undisturbed clean surface over night in dark. Bottles were lined up without their lids. A group of 20 non-blood fed female mosquitoes (laboratory reared) were first introduced into the control bottle and then into the experimental ones. After the determination of diagnostic dose, the experiment was performed in the same manner with wild collected mosquitoes. Data analysis

Data collected from bottle bioassay was analyzed by using SPSS computer software for Probit-regression analysis. In each group LT50 (lethal time for 50% death) and LT95 (lethal time 95% death) was calculated. Resistance ratio (RR) was calculated by dividing lethal time for wild strain (field population) with the lethal time for laboratory reared susceptible strain.

RESULTS AND DISCUSSION

The results indicated that 100% mortality occurred with deltamethrin at

concentration of 1.25 μg/ml (diagnostic dose) in the laboratory reared susceptible Ae. aegypti adult females post 30 minutes exposure (Fig. 1) The Aedes population collected from Government Islamia College for Women Cooper Road, Lahore showed low levels of resistance with respect to percent mortalities in the specific time post exposure, since 100% mortality required 40 minutes as compared to 30 minutes in laboratory reared susceptible population. In contrast population collected from GCU was found susceptible since 100% mortality occurred in 30 minutes post exposure comparable with laboratory reared susceptible population (Table.1; Fig. 2). However, Lethal times for 50% and 95% deaths i.e. LT50-LT95 were 13.137 and 25.114 minutes respectively with deltamethrin 2.5 % EC in laboratory reared (susceptible) adult females. For populations from GCU and Government Islamia College For Women Cooper Road Lahore, the LT50-LT95 were 13.599 and 25.219, 17.329 and 34.56 respectively (Table. 2). Synthetic Insecticides are used worldwide to control the vector population. Regular use of insecticides has led to the development of high rate of resistance


in insect vectors (Nauen, 2007). Thus the development of resistance has become a critical issue in all groups of insect vectors (Hemingway & Ranson, 2000). Resistance / susceptible status play an important role in any vector control program and the knowledge of this status helps to control any vector population in a particular locality. Chemical insecticides such as DDT (dichlorodiphenyltrichloroethane) and Malathion were used to control mosquitoes in the last three decades in Pakistan (Country Report, 2003). These were replaced by Deltamethrin, Fenthion, Temephos etc. due to resistance reported against DDT and Malathion in Anopheline and Culex species in Punjab, Pakistan (Rathore et al., 1980); Malcolm & Boddington, 1989). Resistance against 5% Deltamethrin was also reported in Culex quinquefasciatus from Lahore, Pakistan (Tahir et al., 2009).

In our laboratory, diagnostic dose against deltamethrin 1.5 % EC was found 2.5 µg / ml (unpublished observation) more than current study (1.25 µg/ ml) against deltamethrin 2.5 % EC post 30 minutes exposure. It appeared that more active ingredients in a formulation required lesser amount to kill 100 % populations of Aedes aegypti as compared to the formulation with fewer active ingredients. The strategy for the control of vector population with the restricted group of insecticides is very crucial and facing challenge now a days. Rotational use of different groups of insecticide rather than the use of different members of same group of insecticides is more effective to reduce and deal with the resistance problem. Carbamates and organophosphates must be used in rotation in order to maintain the pyrethroids susceptibility (Nauen, 2007). An effective long term resistance management strategy is needed in order to sustain insecticides susceptibility in nature and this is a target to be achieved in Pakistan to control vector borne diseases.

Table 1: Percent mortalities in susceptible (laboratory reared) and field collected populations against diagnostic dose (1.25 µg/ml) of Deltamethrin 2.5% EC

Time (min)

*Susceptible population

**Field population GCU

**Field population (cooper road)

Control

% mortality % mortality % mortality % mortality

10 45 40 35 0

20 75 75 60 0

30 100 100 80 0

40 100 100 100 0

50 100 100 100 0

60 100 100 100 0


*Hundred percent mortality in susceptible population in thirty minutes, ** hundred % mortality in field collected populations (GCU & Cooper road) in thirty and forty minutes respectively

Table 2: Probit regression analysis for LT50 and LT95 of susceptible

(laboratory reared) and field collected populations

Fig., 1: Evaluation of diagnostic dose against different concentrations of Deltamethrin 2.5% EC as percent mortalities in susceptible (laboratory reared) Ae. aegypti female *Diagnostic dose (1.25 µg/ml) has given 100% mortality post 30 minutes exposure

Lethal time for 50% death

Susceptible population (laboratory reared)

Field collected population (GCU, Lahore)

Field population (Cooper Road, Lahore)

Resistance ratio of field collected population (GCU)

Resistance ratio of field collected population (Cooper Road, Lahore)

LT50 13.137 13.599 17.329 1.03 1.31

LT95 25.114 25.219 34.566 1.004 1.37


Fig., 2: A comparison of susceptible (laboratory reared) and field collected populations (GCU and Government Islamia Cooper Road College, Lahore) as percent mortalities against diagnostic dose (1.25 µg/ml) of Deltamethrin 2.5% EC

REFERENCES

Benedict, M.Q., 2007. Methods in Anopheles Research. CDC Manual. Atlanta,

USA. Brengues, C., Hawkes, N.J., Chandre,F., McCarroll, F., Duchon, S., Guillet, P.,

Manguin, S., Morgan,C.J. & Hemingway, J., 2003. Pyrethroid and DDT cross resistance in Aedes aegypti is correlated with novel mutations in the voltage- gated sodium channel gene . Med. Vet. Entomol., 17: 87-94.

Brogdon, W.G. & McAllister, J.C., 1998a. Insecticide resistance and vector control. Centre for Disease Control and Prevention, Atlanta Georgia USA. Emerg. Infect. Dis., 4 (4).

Brogdon, W.G. & McAllister, J.C., 1998b. Simplifications of adult mosquito bioassays through the use of time mortalities determinations in glass bottles. J Am Mosq Control Assoc., 14: 159-164.

Chandre, F., Darrier, F. & Manga, L.., 1999. Status of pyrethroid resistance in Anopheles Gambiae sensulato. Bull. World Health Organ., 77: 230-234.

Chareonviriyaphap, T., Akratanakul, P. & Nettanomsak, S., 2003. Larval habitats and distribution patterns of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), in Thailand. Southeast Asian J. Trop. Med. Public Health., 34(3): 529-535


Country Report Pakistan 2003. Vector-Borne Diseases in Pakistan. Directorate of Malaria Control Islamabad. 11-12

Gratz, N. G., 1967.The Control of Ae.aegypti In South-East Asia and the Western Pacific. Bull.Org. mond.Sante., 36:614-617.

Hemingway, J. & Ranson, H., 2000. Insecticide resistance in insect vectors of human disease. Ann Rev Entomol., 45: 371-391.

Liu, N., Xu,Q., Zhu,F. & Zhang, L., 2006.Pyrethroid resistance in mosquitoes. Insect Sci.,13: 159-166.

Malcolm, C.A. & Boddington, R.J., 1989. Malathion resistance conferred by a carboxylisterase in Anopheles culicifacies Giles (Species B) (Diptera: Culicidae). Bull. Entomol. Res., 79: 193-199

McCaffery, A. & Nauen, R., 2006. The Insecticide Resistance Committee (IRAC): Public responsibility and enlightened industrial self-interest. Outlook Pest Manag., 17: 11-14.

Nauen, R., 2007. Perspective Insecticide resistance in disease vectors of public health importance, Pest Manag Sci., 63: 628-633.

Rathor, H.R., Toqir, G. & Reisen, W.K., 1980. Insecticide resistance in Anopheline mosquitoes of Punjab province, Pakistan. Southeast Asian J. Trop. Med. Public Health., 11(3): 332–340.

Rueda, L.M., 2004. Pictorial keys for the identification of mosquitoes (Diptera: Culicidae) associated with Dengue Virus Transmission. Zootaxa., 589:1-60.

Tahir, H.M., Butt, A. & Khan, S.Y., 2009. Response of Culex quinquefasciatus to Deltamethrin in Lahore, District. J. Parasitol. Vect. Biol.,1 (3): 019-024.

Tewari, S.C., Munirathinam, A. & Ganjanana, A., 2004. Dengue vector prevalence and viral infection in a rural area in South India. Trop. Med. Int. Health., 4: 499-507

Westaway, E.G. & Blok, J., 1997. Taxonomy and evolutionary relationships of flavivirus: Pp. 147-173.

WHO., 2008. World Health Organization. WHO position statement on integrated vector management. Wkly Epidemiol Rec., 83: 177–81.

http://www.mosquitocatalog.org/files/pdfs/wr385.pdf




16 BIOLOGIA (PAKISTAN)

BIOLOGIA (PAKISTAN) 2010, 56 (1&2), 17-21 PK ISSN 0006 – 3096

Diospyros malabarica (Desr.) Kostel. of Family Ebenaceae

and Ochna serrulata (Hochst.) Walp. of Family Ochnaceae:

Addition to the flora of Pakistan

MUHAMMAD AJAIB & ZAHEER-UD-DIN KHAN

Department of Botany, GC University Lahore, Pakistan

ABSTRACT

During the field survey of Punjab province two pretty trees were identified as

Diospyros malabarica (Desr.) Kostel., “Gaub” of family Ebenaceae and Ochna serrulata

(Hochst.) Walp. “Mickey Mouse Plant” of family Ochnaceae. They were found growing at different locations in Districts Lahore & Faisalabad for the last 10 years. Diospyros malabarica (Desr.) Kostel. was characterized by its globose fruit upto 3.5cm in diameter with persistent calyx. Young fruit of the tree was green, tinted red while Ochna serrulata

(Hochst.) Walp. was characterized by its round, succulent fruit initially green but turning red to black when ripened on the red expanded part of the stalk (receptacle) and being surrounded by the bright red petal-like structures (sepals). These plant species have not been reported in Flora of Pakistan, hence it is an addition into it. Key words: Diospyros malabarica, Ochna serrulata, family Ochnaceae, family

Ebenaceae, Gaub, Mickey Mouse Plant, Flora of Pakistan

INTRODUCTION

The genus Diospyros of family Ebenaceae, first described by Linnaeus in

1753, was documented in the Flora of Pakistan by Ghazanfar (1978) but its species Diospyros malabarica (Desr.) Kostel., was distributed in India, Thailand and North Malaysia (Wiart 2006). Diospyros malabarica (Desr.) Kostel. introduced as an ornamental tree in Pakistan, adopted well in this climate producing flowers and fruits. The ethnomedicinal information gathered from well known herbal practioners (Hakims) of District Lahore indicated that all parts of this plant specially bark, leaves, flowers and fruits are used in different medicinal preparations. The bark and leaves are anti-inflammatory, febrifuge, depurative, constipating, acrid, astringent, cooling and are used in dyspepsia, leprosy, diarrhoea, dysentry, haemorrhages, skin burning, diabetes, spermatorrhea, vaginal diseases, wounds, flatulence, prolepsis, scabies and as carminative, laxative and tonic. Fruits being bitter in taste are used as carminative, aphrodisiac and in digestive disorders. Similar medicinal uses of this plant have also been reported by Warrier et al., (1996).

Ochna serrulata (Hochst.) Walp. belonging to the Family Ochnaceae DC. having South African origin was cultivated in most parts of the world because of its showy flowers, though it is distributed to tropical zones, mainly in the Neotropics. About 37 genera and 460 species are reported so far in this family (Zhang et al.1984; Mabberley 2008). According to Brickell & Zuk (1997) the plant is resistant to spiders & mites. Ethnobotanically the plant root is used to treat enema, gangrenous rectitis and bone diseases in children (Joslyn et al., 2003).

M. AJAIB & Z. KHAN BIOLOGIA (PAKISTAN)

18

Both of the tree species are growing well in Lahore, the Capital of Punjab and the popular city of Pakistan, situated along the left bank of river Ravi and is greatly expanding due to profuse urbanization and high population growth rate. Climatically Lahore falls in subtropical arid type in which summers are really hot and the winters are mild cold (Anonymous 2009).

Lahore is known as the City of Gardens. Diospyros malabarica (Desr.) Kostel. is found almost in each garden of Lahore while Ochna serrulata (Hochst.) Walp. is growing at Badshahi Mosque or "Emperor's Mosque" in one of its lawns, opposite to Iqbal Park. Description of Diospyros malabarica (Desr.) Kostel. (Syn.: Diospyros peregrina (Gaertn.) Gurke)

Medium sized spreading evergreen ornamental tree, about 15m in height with dark gray or black bark, young parts silky and covered with gray tomentum. Leaves simple, elleptic, ovate, rounded at base, 13.5cm long and 4.5cm broad, alternate, exstipulate, coriacious, glabrous, reticulation prominent, petiolate; petiole upto 1cm long. Male flowers 3-5 in paniculate drooping cymes with pedicillate creamy white small flowers. Female flowers solitary with 4 styles and ovary 8 celled. Fruit globose, 3.5cm in diameter with persistent calyx, the young fruit, green and tinted red (Pl. 1a,b,c and d).

Fl. per: July-September. Vern. Guab,Gaabh Vern. Eng. Indian Persimmon, Voucher Specimen No. SAH: 0719.

This species was not reported by Parker (1956), Chaudhary (1969), Stewart (1972) and Ghazanfar (1978) in the Flora of Pakistan. Therefore, the key prepared by Ghazanfar (1978) needs revision to include Diospyros malabarica (Desr.) Kostel. Key to the species: Flowers sessile to shortly pedicellate. Berry 13-22mm in diameter, dark purple to black. ………………………………………………………………………….D. lotus Flowers pedicellate Pedicels 10-15mm long. Berry 3-7cm in diameter, orange to red. …......D. kaki Pedicels not more than 5mm long. Berry upto 3.5cm in diameter, young fruit green, tinted red………………………………………………….…….D. malabarica Description of Ochna serrulata (Hochst.) Walp.

An erect, evergreen shrub upto 3.5 m tall with slightly rough and much branched stem having numerous lenticels on branches. Leaves alternate, oblong to elliptic, slightly undulate, stipulate, finely serrulate, pinkish-bronze when young, glossy green when matured, 10 cm long and 5 cm broad; petiole upto 0.5 cm long. Flowers solitary or in clusters on lateral branches with stalks 1-2 cm long . Sepals 5 broadly elliptic, yellow-green, becoming enlarged and oblong with age and upto 2 cm long, strongly reflexed and bright red on maturation, persistent. Petals 5, 1.5-2 cm long, bright yellow, soon falling, fragrant. Carpel numerous, apocarpous, style common, ovary superior. Fruit (drupe) upto 15 in number, rounded to ellipsoid, green initially, turning red to black on ripening, succulent on the red expanded part of the stalk where fruit attaches (receptacle) and surrounded by the bright red petal-like structures (sepals), becoming black when mature, one seeded (Pl. 2 a,b and c and fig 1).

Vol. 56 (1&2) Diospyros malabarica and Ochna serrulata

19

Propagation: By seeds which probably are dispersed by birds (Csurhes & Edwards 1998; Brickell & Zuk 1997). Native Range: Ochna serrulata is native to Eastern Cape of Good Hope area in South Africa (Palgrave 1988 & Bailey and Bailey 1976). Fl.Pr.: March-April. Vern. Eng.: Bird's-eye bush, Mickey Mouse plant. Voucher Specimen No. SAH: 0720. The presence of these two species was a new record to the flora of Pakistan as indicated by Ajaib et al., 2010.

a b c

d e Plate 1: Diospyros malabarica (Desr.) Kostel.

a. Leaves & fruits b. Male plant preserved on SAH Herbarium sheet

c. Fruit with persistent calyx d. Shoot with female flowers e. Shoot with male flowers


20

a b c

Plate 2: Ochna serrulata (Hochst.) Walp. A, Habit; B, Flower; C, Fruit with persistent calyx

Fig., 1: Ochna serrulata: A, Habit; B, Flower; C, Druplets; D, Seed

REFERENCES

Ajaib, M., Khan, Z., Khan, N. & Wahab, M., 2010. Euryale ferox Salisb. of the

Family Nymphaeaceae: An Addition to the Flora of Pakistan. Pak. J. Bot., 42(4): 2973-2974.

Anonymous. 2009. Annual rainfall, relative humidity and temperature of Lahore 2004-2009. Pakistan Metrological Department Jail Road Lahore, Pakistan.

Vol. 56 (1&2) Diospyros malabarica and Ochna serrulata

21

Bailey, L. H. & Bailey, E. Z., 1976. Hortus third: A concise dictionary of plants cultivated in the United States and Canada, MacMillan, New York. pp:775.

Brickell, C. & Zuk, J.D., 1997. The American Horticultural Society A-Z Encyclopedia of Garden Plants. DK Publishing, Inc., NY.

Chaudhary, S.A., 1969. Flora in Layallpur and adjacent Canal Colony. W. Pak. Agricultural University Lyallpur.pp:167.

Csurhes, S. & Edwards, R., 1998. Potential Environmental Weeds in Australia: Candidate species for preventative control. Canberra, Australia. Biodiversity Group, Environment Australia. pp:208.

Ghazanfar, S.A., 1978. Flora of West Pakistan. No. 116. Ebenaceae. (E. Nasir and S.I.Ali Eds.) National Herbarium, Agriculture Research Council, Islamabad, Pakistan.

Joslyn, L., Taylor, S., Esameldin, E.E., Maes, A, Gorp. U.V., Kimpe, N.D., Staden, J.V. & Verschaeve, L., 2003. Investigating the safety of plants used in South African traditional medicine: Testing for genotoxicity in the micronucleus and alkaline comet assays. Environ. Mol. Mutagen, 42: 144-154.

Mabberley, D.J., 2008. Mabberley's plant-book: a portable dictionary of plants (3

rd Ed.). Cambridge University Press. pp: 594.

Palgrave, K.C., 1988. Trees of Southern Africa. Struik Publishers, Cape Town, South Africa.

Parker, R.N., 1956. A Forest Flora for the Punjab with Hazara and Delhi, Ed. 3. Superintendent, Govt. printing press Lahore. pp: 307-310.

Stewart, R.R., 1972. An Annotated Catalogue of the Vascular Plants of West Pakistan and Kashmir. Flora of West Pakistan E. Nasir and S.I. Ali, (eds.) Fakhri Printing Press, Karachi.

Warrier, P.K., Narrbiar, V.P.K., Ramankutly, C. & Nair, R.V., 1996. Indian Medicinal Plants: a compendium of 500 species. Orient Longman Private Limited, 160 Anna Salai Channai, India. 2. pp: 339.

Wiart, C., 2006. Medicinal Plants of Asia and Pacific. Taylor & Francis group, LLC. CRC Press, USA. pp: 75-76.

Zhang, Z., Maria, D.C. & Amaral, E., 1984. Flora of China. FOC. 12. pp: 361.

http://www.hear.org/pier/references/pierref000038.htm

http://www.hear.org/pier/references/pierref000014.htm

http://www.efloras.org/person_profile.aspx?person_id=8462

http://www.efloras.org/person_profile.aspx?person_id=8576

BIOLOGIA (PAKISTAN) 2010, 56 (1&2), 23-30 PKISSN 0006 – 3096

Effect of livol (herbal polysaccharide) as an

immunomodulator in commercial broilers

MUHAMMAD FIAZ QAMAR, MUHAMMAD ZAFAR, AFTAB AHMAD

ANJUM, MUHAMMAD IMRAN NAJEEB & AZHAR MAQBOOL

1Department of Zoology, Govt. College University, Lahore (MFQ)

2Faculty of Veterinary Science, University Of Veterinary and Animal Sciences, Lahore

(MZ, AAA, MIN, AM)

ABSTRACT

In commercial broiler production, immune status of the birds has direct influence

on the morbidity and mortality due to various contagious and infectious diseases of poultry. At different ages, outbreaks of different infectious diseases reduce the profitability of farmers. The obvious cause of such ailments is the depression in antibody titers which may be increased by vaccination. Recently failure of vaccines has been noticed very frequently and to overcome such situations use of herbal polysaccarides has been a routine practice. The present study has been undertaken to validate the role of herbal products. The livol increased immunity (%) and heptoprotective (%) ability in comparison to control group. Compared to the birds in vaccinated groups that were kept without feeding Livol, the sera of Infectious Bursal Disease IBD (Hot), Infectious Bursal Disease IBD (Intermediate) and Newcastle Disease (NDV) vaccinated birds kept on Livol had higher antibody titers on day 42. Amongst various treatment groups the highest haemagluttination inhibition titers was recorded in IBD (Hot) feed with Livol treated birds as compared to the other groups. These findings suggested that Livol (Herbal Polysaccaride) can effectively stimulate/enhance the immunity in broiler chicks and Livol (Herbal polysaccarides) can be potential ameliorator against various vaccines and its adverse/suppressive effects in broiler chicks.

KEY WORDS: Livol, Herbal Polysaccharides, Immunomodulators, Commercial Broilers.

INTRODUCTION

Research interest has focused on various herbs that possess hypolipidemic, antiplatelet, antitumor, or immune-stimulating properties that may be useful adjuncts in helping reduce the risk of cardiovascular diseases and cancer. In addition to their antimicrobial activity (Valero & Salmeron, 2003), many chemicals, drugs and antibiotics being used as therapeutic agents for the treatment of various disease problems in man, poultry and others have immunomodulatory effect. Suppression of immune responses may be due to temporary or permanent damage to primary lymphoid organs (bursa of Fabricius and thymus) which might result in increased susceptibility of the host to various bacterial, viral and parasitic infections (Muneer et al., 1988).

In poultry production, it is very important to improve immunity so as to protect the birds from infectious diseases. A variety of factors can induce immunodeficiency, such as vaccination failure, infection by immune suppressive diseases, and abuse of antibiotics. Utilization of immunostimulants is one solution to improve the immunity of animals and to decrease their susceptibility to

BIOLOGIA (PAKISTAN) M. F. QAMAR 24

infectious diseases (Liu et al., 2006). Polysaccharides are considered natural immunostimulants that have been shown to promote the secretion of cytokines and antibodies, as well as enhance the function of natural killer cells, T and B lymphocytes (Nie & Zhang 1999). In clinical practice, there are two basic ways to administer polysaccharides: oral administration and injection. Oral administration is the preferred way because of its convenience to use but it is argued that oral administration may decrease the immunostimulating effects of polysaccharides because of the possibility of being destroyed by intestinal enzymes (Wang & Hu.2000).

In the current study, two Chinese herbal polysaccharides: astragalan (APS) and achyranthan (ACH), were investigated. APS is extracted from the Chinese herb Astragalus membranaceus, which belongs to the Fabaceae (Le- guminosae) family. Astragalus membranaceus is a frequently used herbal medicine in China for its activity to treat fatigue, sweating, poor appetite, heart disease, hepatitis, etc. APS is the main ingredient of Astragalus membranaceus. It is a large-molecular- weight polysaccharide that has been intensively studied in human medicine, but rarely utilized in animal production (Shan et al., 2000). ACH is a novel low-molecular weight polysaccharide extracted from the Chinese herb Achyranthes bidentata, which has shown potent immunostimulating effects in human and rats when administered orally or by injection (Tian & Feng 1994). The present study was designed to evaluate the effect of herbal polysaccharides on humoral immune response of ND vaccine in broiler chicks.


Experimental brioler chicks A total of 150 day-old-chicks were reared in the experimental poultry

shed, Department of Microbiology University of Veterinary Sciences Lahore under optimal managemental conditions. The chicks were reared until 42 days of age in experimental broiler houses. Birds in all the groups were grouped and treated as shown in Table 1.

Table 1: GROUPING OF EXPERIMENTAL CHICKS

Groups No. of birds

Dose rate of herbal Polysaccharide (Livol)

Herbal Polysaccharide (Livol) Schedule of birds in days

Vaccination Schedule (Age of Birds in days)

Challenge With virulent ND virus

Group A

A1 5 Control No treatment ND=6 &21 Not challenged

A2 5 Control No treatment ND=6 &21 challenged

Group B

B1 25 1ml/liter In drinking water

throughout the experiment

ND=6 &21 Not challenged

B2 25 1ml/liter In drinking water


ND=6 &21 challenged

Group C

CC1

25 2ml/liter in drinking water


ND=6 &21 Not challenged

Effect of Livol as an Immunomodulator Vol. 56 (1&2) 25

CC2

25 2ml/liter in drinking water


ND=6 &21 challenged

Blood/serum collection

Ten birds were randomly selected for blood collection from each group. Blood sample were collected from each group on 1, 7, 14, 21, 28, 35 and 42 day of their age. The blood was collected from each bird separately and was allowed to clot at room temperature in order to separate the serum. The serum was then pipetted out and stored at -20°c until further processing. Half of the samples were collected in tubes containing EDTA to prevent the blood from clotting for hematological analysis. Serological tests

The serum samples were analyzed by Haemagglutination Inhibition (HI) test for the detection of antibodies against NDV (Allan et al., 1978). Determination of specific antibody level against newcastle disease virus

The maximum dilution of each serum sample causing inhibition of hemagglutination was noted. The HI titre of each serum sample was expressed as reciprocal of the serum dilution. Geometric Mean Titer (GMT) for the various groups was calculated by using method as described by Brugh (1978). Statistical analysis

Data for all response variables were subjected to Complete Randamized Block Design analysis (Burg, 1998).


This study was conducted to evaluate whether Livol (herbal

polysaccharide) had any effect on the immune system of broiler chickens raised in stress conditions by different vaccines. The effects of recommended dosage levels of Livol on immune system of birds were evaluated by recording their body, bursal, splenic and thymic weights response of birds to NDV and IBDV vaccination and the ability of birds to resist the immunosuppressive effects of different vaccine, therefore the effects of Livol (Herbal Polysaccharide) on immune system, bursal, splenic and thymic weights, responses of the birds to NDV and IBDV vaccination and the capacity of the vaccinated birds to resist the immunosuppressive effects of these vaccination, were compared to the non-vaccinated and control groups.

After dividing the birds into their respective groups according to experimental design given in the materials and methods, the groups that were offered Livol (Herbal Polysaccarides) alone in the feed showed significantly higher BF, Splenic and Thymic weights compared with the rest of the groups at (p<0.05). Additionally, this higher BF weights were consistently present throughout the monitoring period in this group. Interestingly, the BF Splenic and Thymic weights of all other groups were almost the same (Fig. 1). Group A1: These birds were vaccinated against ND Vaccines on days 6

th & 21

st;

Infection was given to this group on 35th day of Age, which acted as a control

group reared without Herbal Polysaccharides. Three birds out of twenty-five birds were recorded as dead. Mortality was 12% and Protection was 88%.


Group A2: Similar to group A1, Two birds out of twenty-five birds were recorded as dead. Mortality was 8% and Protection was 92%. Group B1: These birds were vaccinated against ND Vaccines on days 6

th & 21

st;


group reared without Herbal Polysaccharides. One bird out of twenty-five birds was recorded as dead. Mortality was 4% and Protection was 96%. Group B2: Similar to group B1, One bird out of twenty-five birds was recorded as dead. Mortality was 4% and Protection was 96%. Group C1: These birds were vaccinated against ND Vaccines on days 6

th & 21

st;


group reared without Herbal Polysaccharides. No bird out of twenty-five birds was recorded as dead. Mortality was 0% and Protection was 100%. Group C2: Similar to group C1, No bird out of twenty-five birds were recorded as dead. Mortality was 0% and Protection was 100%.

Fig., 1: The protection profile of all groups


EFFECT OF HP ON BURSAL WEIGHT

0

0.5

1

1.5

2

2.5

3

3.5

A1 A2 B1 B2 C1 C2

GROUPS

WE

IGH

T I

N G

RA

MS

14

21

28

Fig., 2: Comparison of mean bursal weights of chickens in different groups at days 14, 21 & 28.

The results presented in Fig., 3 explained that Livol (herbal

polysaccharides) has affected the bird’s health performance and resulted in high BF Splenic and Thymic weights. Moreover, the uses of Livol improve the immunosuppressive effects of Vaccines in groups vaccinated with IBD (I) +ND, IBD (H) +ND (Fig. 2).

Geometric mean titers against ND virus on day one were calculated as

36.76 & 39.4 in A1 & A2, 42.22 & 36.76 in B1 & B2 and 39.4 & 36.76 in Groups C1 & C2 respectively, which declined until 14

th day to a level of 17.15&17.15 in

A1 & A2, 16 &13.93 in B1 & B2 and 17.15 & 16 in Groups C1 & C2 respectively (Fig. 2). The groups that were treated with Livol (herbal polysaccharides) in the feed showed an increased antibody titer response compared with the rest of the groups. Additionally, these high titers were consistently present throughout the monitoring period in this group. Interestingly, the antibody profiles of all other groups were almost the same (Fig. 2). Overall, antibody titers declined from 1

st to

14th day of age in all groups followed by an increase at 21

st day. Another slump in

antibody titers became evident on 28th day of age. This was again followed by a

gradual increase from 35th day onwards which continued even on 42

nd day of

observation. These results indicated that Livol (herbal polysaccharides) supported the immune system of the birds and resulted in high antibody titer against ND, vaccine. Moreover, the use of commercially available Livol ameliorated the immunosuppressive effect of Vaccines (Fig. 2).


Effect of HP on AntibodiesTiter

0

50

100

150

200

250

300

1 7 14 21 28 35 42

Age in Days

GM

T

A1

A2

B1

B2

C1

C2

Fig., 3: Comparison of gm (hi titers) of chickens in various treatment groups at day 42.

Mean antibody titer against ND was significantly (P<0.05) higher in-group

C1 & C2 among the treatments. While lower antibody titers were observed in the groups that were fed high concentrations of herbal polysaccharides. Findings of our research study are correlated to the findings of previous researchers who reported that serum antibody titer against new castle disease were increased by feeding Aloe Vera (Ziauddin et al., 1995; Bilal et al., 2002; Jinag et al., 2005; Valle et al., 2005). Our study had contrast results to the above mentioned findings of some poultry scientists, who reported that there is no influence (P<0.05) on antibody titer against ND while feeding with aniasomnifera to the broiler chicks (Mushtaq et al., 2011). There was significant increase in “relative organ body weights ratio” of spleen, thymus and bursa of Fabricius weights for the chicken fed Livol (Herbal polysaccharides) (Figure 3). These changes in relative organ body weights were consistent with previous reports (Kubena et al., 1993), who also documented similar changes as of this study. However, the “relative organ body weights ratio” of the chicks untreated with Livol (Herbal polysaccharides) were significantly less as compared to the previous one, which indicated that the addition of Livol (Herbal polysaccharides) to the feed had protective/stimulating effects against the stress produced by vaccination in broiler chickens.

In conclusion, use of commercially available Heptoprotective, growth stimulant and immunity enhancer Livol (Herbal Polysaccharide) is beneficial for poultry farming. Economic gains (body weight) not only outweigh the cost of use of the product (Livol), the very use of Livol can also result in production of better quality chicken meat for human consumption. It was clear from the results of the HI tests, that feeding of Livol (herbal polysaccharides) caused an improved immune-response in the experimental broiler chickens to ND, vaccines. On the


other side, the vaccinated groups that were kept control to Livol (herbal polysaccharides) showed depressed immune response as compared to the Livol (Herbal polysaccharides) fed groups.

REFERENCES

Valero. M. & Salmeron, M.C. 2003. Antibacterial activity of 11 essential oils against Bacillus cereus in tyndallized carrot broth. Int. J. Food Microbiol., 85: 73-81

Muneer, M. A., Newman. J. A., Farah, I. O. & Goyal, S. M., 1988. immunosuppression in animals. British Vet. J., 144:288-301.

Liu, Yuqin; Liu, Shengwang; Shan 2006. Anshan Influence of mannan oligosaccharide on parameters of antioxidative and immunological status of broilers. Arch. Anim. Nutr., 60 (6) (10): 467-476.

Nie, W, & Zhang, Y. X., 1999. Progress of the immunomodulating effect of Polysaccharides and their mechanism. Chinese Pharm. Bull., 15:3–5.

Wang, D. Liu, & Hu. Z. B., 2000. Progress of chemical and pharmacological study of astragalus polysaccharide. Acta Shanghai Univ. Trad. Chin. Med., 14:61–65.

Shan, J. J., Wang, S. C., Liu, D. & Hu. Z. B., 2000. Progress of chemical and Pharmacological study of astragalus polysaccharide. Acta Shanghai Univ. Trad. Chin. Med., 14:61–65.

Tian, G. Y, & Feng, Y. C. 1994. Progress in the research and application of polysaccharides. Chem. Prog., 6:44–48.

Allan, W. H., Lancaster, J. E., & Toth, B. 1978. Newcastle disease vaccines production and their use. FAO United Nations, Rom: 57-62.

Burg, M. A., 1998. Simple method for recording and analyzing serological data. Avian Dis., 2:362-365.

Buxton, A. & Fraser, G. 1977. Animal Microbiology. Vol. 2. Blackwell Scientific Publications, London, UK. pp: 481

Jinag, L., Zhang, F.Y., Xu, Y., Xinting, Z., & Yang, D.P., 2005. Effect of gel. Polysaccharide and acemannan from Aloe vera on brioiler gut flora, microvilli density, immune function and growth performance. Chinese J. Vet. Sci., 25 (6): 668-671

Bilal. B. haque, H., R., Parvez, S. Pandey, S., saeed I., & Raisudin, S. 2002. Immunomodulatory effects of fenugreek extract in mice. Department of Medical Elementology and Toxology, Jamia Hamdard University New Delhi, India.

Ziauddin, S. K., Rao, D. N. & Amla, B. L., 1995. Effect of lactic acid, ginger extract and sodium chloride on electrophoretic pattern of buffalo muscle proteins. J. Food Sci. Technol., 32:224–226.

Valle, P.M., Vidamo, G.R., Anunciado, P.J.S, & Lapitan, R.V.P., 2005. Effects of aloe vera (Aloe barbadensis) on the white blood cell count and antibody titer of broiler chickens vaccinated against Newcastle disease. Phil. J. Vet. Med., 42 (1): 49-52

Mushtaq, M., Durrani, F.R., Imtiaz, N., Sadique, U., Hafeez, A., Akhtar, S., & Ahmad, S., 2011. Effect of administration of Withania somnifera on


hematological and immunological profile of broiler chicks. Pak. Vet. J., 31(x): xxx.

Kubena, L.F., Harvey, R.B., Phillips, T.D. & Clement, B.A., 1993. Effect of hydrated Sodium calcium aluminosilicates on aflatoxicosis in broiler chicks. Poultry sci., 72(4); 651-657


Effect of artificial diets on the growth and survival of

rotifers

ABDUL QAYYUM KHAN SULEHRIA, IFFAT YOUNUS & ALTAF HUSSAIN

Department of Zoology, GC University, Kachery Road, Lahore, Pakistan.

ABSTRACT

The aim of present study was to culture the rotifers in laboratory on different

combinations of artificial diet media i.e., Baker’s yeast, starch and albumen. Rotifers were collected from “Manawa” ponds and cultured on six different combinations of diet media. Experiment was carried out at room temperature and continued for six days. Different parameters were determined and maintained during experiment and these were pH, Temperature, Count of rotifers and Change in biomass. Results showed that, among these six diet media, the rotifer number increased in those media which contained either starch or a combination of starch with other media. Statistical analysis showed the significant difference in rotifer count among these diet media. Keywords: rotifers, yeast, rotifer diets, rotifer culture

INTRODUCTION

Zooplanktons plays important function in the food chain as animal food, which is a source of amino acids, fatty acids, vitamins, minerals, etc. (Watanabe et al., 1983). It has been attempted to substitute live food by commercial diets, to rear fry and juvenile of fishes in hatcheries, however, it still depends on the adequate supply of zooplanktons (Habib et al., 1988). The use of the proper food for fry and juvenile stages is essential for proficient and economical hatchery techniques. Generally, rotifers have ample nutrient content and a high rate of reproduction (Lubzens, 1987). The rotifers serve as the first food to fish fry and other organisms during first few days after hatching (Lubzens et al., 2001). Rotifers provide essential nutrients, vitamins and even antibodies to fish fry (Gastesoupe, 1982). The amount of fatty acid in rotifer is thought to control both survival and growth rate of fish larvae (Koven et al., 1990). The culture of freshwater fish fry requires a suitable food source, such as living rotifers. In freshwater aquaculture, Brachionus calyciflorus. B. patulus and B. rubens have been used as food for fish fry (Groeneweg & Schluter, 1981; Mookerji & Rao 1994). To improve larval rearing techniques, a good understanding of larval morphology, behaviour, live food and artificial diet requirements, and environmental conditions is fundamental (Liao et al., 2001). Rotifers are cultured by feeding them microalgae. Rotifers can also be fed yeast, but are more nutritious when fed algae, as they have higher protein and lipid contents. Rotifers also have been shown to be more digestible. (Nadolny, 2003). Apart from fresh Baker’s yeast, instant baker’s yeast, marine yeast or cake yeast may also be used.

http://www.scielo.sa.cr/scielo.php?script=sci_arttext&pid=S0034-77442001000100008&lng=es&nrm=iso#Mookerji



A. Q. K. SULEHRIA ET AL BIOLOGIA (PAKISTAN)

32

The aim of this work was to study the growth rate of mixed culture of rotifers at room temperature, on various combinations of artificial diets such as starch, yeast and albumen.


Sample Collection Water sample containing rotifers was collected using Wisconsin

Planktonic net of 37m. Water sample was taken from the bank of “Manawa” ponds, just below the water surface (about 15-25cm) (Pennak, 1978). Identification of rotifers was carried out by observing their body shape, morphological features and behavior (Hyman, 1951; Ward & Wipple, 1959; Segers, 2007). Culture Media To culture the rotifers on artificial diet media, six combinations of diet media were used, which were as follows: 1. Diet medium 1: 10 ml Distilled H2O + 0.25 gm Yeast +

0.25 gm Starch 2. Diet medium 2: O + 0.25 gm Yeast 3. Diet medium 3: 10 ml Distilled H2O + 0.25 gm Starch 4. Diet medium 4: 10 ml Distilled H2O + 0.25 gm Yeast +

0.25 gm Albumen 5. Diet medium 5: 10 ml Distilled H2O + 0.25 gm Yeast +

0.25 gm Starch+ 0.25 gm Albumen 6. Diet medium 6: 10 ml Distilled H2O + 0.25 gm Albumen 7. Control medium Pond water and natural algae For the preparation of the above-mentioned media seven sets of petri-plates were used, each set containing five petri-plates. After the preparation of culture media, one ml mixed rotifer sample was added to each petri-plate. The mixed culture of rotifers contained Brachionus calyciflorus, Brachionus angularis, Filinia Iongiseta, Philodina roseola, and Keratella valga. The counting of rotifers was carried out by haemocytometer and OLYMPUS microscope (Taylor et al., 1997). Rotifer count of each diet media was compared with other diet media and control media by applying t-test to investigate its significant difference at 0.05 probability.

RESULTS

Diet Medium I (Yeast + Starch)

This medium was found to be effective for the growth of rotifers. Number of rotifers increased during first three days and their maximum number was observed on third day, i.e. 50% increase was observed in their number. But there was a decline in number of organisms in last two days.

Vol. 56 (1&2) Effect of artificial diets on rotifers 33

Initially pH was 8, but on last day of experiment it was 9 - 9.5. The decrease in biomass was observed in this diet medium. Comparison of rotifers count in diet medium 1 with other diet media by applying t-test showed that, it had significant difference from diet medium 2, 4 and 6, at 0.05 probability and degree of freedom 8 (Table 1, 2). Some species survived till end of the experiment which were Brachionus calyciflorus, Brachionus angularis, Filinia Iongiseta.

Table 1: Comparison of change in Bimass of six different Diet Media

No. of

Days

No. of Petri-plates

% Change in wt. in

Diet Medium

1

% Change in wt. in

Diet Medium

2

% Change in wt. in

Diet Medium

3

% Change in wt. in

Diet Medium

4

% Change in wt. in

Diet Medium

5

% Change in wt. in

Diet Medium

6

1 A -3.5 -2.7 -3.1 -1.7 -3.6 -3.3

2 B -3.9 -4.1 -2.8 -2.5 -3.6 -4.4

3 C -3.5 -1.7 -2 -2.8 -3.6 -2

4 D -4.1 -3.4 -3.7 -3.2 -3.7 -3.4

5 E -4.7 -3.5 -3.8 -2.3 -3.6 -2.4

Mean % change

-3.9 -3.1 -3.1 -2.5 -3.6 -3.1

Diet Medium 2 (Yeast)

In this diet medium there was no increase in rotifer numbers on first day. 5% increase in number was observed on second day. But there was continuous


34

decline in their number in last three days of experiment. Decrease in biomass was also observed (Fig., 1). Comparison of rotifer count of diet media 2 with other diet media showed its significant difference from diet media 6 at 0.05 probability & degree of freedom 8 (Table 1, 2). In this diet medium maximum number of rotifers died after second day, but among the surviving rotifers Brachionus calyciflorus & Keratella valga were prominent.

Table 2: Comparison of t-test values of Rotifer count in six different Diet Media (DM)

Diet Medium 3 (Starch)

Starch was also found effective diet component for culture of rotifers. In this diet medium continuous increase in number of rotifers was observed up to fourth day of experiment i.e., about 60%, increase in number. But number of rotifers decreased on last day (Fig., 1). A decrease in biomass was also determined. t-test showed that this medium has significant difference from diet media 2, 4 and 6, at 0.05 probability & degree of freedom 8 (Table 1, 2). In this diet medium maximum increase was observed in Brachionus angularis and Brachionus calyciflorus. A few Philodina roseola were also present.

Diet Medium 4 (Yeast + Albumen)

This diet medium did not support the rotifers to maintain their growth. They died rapidly & their number decreased very quickly. Biomass of the petri-plates decreased. Comparison of change in biomass of six different diet media showed that in this diet medium minimum mean % change was observed i.e., -2.5% (Table 1).

Comparison of rotifer count of this diet medium with other diet media showed insignificant difference, as the number of rotifers decreased rapidly in this diet medium (Table 2). So there were no particular species which grew better and could be identified.

Diet Medium 5 (Yeast + Starch + Albumen)

This diet medium proved to be very effective for the growth of rotifers, as maximum growth of rotifers was observed in this diet medium. In first three days


about 70 % increase occurred, but number of rotifers decreased in last two days (Fig., 1). Biomass decreased gradually. Comparison of rotifer count of this diet medium with other diet media showed that it had significant difference with diet media 2, 4 and 6 (Table 1, 2). Rotifer species which grew best in this diet medium were Brachionus calyciflorus, Brachionus angularis, Filinia longiseta and Philodina roseola.

Diet Medium 6 (Albumen)

No growth of rotifers took place in this diet medium. Most rotifers rapidly died in first two days and the rest were died on day 3 to onward. Biomass of this diet medium also decreased. Comparison of this diet medium with other diet media showed insignificant difference (Fig.,1; Table 1-2).

DISCUSSION

Rotifers are opportunists which respond very quickly to any change in the food types and levels (Nogrady et al., 1993). Rotifers show almost linear numerical addition with rise in food levels and it has been observed in many rotifer genera of family Brachionidae (Dumont et al., 1995; Lucia-Pavon, 2001; Sarma, et al., 2001; Peredo-Alvarez, et al., 2003).

The results of present work showed that, the artificial diet can be used to culture the rotifers or to maintain their growth (Hoff & Snell, 1987; Lubzen, 1987). Out of six diet media, the diet medium which contained starch alone, or in combination with other diet components, was effective for the growth of rotifers. However, maximum growth observed in that medium which contained a mixture of yeast, starch and albumen. On the other hand, the diet medium which contained albumen alone or in combination with yeast didn’t support the growth of rotifers. Their number declined rapidly in these media. It was due to the fact that, the albumen was not a suitable food component for rotifers or the rotifers did not have the ability to digest the albumen.

In the present study, rotifer could not survive more than 4 or 5 days. Because when rotifers were transferred from natural fresh water medium to the artificial diet media, there was a change in their micro-flora i.e., algae and other organic matter present in pond water. The bacteria and other micro-organic particles are important to maintain rotifer culture and to increase their digestibility. (Liao et al., 1993). Other factors may include rise in pH and NH3.

Experiments conducted in present study were very successful, as it provided guide lines for further research in the field of rotifer culture on artificial media. Algae are normally used to culture rotifers throughout the world.

Conclusion

Finally the present work had proved that:

The rotifers can be grown on artificial diet in laboratory, but their life span is short.

The results showed that the rotifers are selective in the choice of medium. it means they may grow on a specific type of diet medium, not on all diet media.


36

Artificial diet media are easily available and easy to maintain.

As an alternate food, the artificial diets should be used on large scale for rotifer culture.

Large scale availability of rotifers might be beneficial to rear fish fry.

Results also indicated that, the presence of specific type of micro-flora are also essential to digest the food for rotifers.

REFERENCES

Dumont, H. J., Sarma, S. S. S. & Ali, A. J., 1995. Laboratory studies on the

population dynamics of Anuraeopsis fissa (Rotifera) in relation to food density. Freshwater Biol. 33: 39-46.

Gastesoupe, F.J., 1982. Nutritional and antibacterial treatments of live food organisms: the influence on survival, growth rate and weaning success of turbot (Scophthalmus maximus). Ann. Zootech., 31:353-368.

Groeneweg, J. & Schluter, M., 1981. Mass production of freshwater rotifers on liquid wastes II. Mass production of Brachionus rubens Ehrenberg, 1838 in the effluent of high-rate algal ponds used for the treatment of piggery waste. Aquaculture., 25: 25-33.

Habib, M. A. B., Moshihuzzaman, M. & Rahman, M. S., 1988. Combined and linear effect of dominant genera of phytoplankton on the abundance of eleven genera of zooplankton. Bangladesh J. Zool., 16: 31-38.

Hoff, F. H. & Snell, T.W., 1987. Plankton culture manual, First edition, Florida Aqua Farms, Inc., Florida. USA. 126 pp.

Hyman, L. H., 1951. The Invertebrates. Vol. III. Acanthocephala, Aschelminthes and Entoprocta. McGraw-Hill, New York. 55 pp.

Koven, W.M., Tandler, A., Kissil, G.V., Friezlander, O. & Harel, M., 1990. The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae. Aquaculture., 1: 131-141.

Liao, P. H., Vizcarra, A. T., Chen, A. & Lo, K. V., 1993. Composting separated solid swine manure. J. Environ. Sci. Health, A28(9): 1889-1901.

Liao, I.C., Su, H.M. & Chang, E.Y., 2001. Techniques in finfish larviculture in Taiwan. Aquaculture. 200:1-31.

Lubzens, E., 1987. Raising rotifers for use in aquaculture. Hydrobiologia, 147: 245–255.

Lubzens, E., Zmora, O., & Barr, Y., 2001. Biotechnology and aquaculture of rotifers. Hydrobiologia, 446: 337–353.

Lucia-Pavon, E., Sarma, S. S. S. & Nandini, S., 2001. Effect of different densities of live and dead Chlorella vulgaris on the population growth of rotifers Brachionus calyciflorus and Brachionus patulus (Rotifera). Rev. Biol. Trop. Dic., 49(3-4): 895-902.

Mookerji, N. & Rao, T. R., 1994. Influence of ontogenetic changes in prey selection on the survival and growth of rohu, Labeo rohita and singhi, Heteropneustes fossilis larvae. J. Fish Biol., 44: 479-490.

Nadolny, 2003. http://mikejacobs.50megs.com/WhatsARotifer.html


Nogrady, T., Wallace, R.L. and Snell, T.W., (eds) 1993. Rotifera-guides to the identification of the microinvertebrates of the continental waters of the world. 4 SPB Academic Publishing. The Hague. pp: 142.

Pennak, R. W., 1978. Fresh-Water Invertebrates of the United States. 2nd

Ed. Wiley, New York. pp:803.

Peredo-Alvarez, M. Víctor, Sarma, S. S. S. & Nandini, S., 2003. Combined effect of concentrations of algal food (Chlorella vulgaris) and salt (sodium chloride) on the population growth of Brachionus calyciflorus and Brachionus patulus (Rotifera). Rev. Biol. Trop., 51(2): 399-408.

Sarma, S. S. S., Susana, P., Jurado, L. & Nandini, S. 2001. Effect of three food types on the population growth of Brachionus calyciflorus and Brachionus patulus (Rotifera: Brachionidae). Rev. Biol. Trop., 49(1):77-84.

Segers, H. 2007. Annotated checklist of the rotifers (phylum Rotifera), with notes on nomenclature, taxonomy and distribution. Zootaxa. 1564:1-104.

Taylor, D. J., Green, N. P. O., & Stout, G. W., 1997. (Reprinted 2008). Biological Science (Cambridge low price editions). 3

rd Ed. Printed in India at

Replika Press Pvt. Ltd., Kundli 131 028. pp: 387-388. Watanabe, T., Kitajima, C., & Fujita, S., 1983. Nutritional values of live organisms

used in Japan for mass propagation of fish: a review. Aquaculture., 34: 115–143.

Ward, H. B. & Whipple, G. C., 1959. Fresh Water Biology.2nd

ed. John Wiley and Sons. New York. 1248 pp.

BIOLOGIA (PAKISTAN) 2010, 56 (1&2), 39-54

PK ISSN 0006 – 3096

Pregnancy and fetal correlations of cypermethrin in mice

(Mus musculus)

KAUSAR RAEES, ASMATULLAH & KHAWAJA RAEES AHMAD

Government College for Women Farooq Colony Sargodha (KR), Department of

Zoology, University of the Punjab, Lahore (A) and University of Sargodha, Sargodha (KRA), Pakistan

ABSTRACT

In the present study Cypermethrin, [(R, S) -cyano-3-phenoxybenzyl (1R, S)-

cistrans-3-(2, 2- dimethylcyclopropane carboxylate) was tested for its capacity to induce pre and peri and pan-gestational losses and teratological changes in albino laboratory mice (Mus musculus). After careful estimation of LD50 for pregnant mice (64mg/kg body weight) 5, 10 and 20% (i.e. 3.2, 6.4 and 12.8mg/kg BW of the dams) of LD50 were chosen as experimental doses. A group of 20 Dams was maintained as control. Experimental doses were applied on GD6 and GD6, 9 and 12 (called single and triple exposures respectively) to a faction of 20 Dams each. Fetuses were exteriorized on GD18 to estimate pre, peri and pan-implantational losses. Fetuses were subjected to study for density/ litter/ faction, Crown Rimp Length (CRL) and Head circumference.

Results indicate a dose and exposure dependent increase in pre, peri and pan-gestational losses. Maximum fetal density was recorded in 3.2mg/kg single exposure sub-group. While in single exposure factions of the remaining two groups it showed dose and exposure related decline. A secondary decline in fetal density was noted in all groups at triple exposure. A dose dependent increased frequency of feto-morphic defects including, microcephaly, hydrocephaly, un-detached pinnae, epinnate ears, skewed neck, meromelia, extradactyly, drooping wrist, round back, hemoregia, torted hid limbs, forked paws, flipper limbs and kinky tail were observed. Fetal head circumference and CRL also showed a dose dependent decline.

Results indicate that cypermethrin is a potent developmental toxicant. It brings about various morphological abnormalities along with significant changes in morphometric data.

Key Words: Cypermethrin, Pan-gestational losses, Fetal derangements

INTRODUCTION

Fetal development is a dynamic process that includes changes in

morphology, anatomy, physiology, biochemistry and general growth (Roger & Kevlock, 2001). Exposure to various environmental chemicals (especially pesticides) during developmental period is liable to give rise to congenial defects (Akhtar, et al. 2006). By now there exist a vast number of evidences that indicate the susceptibility of developing embryos to environmental toxicants especially insecticides (Gupta, 1990; Roy, et al. 1998; Tian, et al. 2005: Slotkin, et al. 2006). The environmental agents interfere with the developmental processes thus derailing them from giving their proper end results (Uggini et al. 2010).

Organophosphates and pyrethroids are the two most commonly used groups of pesticides that are, unfortunately, also known to influence embryonic

K. RAEES ET AL BIOLOGIA (PAKISTAN) 40

development in non-target animals (Ahmad & Asmatullah, 2007, Uggini, et al. 2010). Cypermethrin [(R, S) -cyano-3-phenoxybenzyl (1R, S)-cistrans-3-(2, 2- dimethylcyclopropane carboxylate] is a type II pyrithroid insecticide that is currently being used in agriculture, work places and domestic sectors in developing countries in Asia (Abhilash & Singh, 2009). It is one of the most frequently used insecticides in Pakistan (Ahmad, et al. 2009).

Cypermethrin is a lipophilic substance that forms long term associations with integral membranous proteins (Michelangeli et al., 1990), particularly the ATP dependent ion channels (Prashanth & David, 2010). In this way it enhances the permeability of Na

+ channels (Kumar, et al. 2009) simultaneously altering the

function of transient K+ channels, thus inducing repetitive impulses in neurons

(Smith & Soderlund, 2001; Gowland et al., 2002) causing loss of coordination, muscular tremor, and convulsions (Desi et al., 1986). Cypermethrin has been found to induce DNA damage (Patel et al., 2006) and chromosomal aberrations (Kocaman & Topaktas, 2009), disturb the activities of sex steroids (Waters, et al., 2001; Chen et al., 2002; McCarthy et al., 2006) and cause hormonal disruptions (Xu et al., 2008). Evidences indicate that cypermethrin exposure inflicts oxidative stress leading to DNA damage and apoptosis (Jin et al., 2010).

Along with its systemic and cellular toxicological implications (Muthuviveganandavel et al., 2008; Ahmad et al., 2009; Khan et al., 2009, Sharaf et al., 2010); there exist convincing number of evidences that exposure to cypermethrin in males have led to changes in reproductive organs and sperms leading to a decline in the reproductive success (Elbetieha et al., 2001; Kumar et al., 2004; Ahmad et al., 2009; Rodriguez et al., 2009; Wang et al., 2009, 2010; Al-Hamdani & Yajurvedi, 2010).

According to Shukla & Taneja (2002) cypermethrin brings about germ cell mutations and chromosomal aberrations that lead to death and resorptions of embryos in mouse. There are relatively few studies on cypermethrin dealing directly with gestational exposure leading to various damages to the developing fetuses (Gupta, 1990; Ullah et al., 2006, Farag et al., 2007, Uggini et al., 2010). Farag, et al. (2007) have shown significant delay in down appearance and eye opening in fetuses along with delayed development and detachment of pinna at 10mg/kg cypermethrin per-gestational exposure for 4 weeks /5days in a week in virgin mice. Ullah, et al. (2006) have found pre and post-implantation losses following (ip) cypermethrin exposure at 25, 50, and 75 mg/kg BW in rabbit on post-mating day5, 10, 15, and 20. In a recent study Uggini, et al. (2010) have reported alterations in fetal growth especially in the axial and appendicular skeletal development following cypermethrin exposure, given in combination with chlorpyrifos (5 and 50% respectively) in chick eggs, on incubation day0 at 0.5L / egg.

The above cited literature indicates embryonic disruptions. Cypermethrin exposure at high dose levels may be toxic to the adults as well. Thus the present study was designed to explore all kinds of feto-morphic derangements at motherly safe dose exposures.

Vol. 56 (1&2) Pregnancy and fetal correlations of cypermethrin in mice

41


This study was conducted on 140 pregnant Swiss Webster laboratory mice, ageing between 3 to 4 months weighing 30±3 grams. They were assigned randomly to various groups (Fig 1). The Dams were kept under 12-12-hrs dark light cycles, in 12” x 12” x 18” metal gauzed steel framed cages. Fine cuttings of 40g brand new infection free white paper were provided for bedding. The

bedding was replaced twice a week. Ambient temperature 23 2oC and humidity

35-40% were maintained. Pallets of specially prepared rodent food and water were provided ad labitum. Appropriate dilutions (using corn oil) of commercial formulation of Cypermethrin (10EC) imported and marketed by Warble private limited (Batch: 33080712-A) were used for experimental exposure. As no information about LD50 values for pregnant mice available in literature, it was decided to estimate it carefully. A wide range of exposure doses (i.e. 25, 50, 75, 100 and 125mg/kg) was selected and tested for the estimation of the LD50 values for the pregnant females. To determine mortality (number of animals died within 48hours of exposure) at these exposure doses; each dose was tested on a group of 10 pregnant females. Inline with the probit analysis procedure enumerate by Finney, (1971) the data obtained was subjected to “regression curve analysis” for statistical estimation of required LD50 values (Fig 2).

After careful estimation of LD50 for pregnant mice 20, 10 and 5%of this reference dose (i.e. 12.8, 6.4 and 3.2mg/kg BW respectively) were selected as experimental doses. Each experimental dose was applied in two ways to a faction of 20 animals that is as single (on GD6) and multiple (on GD6, 9 and 12). A group of 20 animals was also maintained as vehicle exposed control (Fig 1). All doses to the experimental animals were applied by gavage.

All females were etherized and weighed just before the fetal recovery on GD18. The fetuses were exteriorized by means of a mid ventral incision in each case. Both horns of the uterus were exposed and finally removed intact, in each case, and the number of implantations were counted in situ. Both ovaries were also removed from each animal to count the corpora lutia under a dissecting research binocular microscope at 20X. Finally with help of fine scissors the graved uteri were opened and all fetuses (live, dead and resorbed) were recovered. Data obtained, at this stage, was represented in the result section on the following bases.

1. Total number of implantation sites/ animal/ group 2. Total number of corpus lutia in both ovaries/ animal/ group 3. Number of live fetuses/ litter/ group 4. Number of dead + resorbed fetuses/ litter/ group 5. Pre, peri and pan-gestational losses/ litter/ group 6. Pregnancy indices in each group

The total number of the corpora lutia and implants were counted in situ from the ovaries and the uterus of each experimental animal as explained above. The pregnancy index was calculated by means of the following equations as reported by Kar, et al. (1984) and Shukla & Taneja (2002).


Pregnancy Index (%) = 100 – % Co-gestational losses

Where as

% Co-gestational

losses =

Total No of Corpora lutia / Female – Total No of implants /female x 100

Total No of corpora lutia / female

The morphological study involved a careful observation of each fetus under a binocular dissecting research microscope (Labomed CXM2) at 10 and 20x. Photomicrographs of the selected fetuses were obtained with the help of digital camera (Kyocera MR410) fitted on a mechanically adjustable stage in super macro-mode.

The morphometric studies involved statistical analyses of the data regarding fetal weight, Crown-rump (CR) and head circumference. To obtain mean fetal weight/ litter, all fetuses in a litter were weighed together on a 0.001mg precision digital balance (Sartorius TF 214S) and the value obtained was divided by the total number of fetuses in that litter. On the other hand all fetuses in each litter were subjected individually for the measurements of CR length with the help of a vernier caliper. Similarly the occipito-frontal length and bi-parietal distance for each fetus were obtained to calculate fetal head circumference with the help of the following formula used for the calculation of circumference of an ellipsoid.

______________

P =2 √ (a2+b

2) / 2

Where a = (occipito-frontal length)/2 and b = (bi-parietal distance)/2

Fetal density can provide a direct indication of the effect of gestational cypermethrin exposure on the extant of fetal bone ossification. In order to calculate the fetal density/ litter; mean/ litter values of fetal weight and volume were obtained with the help of a digital balance (Sartorius TF 214S) and measuring cylinder of 10ml, respectively. Mean/ litter values of fetal density were calculated by using the following formula.

Mean fetal Density/ litter = Mean fetal weight in a litter

Mean fetal volume in a litter


43

Fig., 1: Experimental groups and subgroups of cypermethrin treated animals

RESULTS LD50:

Various doses of Cypermethrin (25, 50, 75, 100 and125mg/kg BW) were given to a group of 10 pregnant mice each. Mortality at each of these doses was plotted against a linier regression trend line; for the statistical estimation of the lethal dose for 50% mortality. The calculated LD50 of cypermethrin for the pregnant mice was 64mg/kg BW (Fig. 2).

y = 0.084x - 0.3

x = 5+0.3/0.084

x = 64

0

1

2

3

4

5

6

7

8

9

10

11

25 50 75 100 125

Dose Groups (mg/kg)

Mo

rtali

ty (

ind

ivid

uals

)

64mg/ kg

Fig., 2: Graph showing the LD50 of cypermethrin for the pregnant mice

Pregnancy index

Mean litter size (live fetuses/ litter) and pregnancy index followed the dose regimen in inverse proportionality. Thus the pre, peri and pan-gestational losses increased with increase in dose and times of exposure in cypermethrin treated groups (Table 1).

44 K. RAEES ET AL BIOLOGIA (PAKISTAN)

Table 1: Deta obtained for corpus lutial, implantations and live fetal count recovered and Calculated values of Pre, Peri and pan- gestational Losses in F0 mice following gestational exposure to various doses of cypermethrin

Groups Leutial

count (Dams)

Corpus leutia / Female ±MSE

Total No. of Implants (Resorptions)

No. of implants/ Female± MSE

Live fetuses/ litter

Percent Pre-implantational losses

Pregnancy index

Post implantational losses / litter

Pan-gestational losses /litter

CONTROL 180 (20) 9±0.29 174 (3) 8.7± 0.22 8.55 3.33 96.67% 1.67 5.0

SINGLE 3.2mg

188 (20)

9.4±0.50 176 (4) 8.8±0.35 8.6 6.38 93.62% 2.13 8.51

TRIPLE 3.2mg

172 (20) 8.6±0.23 156 (6) 7.8±0.29 7.55 9.30 90.70% 3.49 12.79

SINGLE 6.4mg

170 (20) 8.5±0.17 152 (5) 7.6±0.24 7.35 10.59 89.41% 2.94 13.53

TRIPLE 6.4mg

176 (20) 8.8±0.26 156 (7) 7.8±0.31 7.5 11.36 88.64% 3.98 15.34

SINGLE 12.8mg

196 (20) 9.8±0.48 172 (6) 8.6±0.26 8.1 12.24 87.75% 3.06 15.31

TRIPLE 12.8mg

192 (20) 9.6±0.41 164 (12) 8.2± 0.28 7.6 14.58 85.42% 6.25 20.83


45

Table 2: Fetal Density of F0 fetuses following gestational exposure to various doses of cypermethrin

Exposure Groups Fetal Density (mg/ml) ± SD (No. of fetuses)

CONTROL 1.099±0.192mg/ml (171)

SINGLE 3.2mg 1.1463±0.077mg/ml (172)

TRIPLE 3.2mg 1.148±.237mg/ml (150)

SINGLE 6.4mg 1.138±0.159mg/ml (147)

TRIPLE 6.4mg 1.084±.202mg/ml (149)

SINGLE 12.8mg 1.029±0.135mg/ml (166)

TRIPLE 12.8mg 1.004±0.072mg/ml (152)

Fetal density

Fetal density was taken as an indirect measure of the extent of ossification in the fetal skeleton. The data obtained shows an overall significant (P<0.05) variation. Surprisingly it was noted that the density remained higher than control in both sub-groups in 3.2mg/kg and single exposure sub-group in 6.4mg/kg exposure group. On the other hand triple exposure sub-group of 6.4mg/kg and both single and triple exposure sub-groups of 12.8mg/kg group a decline in fetal density to that of the control was noted. However in all three cypermethrin (i.e. 3.2, 6.4 and 12.8 mg/kg) treated groups a general decline was noted in the triple exposure than that of their respective single exposure sub-groups (Table 2).

Table 3: Actual number of F0 fetuses in each particular abnormality recovered from various experimental groups a: Number of abnormal

fetuses (Total number of fetuses recovered)

Ab

no

rmali

ties

G

rou

ps

Co

ntr

ol

3.2mg/kg 6.4 mg/kg 12.8mg/kg

Sin

gle

Tre

atm

en

t

Tri

ple

Tre

atm

en

t

Sin

gle

tr

eatm

en

t

Tri

ple

Tre

atm

en

t

Sin

gle

Tre

atm

en

t

Tri

ple

T

reatm

en

t

Microcephaly 0(171)a 1(172) 3(150) 2(147) 4(149) 3(166) 3(152)

Hydrocephaly 0(171) 2(172) 3(150) 3(147) 3(149) 3(166) 4(152)

Un-detached pinnae 0(171) 5(172) 7(150) 4(147) 5(149) 5(166) 8(152)

Epinnate ears 0(171) 0(172) 0(150) 0(147) 1(149) 2(166) 3(152)

Skewed neck 0(171) 0(172) 1(150) 0(147) 2(149) 2(166) 3(152)

Meromelia 0(171) 0(172) 1(150) 2(147) 3(149) 3(166) 4(152)

Extra dectly 0(171) 0(172) 0(150) 1(147) 4(149) 6(166) 7(152)

Drooping wrist 1(171) 2(172) 2(150) 3(147) 4(149) 5(166) 5(152)

Round back 1(171) 2(172) 3(150) 4(147) 4(149) 5(166) 6(152)

Hemoregia 1(171) 3(172) 2(150) 5(147) 5(149) 4(166) 6(152)

Torted hid limb 0(171) 1(172) 2(150) 2(147) 4(149) 5(166) 7(152)

Forked paws 0(171) 0(172) 0(150) 0(147) 3(149) 4(166) 6(152)

Flipper limbs 0(171) 0(172) 0(150) 0(147) 3(149) 4(166) 5(152)

Kinky tail 0(171) 0(172) 0(150) 0(147) 2(149) 3(166) 5(152)


Fetal morphology

Various morphological abnormalities observed in different cypermethrin exposed sub-groups were: 1) microcephaly, 2) hydrocephaly, 3) un-detached pinnae, 4) epinnate ears, 5) skewed cervical region of spine, 6) meromelia, 7) fore limb extra dectly, 8) drooping wrists, 9) round back, 10) abdominal hemoregia, 11) torted hid limbs, 12) forked paws, 13) flipper shaped limbs and 14) kinky tail (Table 3 and Fig. 3.1- 3.6).

Fig., 3.1 and 3.2: Fetuses from Control (A); and 3.2 mg/kg triple exposure sub groups group (B) a: hydrocephaly, b: meromelia c: drooping

wrists, d: abdominal hemoregia, e: round back, f: kinky tail, g: torted hind limbs

Fig., 3.3 and 3.4: Fetuses from Control (A); and 12.8mg/kg triple exposure sub-group (B and D); C: a closer view of selected area from B, E: a closer view of selected area from D a: extra dectyly , b: forked paw c: flipper

limbs, d: epinnate ear


47

Fig., 3.5: Fetuses from Control (A); and 6.4 mg/kg triple exposure sub-group (B

and C) and 3.6: closer views of cranial area from A and B of fig. 3.5. a: microcephaly , b: hydrocephaly c: skewed cervical region of spine , d: normal

ear pinna, e: un-detached pinna

Fetal crown rump length

Crown rump length is an indication of fetal growth rate. Intra uterine growth retardation is shown as comparison of means of crown rump length of cypermethrin exposure groups to that of the control group (Fig. 4). Analyses of variance indicate that among the three cypermethrin treated groups (i.e., 3.2, 6.4 and 12.8mg.kg); there was no significant variation within the single and/or triple exposure categories. On the other hand the three cypermethrin exposed groups showed significant differences (P<0.05) with that of control at both levels of exposures (i. e., single, and triple). Analyses of the means of the respective subgroups of cypermethrin treated groups with that of the control through “Duncan’s Multiple Range Test ” also indicate the significant variation (p < 0.05), as it has been indicated by means of asterisks in Fig. 4.


20.7

1

16.5

5

16.4

6

13.3

8

20.7

1

13.8

8

13.5

9

11.4

3

0

5

10

15

20

25

Control 3.2S 6.4S 12.8S Control 3.2T 6.4T 12.8T

Experimental Groups

Me

an

Fe

tal

CR

le

ng

th(m

m)

* * **

Fig., 4: Average crown-rump length of F0 fetuses in various cypermethrin treated sub groups to that of the control

+bars: SEM; *: mean values differing significantly from control; S: Single exposure; T: Triple Exposure; 3.2, 6.4 and 12.8: respective mg/kg dose groups

Head circumference

A similar trend, as seen in fetal crown rump length, was observed in fetal head circumference analyses. Analysis of variance of the single and triple exposure subgroups separately; with that of the control, indicate a significant variation in the data (p<0.05). The posthoc analysis of the means have shown significant variations between control and 12.8mg/kg single exposure sub-group; while 6.4 and 12.8mg/kg sub-groups differed significantly with that of the control in triple exposure category (Fig., 5).


49

30

.88

25

.6

24

.2

20

.1

30

.88

23

.3

20

.3

18

.61

0

5

10

15

20

25

30

35

Control 3.2(S) 6.4(S) 12.8(S) Control 3.2(T) 6.4(T) 12.8(T)

Experimental Groups

Mean

Head

Cir

cu

mfr

en

ce(m

m)

* * *

Fig5: Average head circumference in F0 fetuses from various cypermethrin treated sub-groups and the control group

+bars: SEM; *: mean values differing significantly from control; S: Single exposure; T: Triple Exposure; 3.2, 6.4 and 12.8: respective

mg/kg dose groups

DISCUSSION

There are only a few studies pertaining embryo toxicology dealing directly with the embryonic exposure of cypermethrin (Gupta, 1990; Farag et al., 2007; Uggini et al., 2010). Thus the results obtained in this investigation are hardly comparable to the previously available literature. The present study was aimed to discover the toxicological effects of cypermethrin in relation with pregnancy, pre, peri and pan-gestational losses and to report all sorts of developmental abnormalities at morphological and morphometric levels.

The estimation of LD50 was a laborious job. But it was done due to the following reason. 1. Although the values of LD50 for mice are available in literature. However

there exist a wide disparity in these reported values of LD50 for mice for example; 50mg/kg body weight (Luty et al., 2000) and 25mg/kg body weight (Varshneya et al., 1992).

2. All such available values were confined to the males or non-pregnant females. In many cases the available literature does not specify the value of LD50 in relation to sex. Moreover there exist no such values in literature dealing especially with the pregnant mice. Keeping in view of these constrains it was decided that a careful

estimation of LD50 in pregnant mice should be conducted for the selection of motherly safe dose profile. Results indicate that the value of LD50 for


cypermethrin in pregnant mice remained 64mg/kg body weight. This is considerably higher than the already reported value that is 50mg/kg for non-pregnant mice (Luty et al., 2000). The natural phenomenon of suppression of immunological and humoral responses must have enhanced toleration limits towards this toxic environmental chemical during pregnancy that seems to be responsible for this increase in LD50 value (Fessler, 2001; Calder et al., 2006). An embryo is kept as an allo-graft in mammals and natural defense mechanisms have to be suppressed to avoid any damage to them by maternal immunological responses. Due to this partial suppression of the immune system pregnant female mice may have tolerated higher toxic doses of cypermethrin than the males and non-pregnant females.

Dose and extant of exposure (single or triple) related increased pre-implantation losses that have led to a decline in pregnancy index in cypermethrin treated mothers to that of the control group was considered as a direct embryocidal impact of cypermethrin. As the results show (Table1) that this increase in pre-implantational losses was clearly dose dependent, it seems partly because of an increased embryonic death (before or just after implantation - leading to complete resorptions in such cases) in a dose dependant manner and partly because of an interference in the process of implantation as cypermethrin has already been accepted as a strong endocrine disruptor of the sex steroids (Waters et al., 2001; Chen et al., 2002; McCarthy et al., 2006) and hormones of gestation particularly LH (Elbetieha et al., 2001).

Fetal resorptions were considerably increased with the increase in exposure dose strength (3.2, 6.4 and 12.8mg/kg). Slight increase was noted in triple exposure sub-groups in 3.2 and 6.4 mg/kg dose levels then there respective single exposure sub-groups, while in 12.8mg/kg group the percent fetal resorption was more than double in the triple exposure sub-group than that of the single exposure sub-group. This finding clearly suggest that higher dose (i.e. 12.8mg/kg (20% of the LD50)) with multiple exposures are far more embryo lethal than the low doses (3.2 and 6.4mg/kg (10 and 5% of LD50)) in the similar experimental conditions (Table 1). On the whole the pregnancy index and fetal resorption data clearly indicating that cypermethrin is highly embryo toxic that interferes with the process of embryonic implantation and intrauterine fetal development. In this context Rutledge, (1997) had argued that chemicals adversely affect the early conceptus causing in-utero mortality and developmental abnormalities. Further more susceptibility of the post-fertilization period differs from exposures of sperms (Elbetieha et al., 2001) or eggs as the later produces massive pan-gestational losses with lesser number of fetal anomalies. In contrast, similar exposure at pre-gastrulational stage induces peri-implantational and pan-gestational death along with various fetal anomalies (Rutledge, 1997). The period of exposure (GD6-12) of this study begins at the end of the critical period of implantation (i.e. GD4.5-6) (Kaufman & Bard, 1999) that has logically to be followed by gastrulation thus the results obtained in terms of co-gestational losses and developmental abnormalities seems justified.

Fetal density was regarded an indirect measure of the extent of ossification. Surprisingly highest fetal density (1.148mg/kg) was noted in 3.2mg/kg triple exposure sub-group while lowest (1.004mg/kg) in 12.8mg/kg triple exposure subgroup. Results indicate that multiple low dose (3.2mg/kg = 5%


51

of the LD50) gestational exposures might have led to an enhanced Ca++

deposition whereas the multiple high dose (12.8mg/kg = 20 % of the LD50) have in inverse impact upon Ca

++ deposition. As cypermethrin is a proven estrogenic

agonist (Chen et al., 2002; McCarthy et al., 2006) at low dose multiple exposures it might have led to an enhanced osteocytic differentiation in fetuses leading to an above normal Ca

++ deposition. The inverse trend in high dose group is clearly

due to ostioblastic necrosis and lesser retention of Ca++

in the fetal bones (Table 2).

Crown Rump (CR) Length comparison is an important parameter that indicate intrauterine growth retardation in response to the fetal exposure to a noxious environmental chemical substance (like insecticides). Results obtained in present study indicate a dose dependent decline in fetal growth rate as compared with the control group. Multiple exposures even at 3.2mg/kg (i.e. 5% of LD50) produced big decline in CR length indicating that the repeat exposures are much more dangerous for in utero fetal growth (Fig4). The results are comparable with similar previous studies (Ahmad & Asmatullah, 2007).

Measurement of fetal head circumference is another parameter that along with the general retardation in fetal growth does also indicate the extant of fetal brain growth retardation (Ahmad & Asmatullah, 2007). The results obtained in this regard have also shown the comparable trend as obtained for CR length (Fig5).

The available literature on the fetomorphic derangements attributable to cypermethrin exposure is relatively skimpy (Gupta, 1990; Farag, et al. 2007; Uggini, et al. 2010). However various feto-morphological abnormalities were observed in this study that include microcephaly, hydrocephaly, un-detached pinnae, epinnate ears, skewed neck, meromelia, extra dectly, drooping wrist, round back, hemoregic spots, torted hid limb, forked paws, flipper limbs and kinky tail (Fig 3 and Table 3).

Conclusively cypermethrin was found to the highly toxic to the developing fetuses on dose levels as low as 5% of the LD50 (3.2mg/kg) for the mothers. Along with general in utero growth retardation, it has also led to a significant increase in pan-gestational losses along with various feto-morphological abnormalities.

REFERENCES

Abhilash, P. C. & Singh, N., 2009. Pesticide use and application: An Indian scenario. J. Hazard. Mater., 165:1–12.

Ahmad, K., R. & Asmatullah., 2007. Teratological effects of chlorpyrifos in mice. Iran. J. Toxicol., 1(2): 91-99.

Ahmad, M., Hussain, I., Khan, A. & Najib-Ur-Rehman., 2009. Deleterious effects of cypermethrin on semen characteristics and testes of dwarf goats (Capra hircus). Exp. Toxicol. Pathol., 61(4): 339-46.

Akhtar, N., Srivastava, M. K. & Raizada. R. B., 2006. Transplacental disposition and teratogenic effects of chlorpyrifos in rats. J Toxicol Sci., 31(5): 521-7.

Al-Hamdani, N. M. H. & Yajurvedi, H. N., 2010. Cypermethrin reversibly alters sperm count without altering fertility in mice. Ecotoxicol. Environ. Saf., 73: 1092–1097.


Calder, P. C., Krauss-Etschmann, S., DeJong, E. C., Dupont, C., Frick, J. S., Frokiaer, H., Heinrich, J., Garn, H., Koletzko, S., Lack, G., Mattelio, G., Renz, H., Sangild, P. T., Schrezenmeir, J., Stulnig, T. M., Thymann, T., Wold, A. E. & Koletzko, B., 2006. Early nutrition and immunity - progress and perspectives. Br. J. Nutr., 96(4):774-90.

Chen, H., Xiao, J., Hu, G., Zhou, J., Xiao, H. & Wang, X., 2002. Estrogenicity of organophosphorus and pyrethroid pesticides. J. Toxicol. Environ. Health A. 65(19): 1419–1435.

Desi, I., Dobronyi, I. & Varga, L., 1986. Immuno-, neuro- and general toxicologic animal studies of a synthetic pyrethroid, cypermethrin. Ecotoxicol. Environ. Saf., 12:220–32.

Elbetieha, A. Da’as, S. I. Khamas, W. & Darmani, H., 2001. Evaluation of the toxic potentials of cypermethrin pesticide on some reproductive and fertility parameters in the male rats. Arch. Environ. Contam. Toxicol., 41:522–8.

Farag, A. T. Goda, N. F. Shaaban, N. A. & Mansee, A. H., 2007. Effects of oral exposure of synthetic pyrethroid, cypermethrin on the behavior of F1-progeny in mice. Reprod. Toxicol., 23:560–567.

Fessler, D. M. 2001. Luteal phase immunosuppression and meat eating. Riv Biol., 94(3): 26-403.

Finney, D. J., 1971. Probit Analysis, 3rd ed., Cambridge University Press, Cambridge, England, pp: 50-80.

Gowland, B., Webster, L., Fryer, R., Davies. I., Moffat, C. & Stagg, R., 2002. Uptake and effects of the cypermethrin-containing sea lice treatment Excis in the marine mussel Mytilus edulis. Environ. Pollut., 120: 805–11.

Gupta, P. K. 1990. Teratogenic effects of cypermethrin in rats. J. Environ. Bio., 11(2):121-126.

Jin, Y. Zheng, S. Pu, Y. Shu, L. Sun, L. Liu, W. & Fu, Z., 2010. Cypermethrin has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult zebrafish (Danio rerio). Chemosphere., 83(3):398-404.

Kar, R. N., Khan, K. & Mukherjee, S. K., 1984. In vivo mutagenic effect of methyldopa, dominant lethal test in male mice. Cytobios., 41:151–159.

Kaufman, M. H & Bard, J. B. L., 1999. The anatomical basis of mouse development.San, Diego,CA: Academic press.

Khan, A. Faridi, H. A. M. Ali, M. Khan, M. Z. Siddique, M. Hussain, I. & Ahmad. M., 2009. Effects of cypermethrin on some clinico-hemato-biochemical and pathological parameters in male dwarf goats (Capra hircus). Exp. Toxicol. Pathol., 61:151–160.

Kocaman, A. Y. & Topaktao, M., 2009. The in vitro genotoxic effects of a commercial formulation of alpha-cypermethrin in human peripheral blood lymphocytes. Environ. Mol. Mutagen. 50:27–36.

Kumar, A., Rai, D. K. B., Sharma, B. & Pandey. R. S., 2009. -cyhalothrin and cypermethrin induced in vivo alterations in the activity of acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch) Pesticide Biochem. Physiol., 93:96–99.

Kumar, S., Gautam, A. K., Agarwal, K. R., Shah, B. A., & Saiyad, H. N,. 2004. Demonstration of sperm head shape abnormality and clastogenic potential of cypermethrin. J. Environ. Biol., 25:187–190.


53

Luty, S., Latuszynska, J., Obuchowska-Przebirowska, D., Tokarska, M. & Haratym-Maj, A., 2000. Subacute toxicity of orally applied alpha-cypermethrin in Swiss mice. Ann. Agric. Environ. Med., 7(1): 33-41.

McCarthy, A. R., Thomson, B. M., Shaw, I. C. & Abell., A. D., 2006. Estrogenicity of pyrethroid insecticide metabolites. J. Environ. Monit., 8:197-202.

Michelangeli, F., Robson, M. J., East, J. M. & Lee, A. G., 1990. The conformation of pyrethroids bound to lipid bilayers. Biochim. Biophys. Acta., 1028:49–57.

Muthuviveganandavel, V., Muthuraman, P., Muthu, S. & Srikumar, K., 2008. A study on low dose cypermethrin induced histopathology, lipid peroxidation and marker enzyme changes in male rat. Pest. Biochem. Physio., 91:12–16.

Patel, S., Pandey, A. K., Bajpayee, M., Parmar, D. & Dhawan, A., 2006. Cypermethrin-induced DNA damage in organs and tissues of the mouse: Evidence from the comet assay. Mutat. Res., 607:176–183.

Prashanth, M. S. & David, M., 2010. Impact of Cypermethrin on Na+–K+, Ca2+ and Mg2+ ATPases in Indian Major Carp, Cirrhinus mrigala (Hamilton). Bull. Environ. Contam. Toxicol., 84:80–84.

Rodriguez, H., Tamayo, C., Inostroza, J., Soto, C., Bustos-Obregon, E. & Paniagua, R. 2009. Cypermethrin effects on the adult mice seminal glands. Ecotoxicol Environ Saf., 72:658–662.

Roger. J. & Kevlock, R., 2001. Casaret and Doull’s toxicology: the basic science of poisons, In (Klassen, C.D., ed.). pp: 351-381 (chapter 10) Dev. Toxicol., McGraw –Hills, New York.

Roy, T. S., Andrews, J. E., Seidler, F. J. & Slotkin, T. A., 1998. Chlorpyrifos elicits mitotic abnormalities and apoptosis in neuroepithelium of cultured rat embryos. Teratology. 58:62-68.

Rutledge, J. C., 1997. Developmental toxicity induced during early stages of mammalian embryogenesis. Mutat. Res-Fund. Mol. M., 396(1-2):113-127.

Sharaf, S., Khan, A., Khan, M. Z., Aslam, F., Saleemi, M. K. & Mahmood. F., 2010. Clinico-hematological and micronuclear changes induced by cypermethrin in broiler chicks:Their attenuation with vitaminE and selenium. Exp Toxicol Pathol., 62(4):333-41.

Shukla, Y. & Taneja, P., 2002. Mutagenic potential of cypermethrin in mouse dominant lethal assay. J. Environ. Pathol. Toxicol. Oncol., 21:259–65.

Slotkin, T. A., Levin, E. D. & Seidler, F. J., 2006. Comparative developmental neurotoxicity of organophosphate insecticides: effects on brain development are separable from systemic toxicity. Environ. Health Perspect., 114:746–751.

Smith, T. J. & Soderlund, D. M., 2001. Potent actions of the pyrethroid insecticides cismethrim and cypermethrin on rat tetrodotoxin-resistant peripheral nerve (SNS/PN3) sodium channels expressed in Xenopus oocytes. Pest. Biochem. Physiol., 70:52–61.

Tian, Y., Ishikawa, H., Yamaguchi, T., Yamauchi, T. & Yokoyama, K., 2005. Teratogenicity and developmental toxicity of chlorpyrifos. Maternal exposure during organogenesis in mice. Rep. Toxicol., 20(2):267-270.


Uggini, G. K., Patel, P. V. & Balakrishnan. S., 2010. Embryotoxic and Teratogenic Effects of Pesticides in Chick Embryos: A Comparative Study Using Two Commercial Formulations. Environ Toxicol. (Epub. Ahead of Print)

Ullah, M. S., Ahmad, M., Ahmad, N., Khan, M. Z. & Ahmad, I., 2006. Toxic effects of cypermethrin in female rabbits. Pak. Vet. J., 26(4):193–196.

Varshneya, C., Singh, T., Sharma, L.D., Bahga, H.S. & Garg, S. K., 1992. Immunotoxic responses of cypermethrin, a synthetic pyrethroid insecticide in the rats. Indian J. Physiol. Pharmacol., 36:123–126.

Wang, H., Wang, Q., Zhao, X., Liu, P., Meng, X., Yu, T., Ji, Y., Zhang, H., Zhang, C., Zhang, Y. & Xu, D., 2010. Cypermethrin exposure during puberty disrupts testosterone synthesis via downregulating StAR in mouse testes. Arch. Toxicol., 84:53–61.

Wang, X. Z., Liu, S. S., Sun, Y., Wu, J. Y., Zhou, Y. L. & Zhang, J. H., 2009. Beta-cypermethrin impairs reproductive function in male mice by inducing oxidative stress. Theriogenology. 72:599–611.

Waters, K. M., Safe, S. & Gaido, K. W., 2001. Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductive tissues of female mice. Toxicol. Sci., 63:47–56.

Xu, L., Liu, L., Ren, X., Zhang, M., Cong, N., Xu, A. & Shao, J., 2008. Evaluation of androgen receptor transcriptional activities of some pesticides in vitro. Toxicology. 243:59–65.

BIOLOGIA (PAKISTAN) 2010, 56 (1&2), 55- 62 PK ISSN 0006 – 3096

Facile regeneration through adventive/somatic

embryogenesis from in vitro cultured immature leaf

segments of elite varieties of sugarcane

(Saccharum officinarum L)

SAFDAR ALI & JAVED IQBQL

Department of Botany, GC University, Lahore (SA), School of Biological Sciences, University of the Punjab, Lahore (JI)

ABSTRACT

This research work was conducted to optimize protocol for in vitro regeneration

through embryogenesis, of nine elite varieties of sugarcane. In all the nine varieties, somatic embryogenesis with and without an intervening callus phase was achieved under the influence of varying concentrations of 2,4-D ranging from 1.5 to 2.5mg

-l. Embryo

development was seen all over the cultured explants within four weeks of culture and the transfer of either adventive embryos or somatic embryos from embryo induction medium to embryo germination medium containing casein hydrolysate (500mgl

-l) with 2,4-D (1mgl

-l) or

without growth regulators favoured embryo maturation thus resulting in plantlet regeneration within 6 weeks and all the regenerated plantlets grew normally in the greenhouse. Embryogenesis and regeneration was not significantly influenced by the variety. The technique for embryo induction involved culturing of leaf discs derived from immature leaf rolls, on medium containing 2,4-D. Plantlets were ready for hardening after 10-14 week. In a comparative study, transformation efficiency for the direct embryogenic morphogenic route was more than for indirect embryogenic morphogenic route. Due to fewer subcultures, the cost of production via direct somatic embryogenesis can be reduced per transgenic plant. This method could be useful to follow and exploit somaclonal variations as well as provide a target tissue for genetic transformation studies. Key words: adventive embryos, 2,4-D, sugarcane, transformation.

INTRODUCTION

Sugarcane is ideal with its high efficiency of solar energy capture, since it

has both high biomass yields and is easily fractionated into highly digestible (sugar) and less digestible fibrous components (fiber or bagasse) for various uses such as fuel and fiber industry. Sugarcane, in addition to furnishing 70% of world sugar (Lakshmanan et al., 2002), is a source of natural pharmaceutical compounds such as Policosanol (Sahelian, 2004). The compelling demand of refined sugar and the use of its by-products for various purposes emphasize the need to increase production of sugarcane crop.

Somatic embryogenesis is a remarkable illustration of the dictum of plant totipotency, and some sporophytic and gametophytic cells undergo embryogenesis in in vitro culture conditions and differentiate into whole plant (Kohlenbach, 1978; Willium & Maheshwaran, 1986; Dodeman et al., 1997). Sweby et al., (1994) were of the opinion that if callus was transferred after four weeks from an initiation medium containing a high level of 2,4-D (3 mgl

-1) to a

S. ALI & J. IQBAL BIOLOGIA (PAKISTAN)

56

maintenance medium containing a low level of 2,4-D (1 mgl-1

), no genetic variations occur. There has been an increasing interest in the application of tissue culture techniques as an alternative means of asexual, propagation and genetic transformation of economically important plants including sugarcane. Therefore the present piece of research work was intended to devise a simple, rapid and reproducible protocol for regeneration through embryogenesis of nine elite varieties of sugarcane

MATERIALS AND METHODS Plant material and explant preparation: The study was initiated with nine elite sugarcane genotypes. Healthy cane tops of sugarcane varieties viz., S96SP-302, S96SP-571, S96SP-574, HSF-240, CP72-2086, CP77-400, SPF-213, S97US-183 and S97US-102 were kindly provided by Sugarcane Research Institute, Faisalabad, Pakistan. The cane tops collected from 9 month old cane plants were used to obtain explant to raise in vitro cultures (Ali et al, 2010). Immature leaf rolls above the first node, cut into about 1-2mm thick cross-sectional slices were used as explants (Heinz & Mee, 1969; Fitch & Moor, 1993). Culture media and embryo induction: MS (Murashige & Skoog, 1962) medium [MS salts, vitamins, myo-inositol (100 mgl

-1) and sucrose 3%)] was used as basal

culture medium with pH 5.8 and various modifications in growth regulator-supplements such as 2,4-D (0.5-3.5 mgl

-l) alone and in combination with Kinetin

(1 mgl-l) for embryo induction for each genotype.

Culture conditions: Cultures were maintained at temperature (27±2oC) for all

treatments. For embryo induction, the cultures were placed in dark as well as in continuous light conditions and during regeneration studies these cultures were exposed to 16h photoperiod with light (2000-3000 lux). Plantlet regeneration: Both types of embryos (adventive and indirect) were employed on germination/regeneration medium (MS salts, vitamins, myo-inositol (100 mgl

-1), casein hydrolysate (500 mgl

-l), sucrose 3%) and with 2,4-D (1 mgl

-l)

or without 2,4-D. Rooted plantlets were shifted to pots or 2x4 inches polythene bags to acclimatize for field plantation.

RESULTS Embryogenesis response to plant growth regulators Kinetin and NAA alone and in combination were found ineffective in initial studies for somatic embryogenesis. Optimum somatic embryogenesis was observed with the treatment of 2,4-D alone in the range of 1-3 mgl

-l in all the varieties under

study (Table 1). The highest percentage (70) of direct somatic embryos was observed at 1.5 mgl

-l 2,4-D level whereas the highest percentage (90) of indirect

somatic embryos was observed at 3 mgl-l 2,4-D in most of the said genotypes.

Direct somatic embryogenesis From the surface of leaf roll explants, initiation of adventive embryos was observed on MS medium with 2 mgl

-l after 8-10 days under either continuous or

16 h photoperiod (Fig. 1A) as well as in dark conditions in all the nine above mentioned varieties (Fig. 1B). Observation under stereomicroscope revealed the appearance of transparent bubble like proembroids, which afterwards

Vol. 56 (1&2) Facile Regeneration Through Adventive/Somatic Embryogenesis

57

transformed into embryos without forming any callus. These embryos could develop into plantlets when transferred to embryo germination medium. The direct embryo induction was exclusively dependent on 2,4-D concentration and the highest level of 2,4-D for direct somatic embryo induction was observed 2.0mgl

-l but the most of the varieties such as S96SP-571, S96SP-302, CP72-

2086, CP77-400, SPF-213, S97US-183 and HSF-240 reflected their best potential for direct somatic embryogenesis at 1.5 mgl

-l except S96SP-574 and

S97US-102 where direct somatic embryo induction occurred at 2.0 mgl-1

, 2,4-D. Frequency of direct somatic embryos i.e. 70% was recorded at 1.5 mgl

-1, 2,4-D

for the following sugarcane varieties, S96SP-571, S96SP-302, CP72-2086, CP77-400, SPF-213 except S97US-183 and HSF-240 where the percentage of direct somatic embryos was 60 (Table 1). No response was observed below 1.0 mgl-l 2,4-D in the varieties tested except SPF-213 where direct embryos induction started at 0.5 mgl

-1. Similarly above 2.5 mgl

-1 2,4-D level no direct

somatic embryo induction was observed except in HSF-240, where direct somatic embryo formation was observed occasionally at 3.0mgl-l 2,4-D also. Var. S97US-102 responded equally well at both the said 2,4-D levels (1.5 and 2.0 mgl

-1) in terms of direct embryo induction percentage. Adventive embryos and E

callus formation occurred simultaneously within the same cultures. The percentage of embryo formation is given in detail in Table 1. Indirect somatic embryogenesis

Role of various concentrations of 2,4-D for indirect somatic embryo induction was studied in aforementioned sugarcane varieties and it was found that none of the sugarcane varieties under study responded efficiently at 0.5- 1.5 mg l

-l 2,4-D, but in some varieties such as S97US-102, S96SP-302 and SPF-213

it was 30% whereas in S96SP-574 and HSF-240 20% induction of indirect somatic embryos was observed at 1.5 mgl

-l 2,4-D. (Table 1). Indirect

embryogenesis in most of the varieties under study was observed between 80 to 90% at 2.5 mgl

-1 2,4-D in S97US-183, S97US-102, S96SP-571, S96SP-302,

CP72-2086, CP77-400 and HSF-240. In S96SP-574 maximum somatic embryogenesis (80%) was observed at 3.0mgl-l whereas in SPF-213 at 2.0 mgl

-1

2,4-D upto 90% somatic embryos were developed. Above 3.0 mgl-1

2,4-D level no indirect embryo induction was recorded except in S97US-183 and CP72-2086 where 10 and 20% indirect embryos were observed. Response of somatic embryos to growth regulators for regeneration

Under continuous or 16 h photoperiod, both adventive (direct) somatic embryos as well as indirect somatic embryos (via callus) present on the surface of compact nodular green or yellowish white E calluses germinated to form complete plantlets in 6-8 weeks. Transfer of either adventive embryos or indirect somatic embryos (Fig. 3, A) from embryo induction medium [(MS salts, vitamins, myo-inositol (100 mgl

-1) with 2,4-D (Table 1) to embryo germination medium [(MS salts,

vitamins, myo-inositol (100 mgl-1

), casein hydrolysate (500 mgl-l)] with 2,4-D (1

mgl-l) or without growth regulators favoured embryo maturation thus resulting in

plant regeneration (Fig. 3 B & C). Plant regeneration potential of compact nodular yellow/yellowish white or green/greenish white E calluses was maintained up to 5 subcultures, i.e. about 24 weeks of callus age. The plantlets thus obtained grew to complete seedlings (ready for hardening process) rapidly when shifted to agar gelled plain MS medium (Fig. 3 D).


58

DISCUSSION

The totipotency of plant cell can be exploited by modulating culture conditions as described by Reinert (1959). The mechanisms of totipotency, however, are not fully understood so far, and are mainly discussed in relation to the concentration and ratio of plant growth regulators (Toonen & De Vries, 1996) particularly auxins. Auxins have been implicated in a disconcertingly large number of developmental events. Their roles in embryonic polarity, organ initiation, and stem cell maintenance have all been extensively reviewed (De Smet & Jurgens 2007; Bowman & Floyd 2008; Vanneste & Friml 2009). In many of these cases, a general model can be discerned. First, auxin accumulates in a subset of cells, triggering a change in cell fate. This is followed by establishment of a graded distribution of auxin, often setting up an axis. Finally, this axis is used, sometimes in combination with other signals, to establish zones of cell identity. In these scenarios, auxin acts both to initiate early events and subsequently to refine the locations of specific programs. In general most of the embryo induction takes place on auxin-containing media. (Nhut et al., 2000) & in most of them the presence of auxins in the culture medium, especially the synthetic auxin 2,4-D, is the factor that determines the induction of embryo formation, as reported in many cereals such as sugarcane (Chengalrayan & Meagher, 2001), wheat (Li et al., 2003), rye (Ward & Jordan, 2001).The impact of growth regulator like auxin changes with the stage of in vitro culture, the same growth regulator has different morphogenic responses at different stages of in vitro cultures as observed in these studies that 2,4-D in higher concentration induces callus formation in immature leaf tissue, lowering the level of 2,4-D induces embryogenesis while further lowering induces regeneration in in vitro induced embryos (Falco et al., 1996; Chencalrayan & Meagher, 2001; Alam et al., 2003). The decrease in 2,4-D level from 3 & 4 mg l

-l seemed to be the major

factor inducing in vitro embryogenesis (Fitch & Moor 1993). The green or greenish white calli formed in light produce embryos early and leisurely and regenerate readily when shifted to regeneration medium as compared to callus, induced in dark which contain more amount of non regenerative part. The early formation of embryos and faster regeneration in this case may be due to the light sensitive degradation of auxins.

The embryos germination is another important step in in vitro regeneration of plantlets. Embryos responded to germination on full as well as half strength MS medium devoid of 2,4-D, however the time duration was longer in case of full strength MS medium. The behaviour of adventive and somatic embryos towards germination was almost same with the only difference of percentage of germination where adventive embryos were observed less responsive as compared to somatic embryos. The protocol described here may be used to follow and exploit somaclonal variations as it reduced time for regeneration and higher percentage of establishment of in vitro derived plantlets at a faster rate to make in vitro regeneration system cost effective minimizing the time span for plant regeneration. This culture system is the underlying technology for efficient sugarcane transformation system as this technique has been already employed for improved plant regeneration from in vitro cultures transformed in

http://cshperspectives.cshlp.org/content/2/2/a001420.full#ref-16






59

sugarcane through particle bombardments (Khan et. al., 2010) and may prove to be a rapid method to provide virus free stock as well.

0

10

20

30

40

50

60

70

80

90

100

S97

US-183

(D)

(InD

)

S97

US-102

(D)

(InD

)

S96

SP-574

(D)

(InD

)

S96

SP-571

(D)

(InD

)

S96

SP-302

(D)

(InD

)

CP72

-208

6(D)

(InD

)

CP77

-400

(D)

(InD

)

SPF-213

(D)

(InD

)

HSF-240

(D)

(InD

)

%a

ge

em

bry

og

en

esis

2,4-D(mg/L) 0.5 1 1.5 2 2.5 3 3.5

Note:

1. D designates direct embryo induction, without the intervening callus phase.

2. InD designates indirect embryos appearing through the intervening callus phase.

Fig., 1: Effect of 2,4-D on direct and indirect somatic embryo induction in MS modified medium containing MS salts, vitamins, myo-inositol (100 mgl-1), and sucrose 3%.

Fig., 1: Direct embryogenesis in cv. HSF-240. A) Direct and indirect embryogenesis on

spindle leaf discs in continuous light after six weeks. B) Direct and indirect

embryogenesis on spindle leaf discs in dark after 4 week. The arrowheads pointing the

direct somatic embryos developed in both the cultures at MS3 medium (1 x).

A

B

A

A

B B

B


60

A

Fig. 3: Beginning of shoot induction after 12 week on MS medium supplemented with 2,4-D

1mg/l and CH 500mg/l HSF-240 (1 x) B) Embryo germination at the age of 15th week on MS

medium basal supplemented with Vitamins. C) Plantlet growth at 20th week on the same

medium (1 x) D) Seedling growth stages before hardening (1 x).

B

C

D

Fig. 2: A) Embryogenic callus with developed somatic embryos in cv. HSF-240. B)

Close view of the somatic embryos from fig 10A, cultures of 8 week at embryo

germination medium (A at 1 x and B at 2.5 x).

B A


61

REFERENCES Alam, A., Mannan, Sk. A., Karim, Z. & Amin, M. N., 2003. Regeneration of

sugarcane (Saccharum officinarum) plantlet from callus. Pak. Sugar J., p 15-19.

Ali, S., Iqbal, J. & Khan, M. S., 2010. Genotype independent in vitro regeneration system in elite varieties of sugarcane. Pak. J. Bot., 42(6): 3783-3790.

Bowman, J. L. & Floyd, S. K., 2008. Patterning and polarity in seed plant shoots. Ann Rev Plant Biol., 59: 67–88.

Chencalrayan, K. & Meagher, Gallo- M., 2001. Effect of various growth regulators on shoot regeneration of sugarcane. In Vitro Cell Dev. Biol-Plant., 37: 434-439.

De Smet, I. & Jurgens, G., 2007. Patterning the axis in plants–auxin in control. Curr Opin Genetics Dev., 17: 337–343.

Dodeman, V. L., Ducreux, G. & Kreis, M., 1997. Zygotic embryogenesis versus somatic embryogenesis J. Exp. Bot., 48:1493–1509.

Falco, M. C., Mendes, B. M. J. & Neto, A. T., 1996. Cell suspension culture of sugarcane: Growth, management and plant regeneration1. R. Bras. Fisiol. Veg., 8: 1-6.

Fitch, M. M. M. & Moore, P. H., 1993. Long-term culture of embryogenic sugarcane cells. Plant Cell Tiss. Org. Cult., 32(3): 335-343.

Heinz, D. J. & Mee, G. W. P., 1969. Plant differentiation from callus tissue of Saccharum species. Crop Sci. 9:346-348.

Khan, M. S., Ali, S. & Iqbal. J., 2010. Developmental and photosynthetic regulation of d-endotoxin reveals that engineered sugarcane conferring resistance to ‘dead heart’ contains no toxins in cane juice. Mol Biol Rep., 38(4) 2359-2369.

Kohlenbach, H. W., 1978. Comparative somatic embryogenesis. In: T.A. Thorpe (ed.), Frontiers of Plant Tissue Cultures. Univ. Calgary Press, Canada, pp59-66.

Lakshmanan, P., Geijskes, R. J., Elliot, A. R., Wang, L., Mckeon, M. G., Swain, R. S., Borg. Z., Berding. N., Grof C. P. L. & Smith.

G. R., 2002. A thin cell

layer culture system for the rapid and high frequency direct regeneration of sugarcane and other monocot species. In Vitro Cell. Dev. Biol., 38: 1411.

Li, B., Caswell, K., Leung, N. & Chibbar, R. N., 2003. Wheat (Triticum aestivum l.) somatic embryogenesis from isolated scutellum: Days post anthesis, days of spike storage, and sucrose concentration affect efficiency. In Vitro Cell. Dev. Biol – Plant., 39(4): 20-23.

Murashige, T. & Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant,. 15: 473-497.

Nhut, D. T., Le, B. V. &. Van, T. T. K., 2000. Somatic embryogenesis and direct shoot regeneration of rice (Oryza sativa L.) using thin cell layer culture of apical meristematic tissue. J. Plant Physiol., 157: 559-565.

Reinert, J., 1959. Uber die kontrolle der morphogenese und die induction von adventivembryonen an gewebeculturen aus karroten. Planta., 53: 318-333.


62

Sahelian, R., 2004. Policosanol research update: Long-term effects of policosanol on obese patients with Type II Hypercholesterolemia. Asia. Pac. J. Clin. Nutr., 13 (Suppl): S102.

Sweby, D. L., Huckett, B. I. & Botha, F. C., 1994. Minimising somaclonal variation in tissue cultures of sugarcane. Proceedings of the Annual Congress S Afr. Sugar Tech Ass., 68: 46-50.

Toonen, M. A. J. & De Vries, S. C., 1996. Initiation of somatic embryos from single cells. In T.L. Wang and A. Cuming (eds.), Embryogenesis, the generation of a plant. BIOS Scientific Publishers, Oxford, UK, pp. 173-190.

Vanneste, S. & Friml, J., 2009. Auxin: A trigger for change in plant development. Cell., 136: 1005–1016.

Ward, K. A. & Jordan. M. C., 2001. Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.). In Vitro Cell. Dev. Biol. –Plant., 37: 361-368.

Williams, E. G. & Maheswaran, G., 1986. Somatic embryogenesis: Factors influencing coordinated behavior of cells as an embryogenic group. Ann. Bot., 57: 443-462.


PK ISSN 0006 – 3096

Effect of water restriction on the lymphoid organs and

production of broilers

YASSER SALEEM MUSTAFA, ABDUL QAYYUM KHAN SULEHRIA, M. A.

MUNEER & RAHAT MUNIR

Provincial Diagnostic Laboratory, 16-Cooper Road, Lahore (YSM), Department of

Zoology, GC University, Lahore (AQKS), Project Director, NPCPAI, Islamabad (MAM) AND City District Government, Lahore (RM)-Pakistan.

ABSTRACT

The effect of water restriction for different time intervals on lymphoid organs and production of broiler chicks was studied. The effect of water restriction on the mean thymus weight (gm) of chicks in group C (0.60+0.07) was adversely effected as the chicks in this group had significantly (P<0.05) lower weight than group D (4.09+0.70). This indicates that increased in the period of water restriction in chicks adversely affected the mean thymus weight as compared to chicks reared on ad-libitum water. The mean bursal weight (gm) of group C (0.07+0.02) was significantly (P<0.05) lower as compared to group D (1.37+0.88) indicating that water restriction of 24 hrs had affected the bursal development in the chicks. The mean spleenic weight (gm) of group C (2.64+1.49) was significantly (P<0.05) higher than groups A, B and E. The FCR values were significantly (P<0.05) different among groups in 6

th week and there was non-significant (P>0.05)

difference among groups with treatment of Vitamin C, Vitamin E and glucose treated groups. Key words:Bursa of fabricius, FCR, Glucose, Spleen, Thymus, VC and VE, Water and

Weight.

INTRODUCTION

Poultry industry is an important segment of livestock, emerged as a

cheap source of protein for consumption by humans. In Pakistan, the commercial broiler farming was initiated in the early 1960’s with the import of Shaver broiler chicks (Qureshi, 1993). Ever since, the industry is progressing and growing at a rate of about 10 percent per annum and the present share of poultry meat in overall meat and beef consumption at national level is around 35 percent. This industry in Pakistan consists of over 25 thousand commercial farms, 200 chick hatcheries, and 120 Feed Mills, generating employment for about 1.5 million people. The current investment in Poultry industry is around Rs. 320 billion, and it is regarded as a vital source of eggs and meat for the common man. The present poultry production figures are estimated at 425.92 million day old chicks, of which 407.77 million are broiler chicks, which provide 501.31 metric tons of poultry meat per annum. (Anonymous, 2008-09).

Water is one of the important nutrients which is consumed in greater quantity than any of the other nutrients. Birds may die rapidly by the lack of water as compared to lack of any of the other nutrients. For an optimal growth the birds should have free and convenient access to water. The body requirement of water

Y. S. MUSTAFA ET AL BIOLOGIA (PAKISTAN)

64

varies with the age, health status, ambient temperature and the reproductive status of birds. In poultry production water is needed for sustaining the life of birds, to reduce air temperature (includes evaporative cooling pad and fogging systems), and to clean, wash and sanitize the farm premises.

Chickens are able to survive much longer without feed than without water (Katanbaf et al., 1989). A rule of thumb for water is that the bird consumes from 1.5 to 2 times as much water as it does feed (Kellems & Church, 2002).

In the poultry farming, various vitamin preparations are available and prescribed for alleviating the stress on birds (Tibiletti, 1993). Vitamin E is fat soluble and is essential for maintaining the integrity and optimal functioning of the muscular, circulatory, nervous and immune systems (Gershwin et. al., 1985).

Vitamin C is also prescribed as an anti-stressor for the improvement of growth, feed efficiency, immune responses, and livability performance of birds (Muneer et al., 2001).

In Pakistan, the importance of clean and salt-free water for poultry is usually overlooked by the farmers and provision of unhygienic water to the poultry flocks is a common practice in the field conditions. The present work will further help the farmers to realize the importance of the factor in maintaining and improving the overall health status of poultry.

MATERIALS AND METHODS The effect of water restriction on the performance of broiler chicks was

evaluated by rearing a total of 400, Hubbard X Hubbard chicks. All the experimental chicks were assigned randomly to five experimental groups i.e. A, B, C, D and E, each group consisting of 80 chicks. The chicks were separately housed and were offered the commercial broiler feed and water. The experimental chicks in the group A, B, C and E were reared at water restriction for 6, 12, 18 and 24 hrs/day and then offered water for 24 hrs (alternatively); those in group D were offered water ad libitum. The chicks received vaccination Infectious Bronchitis (IB), Newcastle Disease (ND); Infectious Bursal Disease (IBD) and Hydro-pericardium Syndrome (HPS).

At the age of day 50th lymphoid organs such as bursa of Fabricius,

thymus, and spleen of both live and dead birds in each experimental group were removed and observed for morphometeric analysis. At the age of day 56

th, the

experimental chicks were again slaughtered and weight of above referred lymphoid organs was recorded. These organs were excised and cleared off the fat and extraneous tissue material, each organ was separately weighed on an analytical weighing Sartorius Balance and a group organ mean weight was determined.

To observe the effect of various anti-stressors, each experimental group (A, B, C and D) were further sub divided in three sub-groups. The vitamins and glucose treatment were continued from days 36

th to 56

th and of the 03 subgroups

in each experimental group (A1, A2, A3; B1, B2, B3; C1, C2, C3 and D1, D2, D3). These vitamins and glucose were administered via the drinking water as per recommended dosage level of VC 350mg/L, VE 250mg/L and glucose 1gm/L in drinking water. The control chicks in group E did not receive any of the vitamins

Vol. 56 (1&2) Water Restriction on the Lymphoid Organs 65

or glucose. The FCR values of each experimental group were calculated at the age 6, 7 and 8 weeks using the following formula described by Singh & Panda (1992).

The data obtained through the experiment was analyzed using analysis of variance and the statistical differences among the various treatment means were determined using the Least Significant Difference (LSD) test at 05% probability level according to the method described by Steel & Torrie (1980).

RESULTS AND DISCUSSION Water is a necessary agent in all body processes and is the most critical nutrient for the production of poultry. It serves to soften and hydrolyze feedstuff for digestion. Water consumed by the bird is used for nutrient transportation, enzymatic and chemical reactions in the body, body temperature regulation and lubrication of joints and organs. It is eliminated by the bird through feces and respiration. 1. Effect of Water Restriction on the Lymphoid Organs of Chicks

I. Thymus. This investigation indicated significant (P ≤ 0.05) differences in the mean thymus weight among groups. Chicks in group D (4.09±0.70) gained significantly (P ≥ 0.05) higher mean thymus weight than groups A (1.75±0.58), B (0.65±0.08) and C (0.60±0.07) while group E (0.49±0.03) had significant (P ≥ 0.05) lower mean thymus weight than the chicks belonging to groups A, B, C and D (Table-1).

II. Bursa of Fabricius. The chicks in group D (1.37±0.88) gained significantly (P ≤ 0.05) higher mean bursal weight than A (0.69±0.12), B (0.27±0.06) and group E (0.23±0.12) while group C (0.07±0.02) had significantly (P ≤ 0.05) lower mean bursa weight than those of all other groups i.e. A, B, D and E (Table-1).

III. Spleen. There were non-significant (P ≥ 0.05) differences in spleenic weight of groups B (0.99±0.07) and E (0.99±0.01). The chicks in group C (2.64±1.49) had significantly (P ≤ 0.05) higher mean spleenic weight than groups A (1.45±0.22), B (0.99±0.07) and E (0.99±0.01). The mean spleenic weight of the water restricted chicks in the groups A and B was significantly (P ≤ 0.05) lower than the chicks in group D (Table-1).

Pires et al. (2007) reported that water deprivation in post hatching between 48 and 72 hours of age decreased spleenic weight but bursal weight was not affected in broilers which are not in agreement withthe findings of present work.

Offiong et al. (2003) reported decreased spleenic, liver and heart weight in 6-8 hours water deprivation daily in broiler chicks. Negative effect of post hatching water deprivation on liver weight in broiler was also reported by Maorka (2002).

Y. S. MUSTAFA ET AL BIOLOGIA (PAKISTAN)

66

Table 1: Morphometery of Lymphoid Organs of Water Restricted and Unrestricted Chickens

Groupsg

Body weight (gm)

Thymus Weight

(gm)

Bursa Weight (gm)

Spleen Weight

(gm)

Mean ± S.E Mean ± S.E Mean ± S.E Mean ± S.E

Group-A 1466.56±9.89b 1.75±0.58

b 0.69±0.12

b 1.45±0.22

c

Group-B 1398.87±10.21c 0.65±0.08

c 0.27±0.06

c 0.99±0.07

d

Group-C 1444±10.55b 0.60±0.07

c 0.07±0.02

d 2.64±1.49

a

Group-D 1534.25±14.85a 4.09±0.70

a 1.37±0.88

a 2.06±0.14

b

Group-E 1489.12±10.50b 0.49±0.03

d 0.23±0.12

c 0.99±0.01

d

Groups g = Group A, water restriction for 12 hrs; group B, water restriction for 18 hrs,

groupC, water restriction for 24 hrs and ad. libitum water for 24 hrs, alternatively; group D, no waterrestriction (ad libitum water available throughout experimental period) and group

E, water restriction for 6 hrs. a, b, c, d

Any two means carrying the same superscript are not significantly different from each other at 5% probability level using LSD test.

2. Effect of Water Restriction on the Feed Efficiency of Chicks

In the first week of their age, birds in all experimental groups had same FCRs (1.58±0.08) indicating no effect of any type on their feed conversion efficiency. However, in the 6

th week of their age, the effects of water restriction on

FCR were quite evident. The mean FCR values at week 6 of age of chicks in the experimental groups A, B, C, D and E were recorded as 2.37±0.04, 2.50±0.09, 2.38±0.14, 2.25±0.37 and 2.42±0.07, respectively, indicating that the water restriction in chicks caused poor FCR as compared to the birds reared under ad libitum water availability. The differences in the FCR values of chicks in various groups were significant (P≤0.05) at the age of 6 wks. The use of vitamin C, vitamin E and glucose did not indicate any improvement in FCR values of the treated groups (Table-2).

In the 7th week the effect of VC showed significant differences among

groups and group B (2.58±0.16) has significantly high FCR than group A (2.38±0.03) and C (2.38±0.09). The effect of VE showed significant (P ≤ 0.05) differences among groups A (2.37±0.05), C (2.46±0.09), D (2.28±0.18) and E (2.66±0.12). The treatment of glucose showed no differences between groups B (2.58±0.08) and C (2.50±0.09) but differences in groups A (2.33±0.05) and E (2.68±0.15). In 8

th week the effect of VC showed no significant differences in

groups A (2.44±0.08) and C (2.49±0.06) but differences from groups B (2.60±0.07) and E (2.65±0.11). The effect of VE showed no differences in groups A (2.42±0.17) and C (2.54±0.02) but significant (P ≤ 0.05) differences from groups C (2.58±0.13) and E (2.67±0.17). The effect of glucose showed no significant differences in groups B (2.65±0.15) and E (2.63±0.04) but significant (P ≤ 0.05) differences from group A (2.47±0.23) (Table-2).

Vol. 56 (1&2) Water Restriction on the Lymphoid Organs 67

Table 2: Weekly Body Weight/FCR (Mean ± S.E) of Birds of Water Restriction Stress and Control Groupsg

Groupsg

Age in Weeks

6 7 8

Vitamin C Vitamin E Glucose Vitamin C Vitamin E Glucose

Weight(FCR) Weight(FCR) Weight(FCR) Weight(FCR) Weight(FCR) Weight(FCR) Weight(FCR)

Group-A 1466.56±9.89 (2.37±0.04)

b

1543.75±12.45 (2.38±0.03)

a

1520±10.09 (2.37±0.05)

b

1535±14.26 (2.33±0.05)

a

1625±13.01 (2.44±0.08)

a

1535±9.76 (2.42±0.17)

a

1558±12.21 (2.47±0.23)

a

Group-B 1398.87±10.21 (2.50±0.09)

c

1472.50±11.11 (2.58±0.16)

c

1380±10.11 (2.60±1.41)

d

1455±12.62 (2.58±0.08)

b

1550±13.21 (2.60±0.07)

c

1432±10.01 (2.58±0.13)

b

1512±12.21 (2.65±0.15)

c

Group-C 1444±10.55 (2.38±0.14)

b

1520±11.20 (2.38±0.09)

a

1478±12.35 (2.46±0.09)

c

1500±12.38 (2.50±0.09)

b

1600±13.09 (2.49±0.06)

b

1530±10.9 (2.54±0.02)

b

1578±12.32 (2.58±0.11)

b

Group-D 1534.25±14.85 (2.25±0.37)

a

1615±15.21 (2.31±0.18)

b 1500±2.35

(2.42±0.11) a

1565±10.3 (2.36±0.19)

a

1700±15.68 (2.55±0.14)

a 1580±9.35

(2.56±0.11)b

1678±12.35 (2.45±0.09)

a

Group-E* 1489.12±10.50

(2.32±0-07) 1567.50±9.35 (2.35±1.09)

1660±12.35 (2.42±0.12)

Groups g

= Group A, water restriction for 12 hrs; group B, water restriction for 18 hrs, group C, water restriction for 24 hrs and ad. libitum water for 24 hrs, alternatively; group D, no water restriction (ad libitum water available throughout experimental period) and group E, water restriction for 6 hrs. *

Chicks note given treatment with Vitamin. C, Vitamin. E or glucose

a, b, c ,d, Any two means carrying the same superscript are not significantly different from each other at 5% probability level using

LSD test.

68 Y. S. MUSTAFA ET AL BIOLOGIA (PAKISTAN)

The findings of present study are different from Offiong, et al. (2003) who reported no significant changes in FCR in chicks that were water deprived daily for 6-8 hrs.

REFERENCES Annonymus, 2008-09. Economic Survey of Pakistan.Finance

Department,Government of Pakistan, Islamabad. Gershwin, M., Beach, R. & Hurley, L., 1985. The potent impact of nutritional

factors on immune response. In: Nutrition and Immunity. Academic Press, New York. pp. 1-7.

Katanbaf, M. N., Dunnington, E. A. & Siegel, P. B., 1989. Restricted feeding in late-feathering chickens.1.Growth and physiological response. Poult. Sci., 68:344-351.

Kellems, R. O. & Church, D. C., 2002. Livestock Feeds and Feeding. 5th Ed.

Prentice Hall, Upper Saddle River, New Jersey. Maiorka, A., 2002. Desenvolvimento do Trato Gastrointestinal de Frangos

[Dissertacao]. Jaboticabal (SP): Universidade Estadual Paulista. Muneer, M. A., Bahram, A. M., Munir, Z., Hussain, I,Muhammad, K., Rabbani,

M., Akhtar, S., Aleem, M., Sultan, B., Tariq, M. A., & Naeem, K., 2001. Isolationand characterization of avian influenza (H9N2) virus from an outbreak at poultry farms in Karachi (Pakistan). Pak. Vet. J., 21(2): 87-91.

Offiong, S. A., Akpan , F. S & Ojebiyi, O. O., 2003. Effect of partial water deprivation on the performance and carcass characteristics of broiler chickens exposed to seleted durations of water deprivation in the humid tropical environment. Global J. Pure Appl. Sci., 9(1):1-6.

Pires, D. L., Malheiros, E. B & Boleli, I. C., 2007. Influence of sex, age and fasting on blood parameters and body bursa, spleen and yolk sac weights of broiler chicks. Rev. Bras. Ciencc.Avic., 9(4):166-170.

Qureshi, S. R., 1993. A study on the prevalence of different disease in broiler flock monitored in Distt. Sargodha, Annul Progress Report. Poultry Research Institute, Rawalpindi.

Singh, K. S. & Panda, B., 1992. Feed efficiency, In: Poultry Nutrition, 2nd

Ed. Kalyan Publ., Rajinder Nagar, Iughiana, India. pp. 199.

Steel, R. G. D. & Torri, J. H., 1980. Principles and Procedures of Statistics. 2nd

Ed. McGraw Hill book Co. Inc, New York.

Tibiletti, E., 1993. Combating stress with probiotics. Rivista di Avicoltura,

62(6):19-2


PK ISSN 0006 – 3096

Comparison of spider guilds found in various oilseed crops

of Pakistan

MUHAMMAD MOHSIN, ABDUL QAYYUM KHAN SULEHRIA, IFTIKHAR

YOUSUF, MUHAMMAD EJAZ, MUHAMMAD JAMEEL YOUSUF & ALTAF

HUSSAIN

Department of Biology, Govt. Post Graduate College, Satellite Town, Gujranwala (MM, MJY); GC University, Lahore (AQKS, AH); AMC, Department of

Physiology, Rawalpindi (IY); Govt. Degree College, Peoples Colony, Gujranwala (ME)

ABSTRACT

Spiders utilize similar resources in different ways in agro-ecosystems. This assemblage of spiders is known as guild. Here spiders have been classified into guilds by using quantitative analysis of ecological characteristics of spider families occurring in six major oilseed crops of Pakistan. The similarity in spider species richness among crops was evaluated with the Sørensen qualitative coefficient while similarity in spider guild composition was calculated with the proportional similarity index. Clusters were constructed using the unweighted average linkage method. While total species richness varied widely among crops, the proportion of the total species within each guild was remarkably even across crops. The relative abundance of guilds varied greatly, which may reflect availability of resources within a crop type. Patterns of resemblance in guild lay out suggest the possibility of plant habitat structure as an influence on the spider community. Key words: Species richness, Spider guild composition, Oilseed crops, Spider guild.

INTRODUCTION

Invertebrate fauna is present in every agro-ecosystem in which insects

and their relatives predominate. Higher spider species diversity is found in natural and agricultural areas, however, their specific diversity is generally lower than that of insects (Marc & Ysnel, 1999). A larger number of spider species are associated with oilseed crops as compared to any other agro-ecosystem. Among oilseed crops cotton shows the maximum spider species richness (Schoeman et al., 1999). Unfortunately spiders have largely been ignored because of the human tendency to favor some organisms over others of equal importance because they lack a universal appeal (Humphries et al., 1995).

Spiders are among the numerically dominant insectivores in terrestrial ecosystem and exhibit a very diverse range of life style and foraging behaviors. They are important predators of insect pests. They form a major component of the generalist predator fauna and are potentially able to restrict pest population growth (Harwood et al., 2001). Though they are mostly generalist predators but some spider species are specialist predators (Whitecomb, 1962). Spiders can be considered as an ideal biological control agents because besides being generalist predators they are capable of propagating their population rapidly (Slansky & Rodriguez, 1989). They feed on a wide range of different animal

M. MOSHIN ET AL BIOLOGIA (PAKISTAN)

70

groups, however, in general they tend to concentrate on insect prey. The significance of spider assemblages as biological control of pest is cosmopolitan (Pearce et al., 2004).

Interest has been developed to help growers and consultants to understand spiders and their true value in crops: so that they begin to incorporate them in IPM strategies. Among beneficial species in crops, spiders are usually ignored and only predatory insects are considered. The information regarding their predatory role would allow spiders to become an integral part of IPM strategies. Favorable results can be achieved by using spiders as biological control agents in combination with selective and non-persistent insecticides and restricting the number of their applications to only specific times so as to protect them (Mushtaq et al., 2003, 2005).

The indiscriminate use of insecticides in agro-ecosystems has been increased many folds resulting in ecological imbalance and appearance of more resistant pest strains in oilseed crops. Spiders as an excellent bio-control agent offer an opportunity and provide an alternative means of control for insect pest.

Prior to this attempt in Pakistan various studies on biodiversity, relative abundance of spiders and ecology of oilseeds crops were carried on cotton, maize and canola by Saleem (1999); Ghafoor (2002) and Tahir (2008) from Faisalabad. The objective of present research is to review the work of various researchers on spider fauna of oilseed crops so that we may be able to identify spiders into families, genera and species that will help us to know about species composition, relative abundance and spider guilds found in oil seed crops after analyzing data statistically that is likely to increase the production of oils from oilseeds crops in Pakistan.


A number of studies have been done in previous years by the workers of

Araneae laboratory, Department of Zoology and Fisheries, U. A. F. on the biodiversity of spiders found in oilseed crops such as cotton, maize, peanut, canola, soybean etc. In the present study the data provided in these research dissertations has been used and is re-evaluated to know about the spider guilds residing in different oil seed crops and highlight the spider assemblage found in the form of spider guilds which will provide an appropriate parameter for the selection and justification of spider species. Guild concept refers to a group of species utilizing the same resource in similar ways (Uetz., 1991). Therefore guilds form the basis of community and reflect taxonomic relationships. Each guild will be comprised of most dominant families, genera and species found in foliage as well as on ground surface. In this way an excellent combination in the form of most appropriate spider species guild found in ground and upper surface of plants will be identified for use in IPM.

Different collection methods for ground and foliage spiders were used. In the case of ground spiders mostly pitfall traps were used in grid and trap line fashion and for the foliage spiders mostly jarring, net sweeping and hand picking methods were employed which resulted into a good number of species recorded by students.

Vol. 56 (1&2) Spider Guilds Found in Various Oilseed Crops

71

The similarity in spider species has been evaluated with Sorensen qualitative coefficient (Southwood, 1992). The spider guild composition was calculated with proportional similarity index (Price, 1984; Smith, 1996). The clusters have been constructed using the un-weighted average method (Pielou, 1984). Cluster analysis was performed with STATISTICA (StatSoft, 1997).

RESULTS

The hierarchical cluster analysis produced a dendrogram which was

used to construct spider guilds (Fig. 1). The breakdown of successive clusters appeared to be based primarily on web use, web-type, and microhabitat, resulting in 5 to 7 clusters of spider families that can be considered as guilds. Araneidae and tetragnathidae are 100% similar. Dictynidae and theridiidae also showed 100% resemblance. Among philodromidae and thomicidae there existed 93.5% similarity. 85.4% is the similarity level between the oxyopidae and salticidae. Corinnidae and erignoidae resemble 72%. Gnaphosidae and lycosidae show 83.3% similarity. Scytodidae, oonopidae, clubionidae and linyphiidae show similarity of 54.6%, 66.6%, 73.2% and 81.4% with their respective spider guild correspondingly.

Cotton, maize and canola support the highest number of spider species followed by peanut and soybean. Sunflower is the most species-poor crop (Fig.2). Besides vast differences in species richness, spider faunas of individual crops were also distinctly dissimilar. Soybean, maize and canola share similarity of 99.7%. Sunflower has similarity of 98.7% with its respective spider guild. 96.4% similarity exists among cotton and its respective spider guild. Peanut share similarity of 82.3% with spider guild formed by all other crops. In soybean a total of 276 spiders were captured, out of which 260 are ground runners and 16 are foliage runners. In sunflower there are 165 specimens of spiders found, out of which are 23 ambushers, 119 ground runners, 20 foliage runners and 3 stalkers. In canola 445 were ground runners, 40 stalkers, 14 foliage runners and 25 ambushers. The total number of spiders caught from canola was 524. In maize a total of 3079 spiders were caught, out of which 2705 were ground runners, 20 foliage runners, 101 stalkers, 39 ambushers, 213 orb-weavers and 1 wandering. In peanut there were stalkers 213, ground runners 127, ambushers 71, orb-weavers 18 and 16 foliage runners. Total number of spiders caught from peanut was 445. From cotton 5769 spiders were collected, in them were ground runners 2899, foliage runners 906, ambushers 291, stalkers 1036, orb-weavers 545, wandering 47 and space web 45.

Proportional similarity in the relative species richness of spider guilds of oil seed crops indicate that there exists 98.6% similarity among soybean and canola as far as their species richness is concerned (Fig. 3). Maize share 97.9% similarity with its respective guild of soybean and canola. Sunflower, soybean, canola and maize share 94.4% similarity. Peanut and cotton share 97.2% similarity and their guild share similarity of 87.3% with the guild formed by all other crops. In soybean there were 88% ground runners and 12% foliage runners. On the other hand in sunflower there were ambushers 26%, ground runners 53%, foliage runners 16% and stalkers 5%. In canola there were 64% ground runners, 12% foliage runners, 11% ambushers and 13% stalkers. Maize


72

had ground runners 59%, stalkers 17%, orb-weavers 9%, foliage runners 7%, ambushers 6% and wandering 2%. In peanut stalkers were 34%, ground runners 40%, ambushers 13%, foliage runner 5% and orb-weaver 8%.

Spider guild structure (proportional abundance) varied among individual crops (Fig. 4). The dendrogram represents that soybean, peanut, canola and sunflower show similarity of 77.3%. Cotton had the similarity of 62.9% with the guild formed by these four crops. On the other hand maize had the similarity of 41.6% with guild formed by all other crops. Spider guild composition represented that in soybean there are 94% ground runners and remaining 6% were foliage runners. In sunflower there are ambushers 13%, ground runners 72%, foliage runners 12% and stalkers 3%. There are 84% ground runners, 7% stalkers, 2% foliage runners and ambushers 7% in canola. In maize there are ground runners 87%, foliage runners 1%, stalkers 4%, ambushers 2%, orb-weavers 5 % and


73

wandering 1%. In peanut there are stalkers 34%, ground runners 35%, ambushers 20%, orb-weavers 5% and foliage runners 6%. In cotton there are ground runners 50%, foliage runners 15 %, ambushers 5 %, stalkers 17 %, orb-weavers 9%, wandering 1 % and space web 3%.

DISCUSSION

Various spider species utilize similar resources in different ways and form an assemblage in an ecosystem. Not only different spider species are associated with different oilseed crops but their composition also varies (Luczak, 1979; Nyffeler, 1982). The occurrence of particular spider species in specific crops is due to their response to particular features of the habitat or complex community interactions (Moran & Southwood, 1982). It has also been observed that habitat structure maintain diverse spider assemblages (Wise, 1993). Neighboring habitats also directly influence spider species and their abundance particularly in patchy agricultural landscapes (Alvarez et al., 1997).


74

Many early workers stated that there was no evidence of a particular prey preference in spiders. However spiders show preference to certain pests (Bristowe, 1941). Some spider species clearly dominate over the others because their preferred prey reside in that particular ecosystem (Pekar, 2005). There are some specialist predators among spiders such as Pardosa milivina feeds predominately on the pink boll worm (Clark & Glick, 1961). It is very good to use spiders as biological control agents because some species are specialist predators. For a particular crop spider guilds can be used as excellent biological control agents for targeting specific pest species. The present study provides an ample evidence that spiders can be used in a better way in IPM strategies.


75

REFERENCES

Alvarez, T., G. K. Frampton & Goulson, D., 1997. Population dynamics of epigeic Collembola in arable fields: The importance of hedgerow proximity and crop type. Pedobiologia., 41:110-114.

Bristowe, W. S., 1941. The community of spiders., Ray Soc., No. 128. Clark, E. W. & Glick, P.A., 1961. Some predators and scavengers feeding upon

pink bollworm months. J. Econ. Entomol., 54: 806-815. Ghafoor, A., 2002. Taxonomic and some Ecological studies of the Cursorial

spiders of cotton fields at Faisalabad. Ph.D. Thesis, Department of zoology and fisheries, University of Agriculture, Faisalabad., pp. 223-235.

Harwood, J. D., K. D. Sunderland & Symondson, W. O. C., 2001. Web location by linyphiid spiders in relation to prey availability in winter wheat. J. Appl. Ecol., 38:88-99.


76

Humphries, C. J., P. H. Wilson & Vanewright, R. I., 1995. Measuring biodiversity value for conservation. Ann. Rev. Col. & Syst., 26: 93-111.

Luczak, J., 1979. Spiders in agrocoenoses. Poli. Ecol. Stud., 5:151 -200. Marc, P. A. C. & Ysnel, F., 1999. Spiders (Araneae) useful for pest limitation and

bio-indication. Agric., Ecosyst. Environ., 74: 229-273. Moran, V. C. & Southwood, T. R. E., 1982. The guild composition of arthropod

communities in trees. J. Anim. Ecol., 51:289 -306. Mushtaq, S., M.A. Beg & Aziz, S., 2003. Biodiversity and temporal variations in

the abundance of cursorial spiders of a cotton fields at Faisalabad. Pak. J. Zoo., 35: 125-131.

Musthaq, S., M. A. Ali, M. Riaz, A. Murtaza & Ahmad, S., 2005. Spider as insect’s natural enemies: evaluation of feeding niche of co-existing foliage species in cotton. Indus Cotton., 2: 193-204

Nyffeler, M., 1982. Field studies on the ecological role of spiders as insect predators in agro-ecosystems (abandoned grasslands, meadows and cereal fields). Ph.D. Thesis. Swiss Fed. Inst. Tech., Zurich, Switzerland.

Pearce, S. W., M. Hebron, R. J. Raven, M. P. Zalucki & Hassan, E., 2004. Spider fauna of soybean crops in south-east Queensland and their potential as predators of Helicoverpa spp. (Lepidoptera: Noctuidae). Australian J. Entomol., 43: 57-65.

Pekar, S., 2005. Horizontal and vertical distribution of spiders (Araneae) in sunflowers. J. Arachnol., 33: 197-204.

Pielou, E. C., 1984. The Interpretation of Ecological Data. Wiley, New York. Price, P. W., 1984. Insect Ecology (2

nd ed.). Wiley, New York.

Saleem, S., 1999. Comparison of diversity and relative abundance of ground dwelling spiders in two varieties of maize at Faisalabad. M.Sc. Thesis, Department of zoology and fisheries, University of Agriculture, Faisalabad.

Schoeman, D. A., V. D. Berg & Clark, A., 1999. Study spiders in South African cotton fields: species diversity and their abundance (Arachnida: Araneae). Afri. plant protec., 5: 93-103.

Slansky, F. & Rodriguez, J. G., 1989. Nutritional ecology of insects, mites, spiders and related invertebrates.

Smith, R. L., 1996. Ecology and Field Biology (5th ed.). Harper Collins.

Southwood, T. R. E., 1992. Ecological Methods. (2nd

ed.). Chapman and Hall, London. StatSoft, Inc., 1997. STATISTICA for Windows (computer program manual).

Tulsa, Oklahoma. Tahir, T., 2008. Ecological studies on foliage spider fauna of cotton agro-

ecosystem. M. Phil. Thesis, Department of zoology and fisheries, University of Agriculture, Faisalabad.

Uetz, G. W., 1991. Habitat structure and Spider foraging., pp. 325-348. In Habitat Structure; the Physical Arrangements of Objects in Space. (eds McCoy, E. D., S. A. Bell, Mushinsky, H.R.) London: Chapman and Hall.

Whitecomb, W. H., 1962. Egg sac construction and oviposition of green lynx spider, Peucetia viridans (Oxyopidae). Southwestern Nat., 7: 3-4.

Wise, D. H., 1993. Spiders in ecological webs. Cambridge Univ. Press.


PK ISSN 0006 – 3096

Evaluation of biodegradation potential of bacteria in crude

oil contaminated soil

CHAND RAZA, ATIF BILAL & NUSRAT JAHAN

Department of Zoology, Government College University, Lahore, Pakistan (CR, AB, NJ)

ABSTRACT

Crude oil biodegradation has emerged as an important paradigm in the environmental toxicology. Various lines of evidence indicate substantial increase in methodologies to counteract environmental contamination. In the present study, bacterial flora of Rajian and Missa Kaswal oil field soils, from Punjab province was characterized and monitored for their crude oil biodegradation potential. Random soil sampling from waste pits was done. Careful phenotypic and biochemical tests, revealed twenty oil degraders belonging to seven genera, such as Pseudomonas, Bacillus, Klebsiella, Acinetobacter, Clostridium, Erwinia and Streptococcus from Rajian oil field. These microbes were introduced separately in crude oil contaminated soil for 30 days. Twenty-four isolates belonging to twelve genera: Acenitobacter, Amphibacillus, Arthrobacter, Bacillus, Corynebacterium, Flavobacterium, Klebsiella, Lactobacillus, Listeria, Moraxella, Providencia and Pseudomonas, were characterized from Missa Kaswal oil field. Two consortia namely Consortium A (05 strains) and Consortium B (08 strains) were designed and applied on crude oil contaminated soil for 15 days. Microbial enumeration, reductions in weight and crude oil contents were recorded. Comparative findings demonstrated greater degradation of crude oil by members of Pseudomonas (20.20-13.34%), followed by Bacillus (16.68-8.40%), Erwinia (11.55%), Clostridium (11.12%), Klebsiella (10.99-7.22%), Acinetobacter (9.68-9.20%) and Staphylococcus (5.99%) genera, respectively.

Effective removals of 13.38% and 15.84% crude oil contents were noted by Consortia A and B, respectively. This study categorized Pseudomonas, Acinetobacter, Bacillus, Corynebacterium and Klebsiella as common microbial genera of both oil field soils and promising biodegradation of crude oil by microbial consortia in relatively shorter time.

Keywords: Crude oil; Biodegradation; Consortium

INTRODUCTION

It is becoming progressively more understandable that hundreds of

millions of liters of petroleum enter the environment from both natural and anthropogenic sources every year. It is appropriate to mention that the input from natural marine oil seeps alone would be sufficient to cover all of the world's oceans in a layer of oil 20 molecules thick. In accordance with this presumption that globe is not swamped with oil is result of the efficiency and multifunctionality of the networks of microorganisms that degrade hydrocarbons. It is worth mentioning that findings obtained through laboratory methodologies have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.

78 C. RAZA ET AL BIOLOGIA (PAKISTAN)

Industrial globalization over the last few decades has led the dramatic increase of prevalent contaminants including polycyclic aromatic hydrocarbons (PAHs) in soil and aquatic environment by anthropogenic ways (Huang et al., 2004). Massive oil spills have resulted in irreversible damage to biodiversity and affected ecosystem structures (Reynolds, 1993). Crude oil is classified into four major groups including: aliphatic hydrocarbons, aromatic hydrocarbons, resins and asphaltenes. In nature, aliphatic and aromatic hydrocarbons are easily degraded by bacteria and fungi but resins and asphaltenes undergo biodegradation in a complicated manner (Rittmann & McCarty, 2001; Xu et al., 2006).

A wide variety of microbes is capable of degrading crude oil fractions and can be applied to rehabilitate hydrocarbon contaminated soil (Chaudhry et al., 2005; Singh & Jain, 2003). Initial degradation of the hydrocarbon is an oxidation process which is catalyzed by the oxygenases and peroxidases. The common

metabolic pathway for alkane degradation is: alkane alcohol aldehydes

fatty acid acetate via oxidation (Ron & Rosenberg, 2002). Via oxidation these crude oil metabolites degrade further (Rittmann & McCarty, 2001). van Beilen et al. (2001) have reported that alkane degradation pathway is regulated by OCT plasmid DNA in Pseudomonas putida by rubredoxin, monooxygenases and rubredoxin reductase complexes, which function to shunt electrons to the hydroxylase through NADH for conversion of an alkane to alcohol.

Different strategies are currently being evaluated to counteract the tremendously growing threat of hydrocarbon contamination. Chemical extraction is expensive process dealing with tremendous use of organic solvents to extract hydrocarbons from contaminated soils. Increasingly sophisticated information is revealing the fact that microbial biodegradation of environmental pollutants is a significant aspect of environmental health. Biostimulation as Namkoong et al., (2002) indicated, the supply of minerals to spill site boosted the rate of hydrocarbon degradation by counteracting high carbon to nitrogen ratio. Kuiper et al. (2004) showed the application of exogenous microbes having greater biodegradation capabilities (Bioaugmentation) resulting in increased biodegradation rates. The efficiency of biodegradation of microbial consortium is not only the result of merely sum total of the capacities of the individual strains but also provide intermediate substrates for other microbes. Alkanes were degraded by Acinetobacter sp. T4 to intermediate metabolites, which were efficiently taken in by Pseudomonas putida PB4 and finally degraded aromatic hydrocarbons (Komukai-Nakamura et al., 1996).

Current study aims to evaluate bacterial community from Rajian and Missa Kaswal oil field soils, and finding their biodegradation potential in isolated and mixed assemblage forms under laboratory conditions in order to understand role of microbial community in crude oil contaminated environment.


The areas under study Missa Kaswal and Rajian oil fields are located in Gojar Khan and Chakwal districts respectively. Soil samples were taken from waste pit of oil field with the help of sterilized spatula and zipper bags sampling was done.

Vol. 56(1&2) Biodegradation Potential of Bacteria 79

Enrichment and Isolation Enrichment of soil microbial community was carried in Mineral Salt (MS)

medium, composed of Na2HPO4, 5.9 g/L; KH2PO4, 1.5 g/L ; NH4Cl, 0.3 g/L; MgSO4.7H20, 0.1 g/L; KNO3, 2.0 g/L. 5 ml of Trace element solution was added, comprising of EDTA, 50.0 g/L; ZnSO4.7H2O, 22.0 g/L; CaCl2, 5.54 g/L ; MnCl2.4H2O, 5.06 g/L ; FeSO4.4H2O, 4.99 g/L; (NH4)6Mo7O24.4H20, 1.10 g/L ; CuSO4.5H2O, 1.57 g/L ; CoCl2.6H2O, 1.61 g/L. From each soil sample, 1.0 gm of soil was suspended in autoclaved 99.0 ml MS medium enriched with 1.0ml of crude oil in 250 ml conical flask. Rotating flask incubator was used to incubate at 37 °C for 96±2 hours at 100 rpm. In freshly prepared MS medium enriched with 1% crude oil, 5.0 ml volume from previously enriched microbial suspension was transferred. After inoculation in laminar air flow cabinet, flasks were incubated. Further isolation was done according to standard procedure of serial dilution on nutrient agar plates.

Identification of Isolates

Identification of isolates was carried on careful phenotypic and biochemical basis. Colony morphology, motility and stainings (Grams, Spore, Capsule and Acid Fast) properties were initially studied. Biochemical characterization of isolates to genus level was carried out according to standard microbiological criterion. Microbial biochemical properties were assessed on the basis of catalase, oxidase, citrate reduction, nitrate reduction, methyl-red, voges proskaur, McConkey agar, EMB agar, indole, urease, starch hydrolysis and triple sugar fermentation tests and identified according to the Bergey’s Manual of Systematic Bacteriology.

Inoculum and Consortium Preparations Standard inocula were prepared by overnight incubation of pure cultures

in nutrient broth (OXOID) at 37 °C and 100 rpm in shaking incubator. Individual inoculum preparation was made at above mentioned conditions for Rajian oil field isolates. Consortia were prepared by mixing equal volumes of pure strains to get 50 ml suspention. Consortium 1 was formulated by two strains of Corynebacterium and one strain each of Arthrobacter, Bacillus and Listeria genera, while consortium 2 was formulated by three strains of Corynebacterium and one strain each of Arthrobacter, Bacillus, Listeria, Flavobacterium and Moraxella genera.

Crude oil contaminated soil preparations Two sets of soil samples were artificially contaminated with crude oil for

studying individual and consortium potential of crude oil biodegradation. Garden soil from Science Block of GC University Lahore was collected and it was sieved through 1mm pore sized mesh. Sorted and sieved soil was sterilized at 121 °C for 60 minutes three times with the gap of 24 hours.

One set of soil samples (S1) contaminated with crude oil to evaluate biodegradation potential of individual bacterium, was prepared by mixing 22 ml of sterilized crude oil in 100 grams of sterilized soil in Petri dish, along with two controls, namely control A with 10 ml of all isolated bacterial strains and control B with 10 ml of nutrient broth only. Second set of crude oil contaminated soil (S2)


was prepared by mixing 150 ml of crude oil in 400 grams of sterilized soil. Two controls were also conducted A with all bacterial suspensions (50 ml) and other B with nutrient broth (50 ml) applied to deliberately contaminated sample.

Microbial Monitoring Microbial monitoring was done by standard procedure of serial dilution in

which a series of six test tubes containing 9 ml of distilled water was sterilized to serially dilute 1 gram of experimental soil then plating 0.5 ml of dilution to nutrient agar plates to find out CFU/gram of soil. Individual isolates from Rajian oil fields were monitored at days 1, 10, 20 and 30. Missa Kaswal isolated, applied in consortia were monitored at days 03, 06, 09, 12 and 15.

Extraction of Residual oil Two approaches namely, reductions in weight and n-hexane extraction

were conducted to find out biodegradation and concentrations of residual oil. Reduction in weight loss was calculated on daily basis in laminar air flow cabinet. For residual oil concentration 1 grams of soil was mixed with 10 ml of n-hexane following centrifugation at 4000 rpm for 5 minutes, four further washes were given to get 50 ml of n-hexane soluble fraction of crude oil per gram of sample. Optical Density (OD) value at 420 nm was read in spectrophotometer (Urum et al., 2003).

RESULTS AND DISCUSSIONS

Microbial Isolation Bacterial isolates from Missa Kaswal and Rajian oil fields soils were

identified. Twenty-one microbial isolates from crude oil contaminated oil field soil revealed a diverse microbial community belonging to twelve different genera namely, Acenitobacter, Amphibacillus, Arthrobacter, Bacillus, Corynebacterium, Flavobacterium, Klebsiella, Lactobacillus, Listeria, Moraxella, Providencia and Pseudomonas majority of which has already been reported by other researchers on the basis of 16S rDNA sequences and have shown biodegradation capabilities (Chaillan, et al., 2004; Toledo et al., 2006; , Mandri & Lin, 2007; Rahman, et al., 2002). Twenty oil degraders, belonging to seven different genera: Pseudomonas, Bacillus, Klebsiella, Acinetobacter, Clostridium, Erwinia and Staphylococcus, were isolated and characterized from Rajian oil field contaminated soil. Members of Pesudomonas, Bacillus, Acinetobacter and Klebsiella are found in soils of both oil fields. Pseudomonas, Acinetobacter and Flavobacterium were identified from oil contaminated soils of South Africa

(Mandri & Lin, 2007).

Biodegradation Studies Two sets of soil samples were devised to find out biodegradation

potential of individual (S1) and mixed microbial assemblage (S2). In S1 samples, crude oil biodegradation was noted by Pseudomonas strains removing crude oil from 20.20 to 13.34%, followed by Bacillus 16.68 to 8.40%, Erwinia 11.55%, Clostridium 11.12%, Klebsiella 10.99 to 7.22%, Acinetobacter 9.68 to 9.20% and Staphylococcus 5.99%, respectively (Fig. 01). Control A for S1 samples,


including equal volumes of all the isolates showed 21.60% removal in 30 days. In S2, net 13.38% and 15.84% crude oil biodegradation was studied in Consortia A and B respectively (Fig. 02). Removal of 16.67 % and 20.35% crude oil were noticed by Controls A and B respectively. Abiogenic degradation of crude oil fractions was observed in Control B. These results were in agreement with already reported microbial biodegradation of hydrocarbons (Rahman, et al., 2002; Saleh et al., 2009).

Fig. 1: Crude oil biodegradation by Rajian Oil Field Bacterial genera

Fig. 2: Crude oil biodegradation by Consortia from Missa Kaswal Oil

Field Biodegradation process depends on conditions under which bacterial

community is inhabiting, concentration of crude oil (Zang et al., 2005), concentration of nutrients, ratio of carbon and nitrogen (Namkoong et al., 2002), environmental conditions, ability to produce biosurfactants to make easy access of hydrocarbons (Mulligan, 2005; Rahman et al., 2006), microbial metabolic capabilities (Gullotto et al., 2008) and levels of hydrocarbon contaminants (Santhini, et al., 2009) all contribute to natural rate of microbial degradation processes. Microbial capabilities of uptake of crude oil fractions with the secretion of biosurfactants is further enhanced and coupled by enzymatic components, namely monooxygenases and dioxygenases. Toluene o-xylene monooxygenase was isolated from Pseudomonas sp. and was applied in microemulsion and micellar systems of crude oil, and was found responsible for conversion of aromatic hydrocarbons into simpler compounds like mono- and di-hydroxylated substrates, susseptible to degradation by other microbial flora (Gullotto et al., 2008).

Biodegradation of the complex hydrocarbons require the cooperation of more than one species, each strain contributing in hydrocarbon transformation process by metabolizing limited range of hydrocarbons (Kim & Crowely, 2007). Bioremediation of hydrocarbons in contaminated soil by different microbial flora / mixed cultures of hydrocarbon degrading bacteria was reported by many authors (Joo et al., 2008; Allen et al., 2007; Ghazali et al., 2004; Sabate et al., 2004; Rahman et al., 2002; Saleh et al., 2009). Engine oil degrading bacteria from South Africa were found belonging to Acinetobacter, Flavobacterium and Pseudomonas genera and showed up to 90% degradation of not only used


engine oil in 4 week duration but also 80% removal of n-paraffin in 2 week duration (Mandri & Lin, 2007).

An attempt to understand crude oil biodegradation by monitoring reduction in weight of experimental samples was made. Reduction in weight of experimental samples when compared with the extent of crude oil biodegradation showed a parallel relation of reduction in crude oil biodegradation with reduction in weight.

Microbial Enumeration Viable cell count of microbes applied to S1 and S2 were determined by

standard plating method. S1 showed initial increase in microbial count at day 10 and further decrease in microbial contents was studied at day 20 and 30. Microbial number showed value of 10

8 to 10

9 cfu/ml in a study of biodegradation

of crude oil (Shkidchenko et al., 2004). Microbial count in mixed assemblage in

S2 samples showed maximum enumeration of 1011

in Consortium B (Table 01).

Table 1: Microbial monitoring of Rajian Oil Field Soil Isolates

SR. # ISOLATES DAY 0 DAY 10 DAY 20 DAY 30

01 Bacillus sp.I 2.8× 108– 1.09 × 10

6 2.3 × 10

10 – 1.7 ×10

6 4.9× 10

5 3.3× 10

4

02 Pseudomonas sp.I 3.7 × 1010

– 2.5 × 107 2.7 × 10

11– 3.4 × 10

6 7.4× 10

6 6.7× 10

5

03 Streptococcus sp. 1.7 × 105 2.5 × 10

4 1.9× 10

4 1.0× 10

3

04 Bacillus sp.II 4.7 × 108 – 3.9 × 10

7 3.4 × 10

9 – 1.3 × 10

5 9.3× 10

4 7.4× 10

3

05 Klebsiella sp.I 2.1 × 107 – 1.3 × 10

4 5.2 × 10

9 2.0× 10

5 1.5× 10

4

06 Pseudomonas sp.II 1.3 × 1010

– 2.9 × 107 3.7 × 10

11– 4.1 × 10

5 8.6× 10

6 4.2× 10

5

07 Klebsiella sp.II 1.3 × 107 – 1.8 × 10

4 1.7 × 10

8– 3.3 × 10

4 3.6× 10

4 1.9× 10

4

08 Pseudomonas sp.III 1.8 × 108 – 4.2 × 10

3 2.9 × 10

9 – 1.5 × 10

3 7.3× 10

6 4.1× 10

5

09 Clostridium sp. 2.9 × 107 2.8 × 10

8 – 1.3 × 10

4 9.2× 10

3 3.9× 10

3

10 Bacillus sp.III 3.1 × 109

– 1.2 × 105 5.7 × 10

10 1.4× 10

6 2.9× 10

5

11 Acinetobacter sp.I 1.2 × 108– 4.2 × 10

6 1.4 × 10

10 – 8.4 ×10

4 2.3× 10

4 1.4× 10

4

12 Pseudomonas sp.IV 6.3× 107 4.9 × 10

8 – 3.2× 10

4 5.6× 10

5 2.4× 10

5

13 Klebsiella sp.III 3.9 × 109– 3.5× 10

4 1.7× 10

9 1.7× 10

3 0.8× 10

3

14 Bacillus sp.IV 1.0 × 1010

– 1.2 × 108 2.1× 10

11 – 8.1 × 10

5 4.3× 10

6 9.1× 10

5


Present study highlighted the role of individual microbial strain and tried to explore the concerted action of mixed strains of Pseudomonas, Acinetobacter, Bacillus, Corynebacterium and Klebsiella genera in both oil fields soils. Further, this study showed higher biodegradation of hydrocarbons by mixed microbial assemblages, suggesting the use of indigenous microflora of biodegrading capabilities in environment cleanup operations. Overwhelmingly increasing technological advancements have unraveled new approaches for engineering microbes with superior degradative and considerably enhanced microbial fitness under the challenging conditions posed by contaminated environments.

REFERENCES

Allen, J.P., Atekwana, E.A., Atekwana, E.A., Duris, J.W., Werkema, D.D. & Rossbach, S., 2007. The microbial community structure in petroleum-

contaminated sediments corresponds to geophysical signatures. Appl. Environ. Microb., 73: 2860–2870

Chaillan, F., Fleche, A.L., Bury, E., Phantavong, Y., Grimont, P., Saliot, A. & Oudot, J., 2004. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. Research in Microbiology., 155: 587-595.

Chaudhry, Q., Blom-Zandstra, M., Gupta, S. & Joner, E.J., 2005. Utilising of synergy between and rhizosphere micrporganisms to enhance breakdown of pollutants in the environment. Environ. Sci. Pollut. Res., 12: 34-48

Ghazali, F.M., Rahman, R.N.Z.A., Salleh, A.B. & Basri, M., 2004. Biodegradation

of hydrocarbons in soil by microbial consortium. Int. Biodeterior. Biodegrad., 54: 61-67.

Gullotto, A., Branciamore, S., Duchi, I., Caño, M. F., Randazzo, D., Tilli, S., Giardina, P., Sannia, G., Scozzafava, A. & Briganti, F., 2008. Combined action of a bacterial monooxygenase and a fungal laccase for the biodegradation of mono- and poly-aromatic hydrocarbons. Bioresour. Technol., 99: 8353–8359.

Huang, X. D., El-Alawi, Y. S., Penrose, D., Glick, B. R. & Greenberg, B. M., 2004. A multiprocess Jonathan, D.V.H., Singh, A. and Owen, P.W. 2003.

15 Bacillus sp.V 1.9× 107 1.0× 10

8 – 1.3 × 10

4 3.0× 10

3 2.1× 10

3

16 Pseudomonas sp.V 3.7 × 108– 3.9 × 10

6 5.6× 10

9 1.2× 10

5 5.4× 10

4

17 Acinetobacter sp.II 9.3 × 107 1.2× 10

8 – 3.4 × 10

3 5.9× 10

4 4.2× 10

3

18 Erwinia sp. 2.9 × 106– 5.2 × 10

5 2.7 × 10

7 6.4× 10

6 2.4× 10

4

19 Klebsiella sp.IV 2.7 × 109– 1.8 × 10

6 4.8× 10

10 2.6× 10

5 1.6× 10

4

20 Klebsiella sp.V 6.3 × 107 3.5× 10

8 – 1.9 × 10

4 7.5× 10

4 3.1× 10

3


Recent Advances in Petroleum Microbiology. Microbiol. Mol. Biol. Rev., 67(4): 503–549.

Joo, H.S., Ndegwa, M., Shoda, M. & Phae, C.G., 2008. Bioremediation of oil-contaminated soil using Candida catenulate and food waste. Environ. Pollut., 156: 891-896.

Kim, J.S. & Crowley, D.E., 2007. Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits. Appl. Environ. Microbiol., 73(14): 4579-4591.

Komukai–Nakamura, S., Sugiura, K., Yamauchi-inomata, Y., Toki, H., Venkateswaran, K., Yamamoto, S., Tanaka, H. & Harayama, S., 1996. Construction of bacterial consortia that degrade Arabian light crude oil. J. Ferment. Bioeng., 82: 570–574.

Kuiper, I., Lagendijk, E.L., Bloemberg, G.V. & Lugrenberg, B.B.J. 2004., Rhizoremediation: A beneficial plant microbe interaction. Mo.l Plant. Microbe. Interact., 17: 6-15.

Mandri, T. & Lin, J., 2007. Isolation and characterization of engine oil degrading indigenous microorganisms in Kwazulu-Natal, South Africa. Afr. J. Biotechnol., 6: 023-027.

Mulligan C.N., 2005. Environmental applications for biosurfactants. Environ. Pollut., 133: 183-198.

Namkoong, W., Hwang, E.Y., Park, J.S. & Choi, J.Y., 2002. Bioremediation of diesel contaminated soil with composting. Environ. Pollut., 119: 23–31.

Rahman, K.S.M., Banat, I.M., Thahira, J., Thayumanavan, T. & Lakshmanaperumalsamy, P., 2002. Bioremediation of Gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant. Bioresource Technol., 81: 25-32.

Rahman, K.S.M., Street, G., Lord, R., Kane, G., Rahman, T.J., Marchant, R. & Banat, I.M., 2006. Bioremediation of petroleum sludge using bacterial consortium with biosurfactant. In: Environmental Bioremediation Technologies, Singh, S.N. and Tripathi, R.D. (Eds.). Springer Publication, pp: 391-408.

Reynolds, R.M., 1993. Physical oceanography of the Gulf, Strait of Hormuz, and the Gulf of Oman: results from the Mt Mitchell Expedition. Mar. Pollut. Bull., 27: 35–59.

Rittman, B. & McCarty, P.L., 2001. Environmental Biotechnology: Principles and Applications. McGraw-Hill, New York.

Ron, E. & Rosenberg, E., 2002. Biosurfactants and bioremediation, Curr. Opin. Biotech., 13: 249–252.

Sabat'e, J.M., Vi˜nas, A.M. & Solanas., 2004. Laboratory-scale bioremediation

experiments on hydrocarbon-contaminated soils. Int. Biodeterior. Biodegrad., 54: 19 – 25.

Saleh, E., Drobiova, H. & Obuekwe, C., 2009. Predominant culturable crude oil-

degrading bacteria in the coast of Kuwait. Int. Biodeterior. Biodegrad., 63: 400-406.

Santhini, K., Myla, J., Sajani, S. & Usharani, G., 2009 .Screening of Micrococcus Sp from Oil Contaminated Soil with Reference to Bioremediation, Bot. Res. Internat., 2 (4): 248-252.


Shkidchenko, A.N., Kobzev, E.N., Petrikevich, S.B., Chugunov, V.P. & Boronin, A.M., 2004. Biodegradation of black oil by microflora of the bay of Biscay and biopreparations. Process Biochem., 39: 1671-1676.

Singh, O.V. & Jain, R.K., 2003. Phytoremediation of toxic aromatic pollutants from soil. Appl. Microbiol. Biot., 63: 128-135.

Toledo, F.L., Calvo, C., Rodelas, B. & Gonza´ lez-Lo´ pez, J., 2006. Selection and identification of bacteria isolated from waste crude oil with polycyclic aromatic hydrocarbons removal capacities. Syst. Appl. Microbiol., 29: 244–252.

Urum, K., Pekdemir, M. & Gopur, M., 2003. Optimum conditions for washing of crude oil-contaminated soil with biosurfactant solutions. Process Saf Environ. 81(3): 203-209.

van Beilen, J.B., Panke, S., Lucchini, S., Franchini, A.G., Röthlisberger, M., & Witholt, B., 2001. Analysis of Pseudomonas putida alkane degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk-genes. Microbiology. 147: 1621–1630.

Xu, P., Yu, B., Li, F.L., Cai, X.F. & Ma, C.Q., 2006. Microbial degradation of sulfur,nitrogen and oxygen heterocycles. Trends Microbiol., 14(9): 398-405.

Zhang, Guo-liang, Yue-ting, W., Xin-ping, Q. & Qin, M., 2005. Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids. Zhejiang Univ. SCI., 6B (8): 725-730.


Evaluation of industrial waste water quality by monitoring

the effect on growth attributes of Pistia stratiotes L. and

Eichornia crassipes Solm.

WAJID JAVED, AMIN U. KHAN & MUHAMMAD UMAR HAYYAT

Sustainable Development Study Centre, GC University Lahore

ABSTRACT

The effluents directly coming out of the chemical industry were subjected to dilution gradients to detect how much dilution will be required before it becomes fit to support the growth of hydrophytes for bioremediation. For this purpose Pistia startiotes and Eichornia crassipes were used as bio-monitors. A preliminary experiment, using undiluted effluent sample and samples with 40% and 60% dilutions resulted in wilting in a matter of hours. Therefore, a series of higher dilutions (above 80%) were prepared to monitor the growth attributes of the two species. The two hydrophytes survived during the experiment with steady decrease in dry matter, increase in necrosis and reduction in number of leaves per plant with decreasing dilution. 90% dilution seems to be the threshold level, below which the plants are more significantly affected and only at 98% dilution, some sign of vegetative reproduction was evident. It indicates that quality of water of this particular industry would need at least 80% dilution before hydrophytes can be efficiently utilized for bioremediation.

Key words: Pistia stratiotes, Eichornia crassipes, Biomonitoring, Industrial effluents.

INTRODUCTION

Biological monitoring in much broader sense is used for measurement of

effect on the growth, metabolism and contamination in a very wide range of living organisms. It can allow the identification of changes in the level of pollution over the course of time (Burton, 1986). The advantage of using living organisms as bio-monitors derives from the fact that they are capable of revealing the presence of toxic substances even at very low concentration through biological effects, which could be difficult to detect and measure directly in environmental samples. In this context the plant, animals and microorganisms can be particularly useful for bio-monitoring. In recent years plant bio-indicator species or strains of species, which are sensitive to particular pollutants have been used to detect elevated environmental contaminants by observing the extent of visible injury. (Lorenzini, 1992). In the present work the quality of industrial waste water from a caustic soda factory was checked with the help of growth attributes of two hydrophytes Pistia stratiotes L and Eichornia crassipes Solms. The industrial complex is situated about 20 Kilometer north of Lahore on the G.T. Road. The discharge point pours the effluents directly into Deg Nullah, which originates from Jammu and ends in river Ravi downstream. Its water is clear and rich in flora and fauna before the discharge point of this industrial complex. Previous works (Shamsi et.al., 1983; Mustafa, 2002) clearly indicates that apart from the high amount of other pollutants, it contains a number of heavy metals, of which

W. JAVED ET AL BIOLOGIA (PAKISTAN)

88

most significant amount is that of Nickel, followed by Copper and Iron. Generally the amount of heavy metals are reported to be lower than the recommended NEQS (Mustafa, 2002), and the amount is not high enough to be toxic to plants (Kovacs & Podani, 1986). Therefore the emphasis in this work is to measure the amount of pollutants other than heavy metals affecting the growth of Pistia statiotes and Eichornia crasspies, in order to ensure that the effluent level is suitable enough to establish hydrophytes for bioremediation. Both species have been popularly utilized for phyto-remediation purposes (Heaton et al., 1986; Maine et al., 2001; Muzaffarrov et al., 1984; O’Keefee et al., 1984; Salt, et al., 1997; Tarlyn et al., 1998; Yan et al.,1990), and they dominate the local wetlands (Marwat et al., 2010; Khan et al., 2010). The growth attributes of the two species are appreciated by comparison with a control to evaluate the suitability of the waste water in terms of its life support capability.

MATERIAL AND METHODS

Collection and analysis of water samples Two trips were made for the collection of effluents of the industry from the Nullah Deg in April 2002. In the first trip general survey was also conducted of the area for selecting sites for the collection of samples. In the consequent trip the samples were collected from the following three sites:

1. Upstream samples, (100 meter before the discharge outlet of effluents). 2. At the spot, where the effluent enter the nullah. 3. Downstream samples, (100 meter after the discharge outlet).

Water samples in three cans of 30 litre capacity were collected for analysis. In addition, tube-well water of the Government College Botanic Gardens, which was used for diluting the effluents, was analyzed. All samples collected from Nullah were brought to the laboratory where they were analyzed for the pH and electric conductivity by Beckmann pH and Orian conductivity meter (model 160) respectively. The methods reported in American Public Health Association (1992) were used to determine the following: settable solids, suspended solids, chlorides, carbonates, bicarbonates, calcium and magnesium. BOD was determined by BOD sensor and inductive stirring system (Lovibond). COD was determined by DOK reactor Et108 and spectrophotometer S-200D by using COD cuvettes (Lovibond).

In addition, 10 cans (300 litres) of effluents were collected from the spot where the effluents were discharged into the Nullah for the experiment.

The experiment was conducted in two phases, in the first phase a small experiment was set up to determine the effective dilutions where both species can survive in the pots for a couple of days. The following concentrations were used for the preliminary experiment: undiluted effluent and three dilutions, 40%, 60% and 80% by using percentage method dilutions with tube-well water (Javed, 2002). The second experiment was designed in 8 pots each for both species, with the following dilutions 98%, 96%, 94%, 92%, 90%, 88%, 86% and a control with tube-well water. Supplementary additions of corresponding dilutions of industrial effluents were added at regular intervals in order to maintain the loss of water due to evaporation. The experimental pots were covered with plastic

Vol. 56 (1&2) Evaluation of Industrial Waste Water Quality

89

cover during the two rains in order to prevent further dilutions during the course of the experiment. Collection of plant material for the experiment The two species P. startiotes and E. crassipes were collected from the aquatic gardens of Botanic Garden, Government College University Lahore. In order to maintain uniformity in the sizes, the following criteria was adopted. Plants at 4 leaves stage were selected for P. stratiotes and 3 leaves for E. crassipes. Eight plants of both species were transferred into each pots in the two sets of dilutions in ceramic pots ( 75&45cm

2) with maximum water capacity of 30 liter.

Monitoring the Effects on Plants The experiment was initiated on 20

th April 2002 and was terminated after 40 days

on 30 May, 2002. Mean dry weight of ten plants at the beginning of the experiment were taken to be used later on as the bench mark for comparison with the plants at the time of harvest. The increase in dry weight and number of leaves were determined by subtracting the reading taken at harvest from the initial mean weight of plants at the start of the experiment. Size of the necrotic area and total leaf area were measured with the help of the leaf meter in order to determine the percentage of necrosis. Plants were packed in labeled paper bags after measuring all the parameters and placed in oven at 80º C for 48 hours. After 48 hours dry weight was measured.

The following observations were made to estimate the effect of dilution treatment on plants.

1. Number of leaves 2. Percentage necrosis of leaves (%) 3. Dry weight of whole plant

RESULTS

Results indicated that the tube-well water and upstream water were more or less of similar characteristics whereas the effluent at the discharge outlet and downstream show high values of all the pollutants (Table 1). There is gradual improvement 100 downstream in the amount of pH, EC, TDS, SS, calcium, sodium and magnesium as compared to carbonates, bicarbonates and chlorides which are at level considered toxic for the growth of plants (Burton, 1986).

A preliminary experiment designed to select the dilution standards of the industrial effluent indicated that in undiluted, 40%, 60% dilutions the species wilted in 4 hours, 20hours, 40 hours respectively, whereas in the 80% dilutions it survived for the duration of the experiment (64 hours). Therefore in the second experiment the plants were grown in the milder dilutions ranging from 88% to 98%. The plants were allowed to grow for 6 weeks and then harvested.

The results clearly show that dry weight gradually decreased with the decrease in the dilution of effluent. The dry weight significantly decreased at 86% and 88% dilution in P. stratiotes and E. crassipes respectively, when compared with the control (Fig.1). This shows that the trend of decline continues with decreasing dilution. It appears that dilution of 90% is the threshold level and below this level, the effects on the measured growth attributes become more


90

pronounced. The dry weight reduction is accompanied with reduction in the number of leaves per plant and percentage of necrosis on the leaves.

The percentage of necrotic patches on the leaves increased with the decrease in dilution of the effluent. The necrosis increased significantly at 96% dilution in both species as compared to the control. It indicates that even highly diluted samples show symptoms of necrosis which gradually speeds up with every additional doze of the concentration of the effluent (Fig. 2). Similarly, the number of leaves per plant decreased with decreasing dilutions. The number of leaves significantly decreased at 92% and 96% dilutions in P. stratiotes and E. crassipes respectively (Fig. 3).

In spite of the fact that duration of experiment coincide with the on set of reproductive growth period, as is evident from control experiment, where single plant produced at least 3 plantlets. Only a single plantlet was observed in E. crassipes in 98% dilution treatment. No vegetative reproduction was observed in any other dilution treatment.

DISCUSSION

The results clearly showed that hydrophytes unlike land plants, where

soil acts as buffer, are totally dependent on contamination in water and therefore are ideal bio-monitors for detecting pollutants. The higher curves of the E. crassipes are due to the reason that its initial weight was higher as compared to P. stratiotes otherwise both species are equally affected by the pollutants. Although the response of two species is the same but the P. stratiotes because of its smaller size is easy to handle and monitor as compared to E. crassipes. The first experiment clearly shows that even 60% dilution resulted in total mortality of both the species within a couple of days. The second experiment shows 50% reduction in dry weight, increase in the percentage necrosis of the leaves and reduction in the number of leaves per plant below 90% dilution; an indication that over longer period of time even higher dilutions would lead to eventual mortality. However, in 98% dilution, E. crassipes produced a single plantlet, whereas P. stratiotes did not produce any even at this dilution, indicating that species might survive but will not be able to reproduce at higher dilutions and it has to be frequently replenished for bio-remediation purpose. The results showed that in order to use the two species effectively for bioremediation the effluents level from such industries have to be diluted to at least 80% level to ensure long term survival of both species. Furthermore, the results clearly shows that even 100m downstream there is less little reduction in the amount of most of pollutants, in fact the level of pollutants keep on increasing (Mustafa, 2002) with discharge from several other industries entering the nullah throughout its course downstream. This clearly shows that under the circumstances setting up water treatment and resource recovery program by aquatic plants (Dieberg et al., 1987) along the nullah will not succeed as the quality of water never achieves dilution levels conducive enough for long term survival of hydrophytes.


91

Table 1: Analysis of water samples from four locations

Parameters

Water Samples

Tube well water Upstream (100m)

Down stream (100 m)

Discharge point

pH 7.5 7.2 8.5 9.0

EC (µS) 490 600 1300 1800

TDS (mgl-1

) 240 390 767 1450

Calcium (meql-1

) 35 40 63 158

Magnesium (meql-1

) 11 15 37 96

Carbonates (meql-1

)

90 93 480 667

Bicarbonates (meql-1

) 1.4 1.99 7.65 12.07

Chlorides (meql-1

) 0.1 0.19 143 149

Sodium (ppm) 5.0 0.005 547 3800

COD (mgl-1

) 7.6 21 100 800

BOD (mgl-1

) 5.5 10 10 000

Fig. 1: Effect of effluent dilutions on dry weight of P. stratiotes ( )and E. crassipes ( ) After 6 weeks with 95% confidence limits


92

Fig. 2: Effect of effluent dilutions on percentage necrosis in P.

stratiotes ( ) and E. crassipes ( ) after 6 weeks with 95% confidence limits

Fig.3: Effect of effluent dilutions on the increase in number of

leaves in P. stratiotes ( ) and E. crassipes ( ) after six weeks with 95% confidence limits

REFERENCES

American Public Health Association, 1992. Standard Mehods for the examination

of water and wastewater. American water works association water pollution control federation Washington, DC.

Burton, M.A.S., 1986. Biological monitoring of environmental contaminants (Plants). A technical report, Monitoring and assessment center, London. pp:175-187.


93

Dieberg, F.E., T.A. DeBusksnd Jr. & N.A. Goulet., 1987. Removal of copper and lead using thin film technique in aquatic plants for water treatment and resource recovery. Magnolia Publishing Inc. Ltd. pp 497-504.

Heaton, E., Frame, J. & Reddy J. K., 1986. Lead uptake by Eichhornia crassipes. Toxicological and Environmental Chemistry USA., 2: 125-135. Khan, M. A., Qureshi, R. A., Gillani, S. A., Ghufran, M. A., Batool, A. & Sultana,

K. N., 2010. Invasive Species of Fedral Capital Area Islamabad, Pakistan. Pak. J. Bot., 42(3):1529-1534.

Kovacs, M. & Podani, J., 1986. Bioindication: A short review on the use of plants as indicators of heavy metals. Acta Biologica Hungarica, 37(1): 19-29. Javid, W., 2002. Monitoring industrial water quality by using two species of

hydrophytes as bio-indicators. M.Sc. Thesis, Botany Department, Government College, Lahore.

Lorenzini, G., 1992. Plants as Biomonitors of air quality. In: Biological indicators for environmental monitoring (Bonotto et al., eds.). Pubs. Aresserono Symposia via Ravenna. 8, Rome, Italy. pp.75-80.

Maine, M. A, Duarte, M. V. & Sune, M. l., 2001. Cadmium uptake by floating macrophytes. Water Res., 35(11):2629-2634.

Marwat, K.B., Hashim, S. & Ali, H., 2010. Weed Management: A Case study from North-West Pakistan. Pak. J. Bot., 42: 341-353.

Mustafa, G., 2002. State of the Pollution Load at Critical Points at Deg Nallah at Kalashah Kaku. M.Phil Thesis. Department of Chemistry, Government College University, Lahore.

Muzafarrov, A.M., Shoiakubov, R. S., Deviatkin, E. L., Rasulev, T. A., Khasanov, A. & Atamukhamedov S. K., 1984. Use of Pistia stratiotes for ground water pollution control. Uzb. Biol. Tashkent. "Fan" 4: 31-32.

O'Keeffee, D.H., Hardy, J. K. & Rao, R. A., 1984. Cadmium uptake by the water hyacinth: Effects of solution factor. Environmental Pollution, UK. 34: 133-147.

Shamsi, S.R.A., Hussain, I., Naheed, S. & Ariz, N., 1983. The quality and Biota of Fresh and Polluted Waters of Deg Nulah (Stream) in Pakistan. Pakistan Journal of Scientific Research, 35(1-2).

Salt, D.E., Pickering, I. J., Prince, R. C., Gleba, D., Dushenkov, S., Smith, R. D. & Raskin I., 1997. Metal accumulation by aquaculture seedlings of Indian mustard. Environ. Sci. Technol., 37: 636-1644.

Tarlyn, N.M., Kostman, T.A., Nakata, P.A. & Keates, S.E., 1998. Axenic culture of Pistia stratiotes for use in plant biochemical studies, Aquat. Bot., 60(2): 161 – 162.

Yan, S., Liang, D. & Peng, D., 1990. A result on the tolerance and purification ability of eight aquatic plants in heavy metal (CV) contaminated sewage. China Environ. Sci. Zhonggou Huanjing Kexue, 10(3): 166-170.


Alterations in mammogenesis: Gestational length and

nurturing behaviors in mice following co-gestational

chlorpyrifos exposure in mice

KHAWAJA RAEES AHMAD, ASMAT ULLAH, SHAFAAT YAR KHAN,

KAUSAR RAEES, TAHIR ABBAS & SHAHZAD AHMAD MUFTI.

Department of Biological Sciences, University of Sargodha(KRA, SYK),

Department of Zoology University of The Punjab, Lahore (AU, T), Government Degree College (for Women) Farooq Colony, Sargodha (KR) and Advisor

BioSciences COMSATS Institute of Information Technology, Islamabad (SAM) PAKISTAN

ABSTRACT

In present study effects of the organophosphorus insecticide chlorpyrifos on

behavioral pattern of pregnancy and lactation were investigated in mice. In 3 groups of 20 bred female Swiss Webster mice each, insecticide was given as 0, 10 and 20mg/kg respectively by gavage on gestation day 10(GD10). No obvious signs of toxicity appeared at these exposures. Dams were subjected to nest building activity on GD15-17. Gestational length, mammary development in terms of number and location of functional nipples and lactational/ incubational behavioral activities of the mothers were also studied.

An obvious decrease in duration of lactation/incubation and gestational period along with an increase in neonatal cannibalization in 10 and 20mg/kg groups to that of the 0mg/kg group were noted. Likewise a significant decrease in number of functional mammary glands (p< 0.001) was noted. Moreover specific dose related alterations in nest building behavior were observed. The results indicate that gestational exposure of CPF directly affects the duration of pregnancy, mammogenesis and disrupt the behavioral patterns of nest building and lactation at motherly safe dose levels. KEY WORDS: Chlorpyrifos, gestational periods, mammogenesis, nest building, lactational

behavior

INTRODUCTION

Organophosphorus insecticides (OPI) are a group of hazardous

environmental chemicals for the non-target organisms and humans. Many epidemiologic investigations related to behavioral toxicology have indicated that prolonged exposure of OPI may lead to various neurological signs (London et al., 1997; Roldan-Tapia et al., 2005 & 2006; Steenland et al., 1994) and psychiatric disorders (Amr et al., 1997; Fiedler et al., 1997; London et al., 2005; Stephens et al., 1995). Studies have described the potential risk of depression and suicide in farm workers (Beseler et al., 2008; Jaga & Dharmani 2007; London et al., 2005; van-Wijngaarden, 2003). Many animal studies have indicated the neurotoxicological implications of the adulthood of developmental (gestational or neonatal) OPI exposures (Costa, 2006; Slotkin et al., 2008; Slotkin et al., 2009; Slotkin & Seidler, 2007; Timofeeva et al., 2008; Venerosi et al., 2006). Studies

K. R. AHMAD ET AL BIOLOGIA (PAKISTAN)

96

also indicate the nature and relationship between behavioral disorders related to OPI exposure at adulthood in animal models (Aldridge et al., 2005a; Levin et al., 2002; Lima et al., 2009; Sánchez-Amate et al., 2001).

Chlorpyrifos [CPF] and its metabolite CPF-oxon are among the most extensively studied OPI for various aspects of toxicological studies especially neurotoxicology (Meyer et al., 2004; Qiao et al., 2003), endocrine disruptions (Hodgson & Rose, 2006; Jeong et al., 2006; Tait et al., 2009), systemic toxicology, oxidative stress related tissue damage (Saulsbury et al., 2008 & 2009; Verma et al., 2007) and teratology (Asmatullah et al., 2004; Farag et al., 2003; Tian et al., 2005). From the standpoint of co-gestational exposure related neurotoxicology CPF has been reported to cause constant inhibition of brain acetylcholinesterase and choline acetyltransferase (Richardson & Chambers, 2003). Moreover the late gestational exposures of CPF has been reported to induce alterations in adenylate cyclase (Meyer et al., 2004; Venerosi et al., 2006) and cholinergic activities (Qiao et al., 2003) along with synaptic functionalities and turnover related to serotonin (Aldridge et al., 2005a) and dopamine (Aldridge et al., 2005b). It was thus concluded that CPF triggers neurobehavioral teratogenicity with a wide range of susceptibility from early embryonic life to postnatal development (Roy et al., 2005). Many recent studies indicate that in-utero and / or neonatal CPF exposure can lead to permanent alterations in adulthood behaviors (Levin et al., 2002; Meyer et al., 2004; Slotkin & Seidler, 2007; Tait et al., 2009; Venerosi et al., 2008).

Investigations have indicated the neurotoxicological implications of adulthood exposure of CPF in laboratory animals; for example it has been claimed that CPF is anxiogenic in rats at dose levels below than that induces cholinergic signs (Sánchez-Amate et al., 2001) whereas a single high dose exposure to CPF in rats has caused anxiety and alterations in locomotor activity (Lo´pez-Crespo et al., 2007). Recently methamidophos is found to induce depressive-like behavior in adult mice without any signs of systemic toxicology or acetylcholinesterase activity (Lima et al., 2009). There is a dearth of information about low dose CPF exposure during pregnancy in relation to the alterations patterns of reproductive and related behaviors in pregnant animals. Although it has been indicated that CPF induces alterations in reproductive steroids such as anti-androgenic (Kang et al., 2004) and weak estrogenic (Andersen et al., 2002) activities, little is known about the disturbances in physiological and behavioral patterns of pregnancy, nest building, parturition and nurturing/lactation of the young ones in mothers exposed to CPF during pregnancy.

As mentioned above, CPF inflicts endocrine disruptions of the sex steroids that may lead to behavioral alterations. In the present study we have evaluated the modifications in modalities of inbuilt neurobehavioral activities of pregnancy and lactation related behaviors in mice under co-gestational exposure of chlorpyrifos.


1.1) ANIMALS AND GROUPS:

Sixty young virgin 70-80 days old females, of Swiss Webster mice were used in this research work. They were kept under 12hr-12hr dark-light cycles, in

Vol. 56 (1&2) Alterations in Mammogenesis

97

12” X 18” steel cages. The room temperature was maintained at 23 2 0C.

Animals were provided free access to food and drinking water. Estrus females were caged with males for successful coitus. The day on which coitus occurred was designated as GD0 (gestation day 0) for each female. Bred females were parted from the males on GD8 and thereafter kept signally in separate cages for the remaining period of study. They were randomly divided into 3 groups, of 20 animals each, called as 0, 10 and 20mg/kg groups representing the dose of CPF given to the members of their respective groups. 1.2) DOSE PREPARATION AND APPLICATION: SHENCHLOR - Chlorpyrifos 40%EC (marketed by Welgreen Chemicals Pakistan Private Limited) was used in this study. Corn oil was used for drug dilutions. Animals in 0mg/kg group were treated with pure corn oil. The doses were applied orally by gavage. 1.3) BEHAVIORAL STUDIES:

Bred females were exposed to CPF on GD10 and thereafter were maintained to give birth to young ones. Gestational length, nest building behavior, litter size, frequency of neonatal cannibalization, extant of mammary development and lactational/ incubational periods were recorded. 1.4) PATTERNS OF REPRODUCTIVE BEHAVIORS: 1.4.1) Nest Building Activity:

A slightly modified method in line with the previous studies (Petruzzi et al., 1995; Schneider & Lynch, 1984) was used to investigate the nest building activity. This activity was studied for three consecutive days (GD15-17). Each animal was provided with 10g of fine cuttings (1cm

2) of nontoxic (50g/m

2) white

paper for nest building activity. Fresh cuttings were supplied to the animals each day. The paper cuttings were placed on a 3 x 3 inches shelf. The activity time started for a female with first pick or pulls a piece of paper with mouth or forelimbs. The cutoff time for this activity for each animal was 30 minutes. Measurement parameters include the size, shape and position of the nest in the cage. The weight of residual paper was taken as a quantitative index of nest building activity. Latency to start nest building in each case was also noted. 1.4.2) Gestational length, Litter size and frequency of neonatal cannibalization:

Gestational length corresponds for duration of pregnancy in number of days; it was estimated separately for all animals in each group and shown in Fig 1. Litter size corresponds to the number of offspring produced by one female. Mean values of litter size were obtained for each group and shown in Fig 3 along with the frequency of neonatal cannibalization. 1.4.3) Mammary Development and Incubational / lactational activity:

Extant of mammary development was a measure of functional mammary glands per animal, counted directly, after 48hrs of parturition. The data obtained is represented in terms of group means +/- SD in Fig 2. Further more the mammary ridges were also keenly observed in anterior-posterior axis to detect the restriction of functional ability to any specific portion.

Incubational and lactational activity was a measure of the number of visits and average time spent per visit along with the overall percentage of time spent in their nest by the mothers. Five randomly selected mothers were subjected to this study from each group. The observations were carried out after


98

48hrs of parturition in each case. Timing of the observation was 8 consecutive hour (i.e. 11-19). The observations were carried out with the help of a computer base analysis of CCD camera system recordings (Fig 4).

0

1

13

6

11

8

1

0

16

3

1

0

0

2

4

6

8

10

12

14

16

18

No

. o

f fe

male

s

Control 10mg/kg 20mg/kg

Experimental Groups

Delivered on GD18 Delivered on GD19 Delivered on GD20Delivered on GD21

Fig. 1: Effect of CPF on preterm birth. Bars represent the actual number of

females (out of 20) against the days of parturition.

ANOVA: P<0.0001

9.6 6.9 5.50

3

6

9

12


Experimental Groups

Avera

ge N

o.

Of

fun

cti

on

al

mam

mary

gla

nd

s *

Fig. 2: Effect of CPF on the No. of functional mammary glands. Data

represents group means +/- SD. (ANOVA appears at the top of the panel and asterisk denotes significant difference, post hoc analysis, from the control value)


99

RESULTS

2) MATERNAL REPRODUCTIVE AND NEUROBEHAVIORAL TOXICOLOGY 2.1) Gestational Period:

It has been observed that CPF treatment led to a dose dependent reduction in gestational period (Fig 1). The mean gestation lengths for 3 groups were as follows: 0mg/kg =20.25, 10mg/kg =18.5 and 20mg =18.25. 2.2) Mammary Development:

A dose dependent decrease in number of functional mammary nipples in 10 and 20mg/kg groups as compared to the 0mg/kg group was noted (Fig 2). Moreover this decrease in number of functional mammary nipples followed an anterio-posterior pattern. As it was observed that this decline in the development of mammary nipples was restricted to the thoracic portion; whereas the development of functional nipples in the lower belly part of the mammary lines remained mostly unaffected. 2.3) Frequency of visits to the nest, Lactato-Incubation periods, Litter size

and Neonatal cannibalization: With a little variation the mean litter size remained almost constant in all

the 3 groups; whereas the frequency of cannibalization of the live fetuses was incased in 10 and 20mg/kg CPF treated groups to that of the 0mg/kg group (Fig 3). On the other hand with slight increase in frequency of visits, mothers gave lesser average time for incubation and lactation in both CPF treated groups as compared to the 0mg/kg group (Fig 4).

8.8 8.728.67

2.3

1.4

0.45

0

2

4

6

8

10

12


Experimental Groups

Me

an

lit

ter

siz

e

Mean Litter size [+ bars: SD]

Group frequency of canebolization

Fig. 3: Effect of Chlorpyrifos on litter size and frequency of neonatal

cannibalization data points are group means +SEM shown against mean group frequency of neonatal cannibalization.

2.4) Nest Building Behavior:

There were no considerable variations in terms of size and position of the nest and the weight of paper cuttings actually used to produce the nest, among the experimental groups. Mean latency period to begin nest building


100

activity in the 0mg/kg group showed a day dependent gradual decline [i.e. 121.47, 111.68 and 85.11 sec. respectively on GD 15, 16 and 17]. No such day dependent effect was noted in 10 and 20mg/kg groups. Rather in these groups, with slight variations, the mean latency period remained at a constantly higher level. The statistical analysis of data indicates significant variations among the groups {F (5.143249) = 13.45114, p< 0.01}, post hoc analysis of the means indicate the significant difference {p<0.05} of both CPF groups to that of the 0mg/kg group on GD17 (Fig 5).

0--1

0--2

0--3

0--4

0--5

10--1

10--2

10--3

10--4

10--5

20--1

20--2

20--3

20--4

20--5

Gro

up

--A

nim

al n

um

be

r

Dark bands= time periods spent by each mother in lactation Light bands = time periods spent by the mothers away from the neonates Fig. 4: Effect of chlorpyrifos exposure on lactating mothers on lactational /

incubational duration and frequency of visits to the nest.

The frequency of nest building and the frequency of producing proper (round) nests remained lesser than the 0mg/kg in both CPF groups on GD15 and were further decreased on GD16 and 17 in a dose dependent manner. The frequency of producing improper (ellipsoidal or irregular) nest remained constantly higher in both CPF groups to that of the 0mg/kg group (Fig 6).

1100 1200 1300 1400 1500 1600 1700 1800 1900

Time of the Day

Mean time spent in the nest = 47.29% Average number of visits to the nest = 7.4 Mean time/ visit = 30.77




101

Fig. 5: Effect of chlorpyrifos on latency period (seconds) to start nest

building activity in pregnant animals, the data points are group means + SEM.

*: significant difference (post hoc analysis of the means) from control (p< 0.05).

Ability of nest building

GD16 GD15 GD16 GD17

GD17GD16

GD17

Proper (round) nest

GD15

GD15

Improper (elliptical or

irregular) nest

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

Ch

an

ge i

n F

req

uen

cy f

rom

co

ntr

ol

10mg/kg

20mg/kg

Fig. 6: Alterations in frequencies of ability of nest building behavior,

production of proper (rounded) and improper (elliptical or irregularly shaped) nest in chlorpyrifos treated groups to that of the control.


102

DISCUSSION

Conflicting claims have been made about the developmental toxicity of CPF. It was once claimed not to be embryolethal, embryo/fetotoxic, or teratogenic, moreover causing no adverse affects on fertility and the structure or function of the reproductive organs (Breslin et al., 1996). Thus it was considered reproductively safe insecticide. This preview was shaken when Farag et al., (2003) claimed that exposure to motherly toxic dose of CPF brings about increased fetal resorptions, low birth weight along with visceral and skeletal abnormalities of the embryos. Feto-toxicity at maternally safe doses of exposure has been claimed by Tian et al., (2005). In the present study we find no signs of toxicological symptoms in Dams and no significant effect on litter size related to CPF at 10 and 20mg/kg maternal exposure on GD10, however decreased gestational period with lesser no of functional mammary gland were noted as obscure indications towards reproductive toxicology.

Along with neuroendocrine physiology of reproduction, pregnancy, parturition and lactation, the peptides of hypothalamus and pituitary in conjunction with gonadal steroid hormones are directly involved in sex related maturational changes in reproductive organs and behaviors in mammals. Moreover the pituitary neuropeptides such as Oxytocin, Arginine, vasopressin and Prolactin are also believed to be the potent modulators of social behavior in mammals. In this context Tait et al., (2009) has found that low dose (3-6mg/kg) late gestational (GD 15-18) and (1-3mg/kg) post natal (PND 11-14) exposure of CPF in mice disrupt the bio-synthesis of these neuropeptides in adulthood in mice as determined by Western blot or enzyme-linked immuno-sorbent assay analysis. Chlorpyrifos has also been found to induce estrogenic effect (Andersen et al., 2002; Hodgson & Rose, 2006; Jeong et al., 2006). Maintenance of pregnancy, mammogenesis, lactogenesis, nest building, maternal care and nurturing the neonates involve progesterone (Voci & Carlson, 1973), prolactin (Bole-Feysot et al. 1998; Miller et al., 2004) and oxytocin. Thus behavioral alterations in nest building (Fig 5) and nurturing the young ones (Fig 4) with considerably high frequency of neonatal cannibalization were considered as out-comes of decreased maternal care that must involve alterations in neuro-hypothalamic, pituitary and ovarian hormonal interplay. Reports from already existing literature on human prenatal exposure to CPF that can be transferred through placenta during pregnancy leading to decreased fetal growth (Whyatt et al., 2004) and reduction in gestation period (Eskenazi et al., 2004) consolidate our preview in this context. It seems that chlorpyrifos induced resetting of neuroendocrine control of pregnancy and parturition must be responsible for decrease in gestational period in this study. Similarly decreased postpartum prolectin secretion from the maternal pituitary in these mothers must at least be a major cause of lesser mammary development, decreased lactation and neonatal incubation by the mothers ultimately leading to the neonatal cannibalization. Bioassay of the important hormones of pregnancy and lactation can give a deeper insight in this connection.

Results obtained in this study clearly indicate that maternal toxicities of CPF are multidimensional. This toxic insecticide seems to cause subtle


103

disruptions in neuro-endocrine-physiology of pregnancy, lactation and associated behaviors in mothers at dose levels as low as 10 mg/kg BW in mice.

REFERENCES

Aldridge, J. E., Levin, E. D., Seidler, F. J. & Slotkin, T. A. 2005a. Developmental

exposure of rats to chlorpyrifos leads to behavioral alterations in adulthood, involving serotonergic mechanisms and resembling animal models of depression. Environ. Health Perspect., 113: 527-531.

Aldridge, J. E., Meyer, A., Seidler, F. J. & Slotkin, T. A. 2005b. Alterations in central nervous system serotonergic and dopaminergic synaptic activity in adulthood after prenatal or neonatal chlorpyrifos exposure. Environ. Health Perspect., 113: 1027-1031.

Amr, M. M., Halim, Z. S. & Moussa, S. S. 1997. Psychiatric disorders among Egyptian pesticide applicators and formulators. Environ. Res., 73: 193-199.

Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M. & Bonefeld- Jørgensen, E. C. 2002. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl. Pharmacol., 179: 1-12.

Asmatullah, Andleeb, S. & Zahra, N. 2004. Teratological effects of chlorpyrifos on chick development. Punjab Univ. J. Zool., 19: 9-16.

Beseler, C. L., Stallones, L ., Hoppin, J. A., Alavanja, M. C., Blair, A., Keefe, T. & Kamel. F. 2008. Depression and pesticide exposures among private pesticide applicators enrolled in the agricultural health study. Environ. Health Perspect., 116: 1713-1719.

Bole-Feysot, C., Goffin, V., Edery, M., Binart, N. & Kelly, P. A. 1998. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev., 19: 225-268.

Breslin, W. J., Liberacki, A. B., Dittenber, D. A. & Quast, J. F. 1996. Evaluation of the

developmental and reproductive toxicity of chlorpyrifos in the rat. Fund. Appl. Toxicol., 29: 119-130.

Costa, L. G. 2006. Current issues in organophosphate toxicology. Clinica. Chimica. Acta, 366: 1-13.

Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. P. & Barr, D. B. 2004. Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ. Health Perspect., 112: 1116-24.

Farag, A. T., El-Okazy, A. M. & El-Aswed, A. F. 2003. Developmental toxicity study of chlorpyrifos in rats. Reprod. Toxicol., 17: 203-208.

Fiedler, N., Kipen, H., Kelly-McNeil, K. & Fenske, R. 1997. Long-term use of organophosphates and neuropsychological performance. Am. J. Ind. Med., 32: 487-496.

Hodgson, E. & Rose, R. L. 2006. Organophosphorus chemicals: Potent inhibitors of the human metabolism of steroid hormones and xenobiotics. Drug Metabol. Rev., 38: 149-162.


104

Jaga, K. & Dharmani, C. 2007. The interrelation between organophosphate toxicity and the epidemiology of depression and suicide. Rev. Environ. Health, 22: 57-73.

Jeong, S., Kim, B., Kang, H., Ku, H. & Cho, J. 2006. Effect of chlorpyrifos-methyl on steroid and thyroid hormones in rat F0- and F1-generations. Toxicology, 220: 189-202.

Kang, H. G., Jeong, S. H., Cho, J. H., Kim, D. G., Park, J. M. & Cho. M. H. 2004. Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology, 199: 219-30.

Levin, E. D., Addy, N., Baruah, A., Elias, A., Christopher, N. C., Seidler, F. J. & Slotkin, T. A. 2002. Prenatal chlorpyrifos exposure in rats causes persistent behavioral alterations. Neurotoxicol. Teratol., 24: 733-741.

Lima, C. S., Ribeiro-Carvalho, A., Filgueiras, C. C., Manha˜es, A. C., Meyer, A. & Abreu-Villaca. Y. 2009. Exposure to methamidophos at adulthood elicits depressive-like behavior in mice. Neurotoxicology, 30: 471-8.

Lo´pez-Crespo, G. A., Carvajal, F., Flores, P., Sa´nchez-Santed, F. & Sa´nchez-Amate, M. C. 2007. Time course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurtoxicology, 28: 541-547.

London, L., Flisher, A. J., Wesseling, C., Mergler, D. & Kromhout. H. 2005. Suicide and exposure to organophosphate insecticides: cause or effect? Am. J. Ind. Med., 47: 308-321.

London, L., Myers, J. E., Nell, V., Taylor, T. & Thompson, M. L. 1997. An investigation into neurologic and neurobehavioral effects of long-term agrichemical use among deciduous fruit farm workers in the Western Cape, South Africa. Environ. Res., 73: 132-145.

Meyer, A., Seidler, F. J., Aldridge, J. E., Tate, C. A., Cousins, M. M. & Slotkin, T. A. 2004. Critical periods for chlorpyrifos induced developmental neurotoxicity: alterations in adenylyl cyclase signaling in adult rat brain regions after gestational or neonatal exposure. Environ. Health Perspect., 112: 295-301.

Miller, K. P., Borgeest, C., Greenfeld, C., Tomic, D. & Flaws, J. A. 2004. In utero effects of chemicals on reproductive tissues in females. Toxicol. Appl. Pharmacol., 198: 111-131.

Petruzzi, S., Chiarotti, F., Alleva, E. & Laviola. G. 1995. Limited changes of mouse maternal care after prenatal oxazepam: dissociation from pup-related stimulus perception. Psychopharmacology (Berl), 122: 58–65.

Qiao, D., Seidler, F. J., Tate, C. A., Cousins, M. M. & Slotkin, T. A. 2003. Fetal chlorpyrifos exposure: adverse effects on brain cell development and cholinergic biomarkers emerge postnatally and continue into adolescence and adulthood. Environ. Health Perspect., 111: 536-544.

Richardson, J. & Chambers, J. 2003. Effects of gestational exposure to chlorpyrifos on postnatal central and peripheral cholinergic neurochemistry. J. Toxicol. Environ. Health Part A, 66: 275-289.

Roldan-Tapia, L., Nieto-Escamez, F. A., del Aguila, E. M., Laynez, F., Parron, T. & Sanchez-Santed. F. 2006. Neuropsychological sequelae from acute poisoning and long-term exposure to carbamate and organophosphate pesticides. Neurotoxicol. Teratol., 28: 694-703.

Roldan-Tapia, L., Parron, T. & Sanchez-Santed. F. 2005. Neuropsychological effects of long-term exposure to organophosphate pesticides. Neurotoxicol. Teratol., 27: 259-66.


105

Roy, T. S., Sharma, V., Seidler, F. J. and Slotkin. T. A. 2005. Quantitative morphological assessment reveals neuronal and glial deficits in hippocampus after a brief subtoxic exposure to chlorpyrifos in neonatal rats. Dev. Brain Res., 155: 71-80.

Sánchez-Amate, M. C., Flores, P. & Sánchez-Santed, F. 2001. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav. Pharmacol., 12: 285-292.

Saulsbury, M. D., Heyliger, S. O., Wang, K. & Johnson, D. J. 2009. Chlorpyrifos induces oxidative stress in oligodendrocyte progenitor cells. Toxicology, 259:1-9.

Saulsbury, M. D., Heyliger, S. O., Wang, K. & Round, D. 2008. Characterization of chlorpyrifos-induced apoptosis in placental cells. Toxicology, 244: 98-110.

Schneider, J. E. & Lynch, C. B. 1984. Investigation of a common physiological mechanism underlying progesterone-induced and maternal nesting in mice (Mus musculus). J. Comp. Psychol., 98: 165–176.

Slotkin, T. A. & Seidler, FJ. 2007. Prenatal chlorpyrifos exposure elicits presynaptic serotonergic and dopaminergic hyperactivity at adolescence: Critical periods for regional and sex-selective effects. Rep. Toxicol., 23: 421-427.

Slotkin, T. A., Levin, E. D. & Seidler, F. J. 2009. Developmental neurotoxicity of parathion: Progressive effects on serotonergic systems in adolescence and adulthood. Neurotoxicol. Teratol., 31: 11-17.

Slotkin, T. A., Ryde, I. T., Levin, E.D. & Seidler, F. J. 2008. Developmental neurotoxicity of low dose diazinon exposure of neonatal rats: Effects on serotonin systems in adolescence and adulthood. Brain Res. Bull., 75: 640-647.

Steenland, K., Jenkins, B., Ames, R. G., O’Malley, M., Chrislip, D. & Russo, J. 1994. Chronic neurological sequelae to organophosphate pesticide poisoning. Am. J. Public Health, 84: 731-6.

Stephens, R., Spurgeon, A., Calvert, I. A., Beach, J., Levy, L. S., Berry, H. & Harrington, J. M. 1995. Neuropsychological effects of long-term exposure to organophosphates in sheep dip. Lancet, 345: 1135-9.

Tait, S., Ricceri, L., Venerosi, A., Maranghi, F., Mantovani, A. & Calamandrei, G. 2009.

Long-term effects on hypothalamic neuropeptides after developmental exposure to chlorpyrifos in mice. Environ. Health Perspect., 117: 112-116.

Tian, Y., Ishikawa, H., Yamaguchi, T., Yamauchi, T. & Yokoyama, K. 2005. Teratogenicity and developmental toxicity of chlorpyrifos. Maternal exposure during organogenesis in mice. Rep. Toxicol., 20: 267-270.

Timofeeva, O. A., Sanders, D., Seemann, K., Yang, L., Hermanson, D., Regenbogen, S., Agoos, S., Kallepalli, A., Rastogi, A., Braddy, D., Wells, C., Perraut, C., Seidler, F. J., Slotkin, T. A. & Levin, E. D. 2008. Persistent behavioral alterations in rats neonatally exposed to low doses of the organophosphate pesticide, parathion. Brain Res. Bull., 77: 404-411.

van Wijngaarden, E. 2003. An exploratory investigation of suicide and occupational exposure. J. Occup. Envern. Med., 45: 96-101.

Venerosi, A., Calamandrei, G. & Ricceri, L. 2006. A social recognition test for female mice reveals behavioral effects of developmental chlorpyrifos exposure. Neurotoxicol. Teratol., 28: 466-471.

Venerosi, A., Cutuli, D., Colonnello, V., Cardona, D., Ricceri, L. & Calamandrei, G. 2008. Neonatal exposure to chlorpyrifos affects maternal responses and maternal aggression of female mice in adulthood. Neurotoxicol. Teratol., 30: 468-474.


106

Verma, R. S., Mehta, A. & Srivastava, N. 2007. In vivo chlorpyrifos induced oxidative stress: Attenuation by antioxidant vitamins. Pestic. Biochem. Physiol., 88: 191-196.

Voci, V. E. & Carlson, N. R. 1973. Enhancement of maternal behavior and nest building following systemic and diencephalic administration of prolactin and progesterone in the mouse. J. Comp. Physiol. Psychol., 83: 388-393.

Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. F., Garfinkel, R., Hoepner, L. A., Diaz, D., Dietrich, J., Reyes, A., Tang, D., Kinney, P. L. & Perera, F. P. 2004. Prenatal insecticide exposures birth weight and length among an urban minority cohort. Environ. Health Perspect., 112: 1125-32.


An overview on nutrition and feeding of prawn

(Penaeus Japonicus)

ANJUM KHALIQUE, NOOR KHAN, MUHAMMAD SHARIF MUGHAL &

KHALID MAHMOOD ANJUM

Department of Animal Nutrition, (AK). Department of Fisheries and Aquaculture, (NK,

MSM). Department of Wildlife and Ecology, University of Veterinary and Animal

Sciences, (KMA)Lahore.

ABSTRACT

This paper presents an overview of the biochemical aspects of shellfish nutrition especially that of prawn (Penaeus japonicus). Although the nutritive requirements of the prawn are same as for other animals (adequate levels of protein, lipids, carbohydrates, minerals and vitamins) for their growth. The study with prawn has revealed its unique aspects of metabolism; especially lipid metabolism. Based on this biochemical knowledge, various formulations of artificial diets have been used for the mass requirement of different growth stages of P. japonicus. Key words: Prawn (Penaeus japonicas), Nutrition, Feeding.

INTRODUCTION

Crustacean farming such as that of shrimps and prawn has made

significant progress during the last few decades in many parts of the world due to luxuriant profitable commodity. The most favored species were those which commanded the highest prices when sold as luxury foods. Increasing demand and benefits have provided sufficient stimulus to promote investment in prawn culture by the private sector. In order to help and solve the problems of the industry which have arisen due to rapid expansion and growth of their culture, studies are required on management techniques, diseases and feeding, etc.

Like other animals, shellfish (crustacean) require nutrients which can support growth, maintain life and resistance against diseases. These substances include proteins, lipids, carbohydrates, vitamins and minerals. The proteins are primarily necessary for growth and defense while, fats and carbohydrates provide energy. When usual energy furnishing components are not provided in the diet in sufficient amount, the protein is used for heat and energy rather for growth. Vitamins and minerals are important for the regulation of body processes. All these nutrients are interrelated and have to be the part of diets to be fully utilized by the body (Falicitas, 1983; Pascual et al., 1983).

Since Hudinaga (1942) succeeded in rearing the prawn under artificial conditions, technique for rearing from hatching to commercial size have been steadily improved in Japan and have been applied to other penacid species in many countries. Studies on the nutritional requirements of P. japonicus were begun about 40 years ago (Kanazawa et al., 1970; Kitabayashi et al., 1971a). As a result, the specific needs of nutrients for growth and survival of prawn have been identified and developed (New, 1976; 1980).

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Compounded artificial diets are presently being used for commercial prawn production. The production of larvae depends primarily on the live feed such as diatoms and Artemia salina L. Production of live feeds requires extensive labour, and facilities that vary with climatic conditions. However, live Artemia nauplii is still considered as one of the best food for rearing of both fish and shellfish larvae. The use of artificial diets can help to reduce feed cost and provide a more consistent nutrition and predictable output (AQUA Culture, 2008). These diets are now successfully used to partially replace live Artemia in commercial shrimp hatcheries. The nutrition of shrimps and prawns requires knowledge about behavioral, mechanical and physiological processes of feeding at larval or post larval stage. One of the key considerations, in this regard, is the development of the gut structure and its functions. Larvae of crustaceans have a simple gut which gradually becomes complex as the organism grows. The gut physiology and its enzymes also changes during short transit times. The manufacturing of nutritious and easily-digestible diet is still a challenge for nutritionists. This is particularly important in early post larval stages, when the increased consumption of Artemia is becoming a high cost in the hatchery operation. It has been demonstrated with different penaeid species that enzyme secretion is particularly limited in post-larvae, which are unable to digest sufficient amounts of full-length proteins and longer peptides in the feed (AQUA Culture, 2008). As for general nutrition, review had been presented by several other workers (New, 1976; Biddle, 1977: Ceccaldi, 1978: Conklin, 1980: Kanazawa, 1980). Several efforts have been carried out to develop artificial diets capable of sustaining good growth (Das et al., 1996; Venkataramani et al., 2002; Anh et al., 2009). This paper represents an over view of the biochemical aspects of penaecid nutrition, especially prawn larvae and juveniles. Micro particulture diets for larval stages of P. japonicas Live foods such as diatoms and Artemia salina L. have been used in general for rearing the Zoeal and mysid stages of prawn. Several types of micro particulture diets have been developed and used as live food substitutes for larval prawns (Jones et al.,1979; Villegas & Kanazawa, 1980; Kanazawa et al., 1982). Three approaches for presenting the test diets have been examined; nylon protein micro capsules, carrageenan- microbond and Zein microbond particles. The results demonstrated that all three forms were found to sustain reasonable growth and survival of prawn from the zoeal to post larval stage when compared to live foods. Proteins and amino acid requirements Generally speaking, the growth rate of cultured shrimp is highest when the amino-acid composition of their feed is maintained the same as that of their own protein. The amino-acid composition of P. japonicus protein is most similar to that of mollusks such as short necked clam, squid and some small shrimps. The original artificial shrimp feeds include squid meal and short necked clam as an important ingredient, but are very expensive. Therefore, the composition of the artificial feed had to be changed to include cheaper ingredients.

Studies revealed that peptides, as well as free amino-acid, are far inferior to protein as a nitrogen source in the prawn diet. Addition of glycine to the diet significantly stimulated feed intake. It has been concluded that optimum dietary protein level for P. japonicus is within the range of 52 to 57%; this level gave

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good results on the basis of weight gain and feed efficiency. The optimum protein level for juveniles ranged from 52% - 57% on the basis of weight gain and feed efficiency (Deshimaru & Youne, 1978a; Boonyaratpalin et al., 1980). In general Juveniles or adult penacids have been shown to attain optimum growth on diets containing upto 60% protein (New, 1976). Several groups of workers have reported the optimum protein level in diets for Penacus indicus to be 43% (Colvin, 1976a), Penaeus morden 40% (Aqacop, 1977: Khannapa, 1977), 35% (Bages & Sloane, 1981) and Panaeus setiferus 28% - 32% (Andrews et al., 1972).

Deshimaru & Kuroki (1975) reported that a diet containing crystaline amino acids mixture instead of protein used to study the essential amino acid requirement of prawn and found that such a diet was unsuitable for sustaining growth and survival. Their results indicated that prawn was incapable of efficient utilization of free amino acids in diet. (Kanazawa et al., 1984), therefore, investigated the incorporation of radio active acetate into the individual amino acids of the prawn in order to determine the requirements for essential amino acids. The prawn was shown to require ten amino acids, arginine, methionine, valine, threonine, isoleusine, leusine, lysine, histidine, phynlalanine, and tryptophane. Such amino acids have also been demonstrated to be essential for Penacus aztecus (Shewbart et al., 1972), P. serratus (Cowey & Forster, 1971, Homarus americanus (Galleghar, 1976), Astacus astecus (Zandee, 1966), Hcrobranchium ohion (Lightner et al., 1977). Teshima (1984) studied the effect of protein levels on growth and survival of prawn larvae with dietary carbohydrate levels but not with dietary lipid levels. The optimum protein levels for prawn larvae were estimated to be around 45% - 55% and 55% or more when the diets contained 25%, 15% and 5% levels of carbohydrates, respectively. The utilization of dietary protein is mainly affected by its amino acid composition, level of protein intake, calorie content of the diet, digestibility of the protein, physiological state of the species, water temperature, and size of prawn (Mukhopadhyay et al., 2003). Carbohydrate requirements Carbohydrates are another source of cheep nutrients that play an important role in the growth of prawns. The nutritive value of monosaccharide, especially glucose, for the prawn, P. japonicus, is inferior to that of the disaccharides, sucrose and maltose, and polysaccharides such as starch, dextrin and glycogen. Diets containing glucose or galactose for 30 days resulted high hepatopancreatic glycogen concentration. The serum glucose level of the prawn increases quickly and remains at a high level for 24 hours. Accordingly, the growth of prawn was inhibited with the increased level of dietary glucose. The optimum disaccharides and polysaccharide level in diet was found to be 20% (Boonyaratpalin et al., 1980). Studies revealed that the addition of glucose to diet has inhibited the growth of Pana aztecus (Andrews et al., 1972), P. duoranum (Sick & Andrew, 1973) and P. japonicus (Deshimaru & Yone, 1978b). Abdel Rehman et al. (1979) also demonstrated the addition of over 10% glucose to the diet markedly inhibited growth of prawn. High weight gain has been obtained on diets containing disaccharides (Abdel Rehman et al., 1979). The addition of 0.52% glucosamine to the diet improved growth of prawn but the inclusion of chitin inhibited growth (Kitabayashi et al., 1971a). Pascual et al. (1983) have demonstrated that the addition of sucrose or dextrin as carbohydrate source for


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P. monodon juveniles proved better than other carbohydrates. They also pointed out that dietary source of glucosamine is necessary for P. japonicus juveniles which inhibits the growth promoting effect of cholesterol. However, the role of dietary glucosamine is still not clear. Lipids and fatty acids requirements Fatty acids have been shown to play an important role not only as energy source but also essential nutrients for both fish and crustacean (Teshima, 1978: Yone, 1978). Animals are generally capable of synthesizing steroids but not sterols from acetate (Teshima & Kanzawa, 1971). Crustaceans are capable of converting C28 and C29 sterol to cholesterol (Teshima, 1971) and utilizing ergosterol and Beta- sitosterol to some extent for growth (Kanazawa et al., 1971). Prawns are able to metabolize parent fatty acids such as linoleic and linolenic acids to ω-6 and ω-3 highly polyunsaturated fatty acids (PUFA). Hence, the speed of these reactions appears to be too slow to meet the requirements for essential fatty acids and the ω-3 series of polyunsaturated fatty acid that must be provided in the diet. Thus 20:5 ω-3 and/or 20:6 ω-3 possess higher activity as essential fatty acids in the prawn P. japonicus than 18:2 ω-6 and 18:3 ω-3 (Boonyaratpalin et al., 1980).

Studies revealed that crustaceans as well as fish have a requirement for specific fatty acids (Teshima, 1978) which has also been confirmed by metabolic studies using radioactive traces. Studies on EFA requirements for crustaceans have suggested that the nutritive value of lipids for prawn and shrimps is probably related to the types and contents of EFA. High nutritive values of lipids rich in ω-3 HUFA, such as pollack liver oil and shrimp liver oil have been demonstrated for P. duorarum (Sick & Andrews, 1973). Kanazawa et al. (1977) have pointed out that the superior dietary value was obtained with marine lipids containing ω-3HUFA such as pollack liver oil and short necked clam oil, indicating that the inferior dietary value of soya bean oil containing 18: 3 ω-3 is possibly due to the shortage of ω-3 HUFA such as 20: 5 ω-3 and 22:6 ω-3. Guary et al. (1976) also reported a high nutritive value of sardine oil and short neck clam oil for P. Japonicus. Aquacop (1978) reported that cod liver oil resulted in sustained growth and survival of P. merguiensis and was considered one of the best sources of lipid. On the other hand Dashimanu et al. (1979) reported that a good lipid source for P. japonicus diet is a mixture of soybean oil and pollack liver oil (6% in diets 1:3 or 1:1 w/w). Colvin (1976b) also reported that a mixture of wheat germ oil and pea nut oil was best for P. indicus among the vegetable oil. A mixture of pollack liver oil and soybean oil in a ratio ranging from 3:1 to 1:1 containing approximately 1.2–1.8% ω-6 and 0.6–1.2% ω-3 fatty acid in diet is considered to be a desirable dietary lipid source for prawn. According to Boonyaratpalin et al. (1980) the suitable dietary lipid level appears to be approximately 6%. The types and content of EFA dominate the nutritive value of dietary lipids. However, other lipid components such as phospholipids and sterols should be considered in evaluating the dietary value of lipids for prawns and shrimps. Phospholipids requirements To determine the reason for the superior nutritive value of short necked clam oil (Tapes oil compared with pollack liver oil for the prawn P. japonicas), Kanazawa et al. (1979) examined the effects of several lipid fractions of tapes oil on the


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growth of P. japonicus. Conklin et al. (1980) have shown that the survival rate of juvenile lobsters was remarkably improved by the diet. D’ Abramo et al. (1981) pointed out the essentiality of phosphaltialylcholin for survival of the juvenile lobster Homarus americanus. However, prawns fed on cholesterol-fortified diets attained a remarkably high growth compared to those on non-fortified diets, and it was easily concluded that cholesterol should be included in the diet as an indispensable ingredient. The optimum dietary level was found to be 1.4–2.1%. There is little information available regarding dietary sources of phospholipids that are effective in enhancing or sustaining growth and survival of larval and juvenile P. japonicus. Kanazawa (1985) assumed that dietary phospholipids may be required for both the smooth transport of dietary lipids, particularly cholesterol, in the hemolymph and a slow rate of phospholipid biosynthesis. Minerals requirements Prawn and shrimps may absorb some minerals from the water to some extent. Conklin et al. (1975) suggested that the mineralization of shell in juvenile lobsters was improved with calcium rich diets. Deshimaru & Yone (1978c) have shown that P. japonicus takes up calcium from seawater and does not require calcium, magnesium and iron. Kanazawa et al. (1984) reported that addition of calcium to diets could be necessary to maintain the ratio of Calcium-Phosphorus (1:1) in diets, although growth of P. japonicus on diets with and with out calcium supplement was compatible. Kitabayashi et al. (1971b) have also pointed out the importance of the Ca/P ratio, indicating an optimum ratio of 1:1 for P. japonicus. Huner & Colvin (1977) have shown Ca/P ratio of 2.2:1 to the optimum for growth of juvenile shrimp, P. californiensis. Shewbart et al. (1973) considered that calcium, phosphorus and sodium chloride were not necessary for P. aztecus, but phosphorus may be essential. The necessity of phosphorus has been manifested with P. japoniocus (Kitabyashi et al., 1971c; Desimaru & Yone, 1978a; Kanazawa et al., 1982). Deshimanu et al. (1979) reported that P. japonicus also requires phosphorus (2.0%), potassium (10%) and trace minerals (0.2%). Kanazawa et al. (1984) have shown that this species requires Ca (1.0%), P (1.0%), Mg (0.3%), K (0.9%) and Cu (0.8%) in the dry diets. There is some conflict on the published values for the requirement of prawn for Ca and Mg. Since, it is likely that the effect of Ca varies according to types of Ca salt used such as primary, secondary and tertiary salts. The Ca requirement of prawn should be reevaluated by a more detailed experiment. The addition of iron (0.006%) and Mn (0.003%) inhibited growth of P. japonicus, juveniles. According to Boonyaratpalin et al. (1980) supplements of calcium, magnesium, and iron did not improve the nutritive value of the diet at levels of 2%, 0.3%, and 0.02%, respectively. While, supplementary iron rather reduced its value. The improved effect of phosphorus supplementation was noticed at a 2% level in the diet. The group fed on the diet supplemented with potassium at a level of 1% showed higher growth and feed efficiency than those of the group fed on the diet without it. The suitable level of trace minerals in the diet was found to be 0.2% while levels over 0.2% resulted in a lower nutritive value than the diet without the supplement.” Vitamins requirements The effect of vitamins on growth and survival of Juvenile lobsters has been demonstrated by Conklin et al. (1980) using a purified diet. The vitamin requirement of the juvenile P. japonicus have been investigated by Kanazawa et


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al. (1976); Guary et al. (1976) and Deshimaru & Kuroki (1979), (Table I). Kanazawa et al. (1989) had examined the use of carrageenan microbound test diets, the vitamin requirements of P. japonicus larvae. These larvae fed on a vitamin free diet did not reach the post-larval stage, suffering 100% mortality in the mysid stage. “The survival of prawn larvae was extremely low when they were fed on diets lacking either tocopherol, calciferol, choline and vitamin C. Addition of vitamin C to a squid based diet for Juvenile P. japonicus accelerated growth rate, however, excess of vitamin C inhibited growth. Prawn grew best at inclusion levels of 0.22% (Kitatayashi et al., 1971b). Iwata & Shigeno (1980) reported the whitening induced by vitamin C deficient diets. Lightner et al. (1977) have found that P. californiensis and P. stylirostyis sometimes showed an abnormal symptom, named “Black Death” with a characteristic blackening of esophagus wall, cuticle, gastric wall, hind gut wall and gills. “Black death” has been recognized as a symptom of Vitamin C deficiency (Margarelli et al., 1979). The desirable level of dietary thiamine hydrochloride (Vitamin B1) was found to be approximately 6 mg per 100 g diet on the basis of growth, or approximately 12 mg based on the thiamine content of prawn at the end of the feeding experiment.”

“Some problems and diseased symptoms associated with vitamin deficiency as reported “by Boonyaratpalin et al. (1980) such as retardation of growth and high mortality occurred early on pyridoxine deficient diets. Prawns maintained on a diet with a high pyridoxine level exhibited low growth. The favorable level of dietary pyridoxine was found to be approximately 12 mg per 100 g diet on the basis of both the growth and the pyridoxine content of prawn. The growth and survival of prawn receiving a choline or inositol deficient diet was found to be inferior to those of the complete diet group.”The required levels for choline chloride and inositol were estimated to be approximately 60 mg and 200 mg per 100 g of the diet, respectively. Dietary ascorbic acid was found to be effective for the survival, growth and molting of prawn. The requirement for ascorbic acid of prawn was about 500–1000 mg per 100 g of dry diet.”

Table 1: Vitamin requirements of Penaeus japonicus

Requirement (mg %)

Vitamin Kanazawa et al. (1976)

Guary et al. (1976)

Deshimasu & Kuroki (1979)

Ascorbic acid -- 1000-2000 300 Choline 60 -- Dispensable Inositol 200 -- 400 Thiamine -- -- 6-12 Pyridoxine -- -- 12


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REFERENCES Abdel Rehman, S. M., Kanazawa, A. & Teshima, S., 1979. Effects of dietary

carbohydrates on the growth and the levels of the hepatopancneatic glycogen and serum glucose of prawn. Bull. Japanese Soci. Sci. Fish., 45: 1491-1494.

Andrews, J. W., Sick, I. V. & Baptist, G. L., 1972. The influence of dietary protein and energy levels on growth and survival of Penaeid shrimp. Aquaculture, 1: 341-347.

Anh, N. T. N., Hien, T. T. T., Mathieu, W. Hoa, N. V. & Sorgeloos, P., 2009. Effect of fishmeal replacement with Artemia biomass as a protein source in practical diets for the giant freshwater prawn Macrobrachium rosenbergii. Aqua. Res., 40: 669-680.

Aquacop., 1977. Reproduction in captivity and growth of Penaeus monodon fabricius in Polynesia. Proc. Word Maricul. Soc., 8:927-945.

Aquacop., 1978. Study of nutrition requirements and growth of Penaeus merguiensis in tanks by means of purified and artificial diets. Proc. Word Maricul. Soc., 9:225-234.

AQUA Culture., 2008. Feed Technology. Asia Pacific Magazine. Bages, H. & Sloane, L., 1981. Effects of dietary protein and starch levels on

growth and survival of Penaeus monodon (fabricus) post larvae. Aquaculture, 25:117-128.

Biddle, G.N., 1977. The nutrition of macrobrachium species. Page 272-291 in J.A. Hanson and H.L. Goodwin (eds), Shrimp and prawn farming in the western hemisphere, dowen, Huchison and Ross Inc., Pennsylvania.

Boonyaratpalin, M., Vorasayan, P., Suksucheep, V., Taechajunta, K. & Chacheongsao, B., 1980. Shrimp Nutrition In Japan A Study Tour Report. Programme for the Expansion of Freshwater Prawn Farming (FAO/UNDP/THA/75/008).

Ceccaldi, J. H., 1978. La nutrition des crustaces, Oceanis (Serie de documents oceanegraphiques, Paris) 4:55-62.

Colvin, P.M., 1976a. Nutritional studies on penaeid prawns: protein requirements in compounded diets for juvenile Penaeus indicus. Aquaculture, 7:315-326.

Colvin, P.M., 1976b. The effect of selected seed oils on the fatty acid composition and growth of Penaeus indicus. Aquaculture, 8:81-89.

Colvin, L. B. & Brand, C.W., 1977. The protein requirement of Penaeid Shrimp at various life cycle stages with compounded diets in controlled environment systems. Proc. World Maricul. Soci., 8:821-840.

Conklin, D. E., 1980. Nutrition. Page 277-800 in J.S. Cobb and B.F. Phillips (eds.), Biology and Management of lobsters, Vol. 1, Academic Press Inc., New York.

Conklin, D. E., D’Abramo, L. R., Bordner, C. E. & Baum, N. A., 1980. A successful purified diet for the culture of juvenile lobsters: The effect of Lecithin. Aquaculture, 21:243-249.

Conkolin, D.E., Devers, K. & Shleser, R. A., 1975. Initial development of artificial diets for the lobster, (Homarus americanus). Proc. Annual Meeting World Maricul. Soci., 6:237-248.


114

Cowey, C. B. & Forster, J. R. M., 1971. The essential amino acid requirements of the prawn Palaemon serratus. The growth of prawn on diets containing protein of different amino acid compositions. Marine Biol. 10:77-81.

D’Abramo, L.R., Bordner, C. E., Conklin, D. E. & Baum, N. A., 1981. Essentiality of dietary phosphatidylcholine for the survival of juvenile lobsters. J. Nutri., 111:425-431.

Das, N. N., Saad, C. R., Ang, K. J., Law, A. T. & Harmin, S. A., 1996. Diet formulation for Macrobrachium rosenbergii (de Man) broodstock based on essential amino acid profile of its eggs. Aqua. Res., 27: 543–555.

Deshimaru, O. & Kuroki, K., 1975. Studies on a purified diet for prawn. IV. Evaluation of protein, free amino acids and their mixture as nitrogen source. Bull. Japan Soci. Fish. 41:101-103.

Deshimaru, O. & Yone, Y., 1978a. Requirements of prawn for dietary minerals. Bull. Japan Soci. Sci. Fish., 44: 207-210.

Deshimaru, O. & Yone, Y., 1978b. Effect of dietary carbohydrate source on the growth and feed efficiency of prawn. Bull. Japan Soc. Sci. Fish., 44:1161-1163.

Deshimaru, O. & Yone, Y., 1978c. Optimum level of dietary protein for prawn. Bull. Japan Soci. Sci. Fish., 44:1395-1397.

Deshimaru, O. & Kuroki, K., 1979. Requirement of prawn for dietary thiamine, pyridoxin and choline chloride. Bull. Japan Soc. Sci. Fish., 45:363-367.

Deshimaru, O., Kuroki, K. & Yone, Y., 1979. The composition and levels of dietary lipids appropriate for growth of prawn. Bull. Japan Soc. Sci. Fish., 45:519-524.

Felicitas, B., 1983. Nutrition and feeding of Penaeus monodon Ext. mandal No. 3, 3

rd ed. Aquaculture Dept. South East Asian Fishries Development

Centre, Philphines. Gallagher, M. L., 1976. The nutritional requirements of juvenile lobsters,

Homarus americanus. Ph.D. Thesis University of California, Davis. Guary, M., Kanazawa, A., Tanka, N. & Ceccaldi, H. J., 1976. Nutritional

requirements of prawn-IV. Requirement for ascorbic acid. Memoris of the Faculty of Fisheries, Kangoshima University, 25:53-57.

Hudinaga, M., 1942. Reproduction, development and rearing of Penaeus japonicus Bate. Japanese. J. Zool., 10:305-593.

Huner, J. V. & Colvin, L. B., 1977. A short term study on the effects of diets with varied calcium: phosphorus ratios on the growth of juvenile shrimp, Penaeus californiensis (Penaeidae: Crustacea). Proc. World Maricul. Soc., 8: 775-778.

Iwata, M. & Shigeno, K., 1980. Vitamin-C deficiency symptoms of prawn, Penaeus japonicus. Meeting of the Japanese Soci. Sci. Fish, Abstract, 8 pp.

Jones, D., Kanazawa, A. & Ono, K.,1979. Studies on the Nutritional requirements of the larval stages of Penaeus japonicus using microencapsulated diets. Marine Biol., 54:261-267.

Kanazawa, A., 1980. Nutritional requirements of lobster, shrimp and prawn. Marine Sci., 12:864-871.


115

Kanazawa, A., Shimaya, M., Kawasaki, M. & Kashiwada, K., 1970. Nutritional requirements of prawn, I. Feeding on artificial diet. Bull. Japan Soc. Sci. Fish., 36:949-954.

Kanazawa, A., Tanaka, N., Teshima, S. & Kashiwada, K., 1971. Nutritional requirements of prawn, II. Requirement for sterols. Bull. Japanese Soci. Sci. Fish., 37:211-215.

Kanazawa, A., Teshima, S., Sasada, H. & Abdel-Rehman, S., 1982. Culture of the prawn larvae with micro-particulate diets. Bull. Japan Soci. Sci. Fish., 48:195-199.

Kanazawa, A., Teshima, S. & Tanaka, N., 1976. Nutritional requirements of prawn. Requirements for choline an inositol. Memories of the faculty of fisheries, Kagoshima University, 25:47-51.

Kanazawa, A., Teshima, S., Tokiwa, S. & Ceccaldi, H. J., 1979. Effects of dietary linoleic acids on growth of prawn. Oceanol. Acta., 2:41-47.

Kanazawa, A., Tokiwa, S., Kayama, M. & Mirata, M., 1977. Essential fatty acids in the diet of prawn. Effects of linileic and linolenic acids on growth. Bull. Japan Soci. Sci. Fish. 43:1111-1114.

Kanazawa, A., Teshima, S. & Sasaki, M., 1984. Requirements of potassium, copper, manganese, and iron. Mem. Fac. Fish. Kagoshima Univ., 33: 63-71.

Kanazawa, A., 1985. Nutrition of penaeid prawns and shrimps. In Taki Y., Primavera J. H. and Llobrera J.A. (Eds.). Proceedings of the First International Conference on the Culture of Penaeid Prawns/Shrimps, 4-7 December 1984, Iloilo City, Philippines (pp. 123-130). Iloilo City, Philippines: Aquaculture Department, Southeast Asian Fisheries Development Center.

Kanazawa, A., 1989. Microparticulate feeds for Panaeid larvae. Advances in tropical aquaculture. AQUACOP IFREMER Actes de Colloque., 9 pp 395-404.

Khannapa, A., 1977. Effect of various protein levels on growth and survival rates of Penaeus monodon. Q. Res. Rep., SEAFDEC Agriculture Dept., 1(11):24-28.

Kitabayashi, K., Kurata, K., Nakamura, K., & Ishikawa, S., 1971a. Studies on formula feed for Kuruma prawn. On the relationship among glucosaamine, phosphorus and calcium. Bull. Japan Soci. Sci. Fish., 65:91-107.

Kitabayashi, K., Shudo, K., Nakamura, K. & Ishikawa, S., 1971b. Studies on formula feed for Kuruma prawn. On the utilization values of glucose. Bull. Japan Soci. Sci. Fish., 65:109-118.

Kitabayashi, K., Shudo, K., Nakamura, K.& Ishikawa, S., 1971c. Studies on formula feed for Kuruma prawn. On the growth promoting effects of both arginine and methionine. Bull. Japan Soci. Sci. Fish., 65:119-127.

Lightner, D. V., Colvin, L. B., Brand, C. & Donald, D. A., 1977. “Black Death” a disease syndrome of penaeid shrimp related to a dietary deficiency of ascorbic acid. Proc. World Maricul. Soc., 8:611-618.

Magarelli, P.C., Hunter, B., Lightner, D.V. & Colvin, L. B., 1979. Black Death an ascorbic acid deficiency diesease in penaeid shrimp. Comparative Biochemistry and Physiology, 68A:103-108.


116

Mukhopadhyay, P. K., Rangacharyulu, P. V., Mitra, G. & Jana, B. B., 2003. Applied nutrition in fresh water prawn, Macrobrachium rosenbergii culture. J. App. Aquacult., 13: 317-340.

New, M. B., 1976. A review of dietary studies with shrimp and prawns. Aquaculture, 9:101-144.

New, M.B., 1980. A bibliography of shrimp and prawn nutrition. Aquaculture, 21:101-28.

Pascual, B.P., Coloso, R. M. & Tamse, C. T., 1983. Survival and some histological changes in Penaeus monodon Fabricius Juveniles fed various carbohydrates. Aquaculture, 19:127-137.

Shewbart, K., Mies, W. I. & Ludwing, P. D., 1972. Identification and quantitative analysis of amino acids present in protein of the brown shrimp, Penaeus aztecus. Marine Biol., 16:46-67.

Shewbart, K., Mies, W. I. & Ludwing, P., 1973. Nutritional requirements of brown shrimp Penaeus aztecus. U.S. Dept., Commer, Res. No. COM. 73-11794, 52pp.

Sick, L.V. & Andrews, J. W., 1973. The effect of selected dietary lipids, carbohydrates and proteins on the growth, survival and body composition of Penaeus duorarum. Proc. World Maricul. Soci., 4:263-276.

Teshima, S., 1971. Bioconversion of beta sitosterol and 24-methylcholesterol to cholesterol in marine cruatacea. Comparative Biochem. and Physiol., 398:815-823.

Teshima, S. & Kanazawa, A., 1971. Biosynthesis of sterol in the lobster, Penulirus japonicus, the prawn, Penaeus japonicus, and the crab, Portunus trituberculatus. Comparative Biochem. and Physiol., 38b:597-602.

Teshima, S., 1978. Essential fatty acids and necessity of sterols in crustaceans. Pages 60-77 in publishing committee of the Japanese Soc. Sci. Fish, (eds.), Fish culture and dietary Lipids, Suisangaku series no. 22, Koseisha-Koseikaku, Tokyo.

Teshima, S., 1984. Nutritional requirements in prawn. Food oil Abst. B. (19):1-10. Venkataramani, V. K., Rajagopalsamy, C. B. T., & Ravi, D., 2002. Effect of

formulated feeds on growth and broodstock development in Macrobrachium rosenbergii. Asian Fish. Sci. 15:357-364.

Villegas, C. T. & Kanzawa, A., 1980. Rearing of the larval stages of prawn, Paneaus japonicus Bate, using artificial diet. Memories of the Kagoshima Univ. Research for the South Pacific, 1:43-49.

Yone, Y., 1978. Essential fatty acid in marine fish and nutritional value of lipids. Pages 43-59 in publishing committee of the Japanese Soc. Sci. Fish., (eds.), Fish culture and dietary lipids, Suisangaku series no. 22. Koeisha-Koseikaku, Tokyo.

Zandee, D. I., 1966. Metabolism in the crayfish Astacus astacus. Biosynthesis of amino acids. Archives internationales de physiologie et de Biochimie, 74:35-44.


Bacteriology of mastitic milk of cattle and In Vitro antibiogram

of the pathogens isolated, in District Hafizabad, Pakistan

AFTAB AHMAD ANJUM, MUHAMMAD FIAZ QAMAR, MUHAMMAD ASAD ALI,

MUHAMMAD USMAN & TARIQ MUHAMMAD KHAN

Department of Microbiology, Faculty of Veterinary Science, University of Veterinary And

Animal Sciences, Lahore, Pakistan Department of Zoology, GC University, Lahore

Pakistan

ABSTRACT

Mastitis is one of the most important diseases of high yielding dairy cows and

lactating animals. Mastitis is a multietiological complex disease; however, Bacteria constitute the common etiological agents of mastitis. In the present study, bacteria associated with mastitis were investigated and their susceptibility to various antibiotics was tested. Fifty two infected milk samples collected were subjected to California Mastitis Test. The samples found positive were cultured on different media i.e; Nutrient agar, MacConkey’s agar, Staph 110 medium, Mannitol agar and Blood agar for bacterial isolation. The isolated colonies were studied for the staining characteristics, morphology and hemolytic activity. Selected isolates were then studied for bio-chemical characteristics and identified as Staphylococci, Streptococci, E. coli, Pseudomonas and Corynebacterium. One isolate was identified as coagulase negative staphylococcus species. The organisms so isolated and identified were subjected to commonly used antibiotics for determination of antibiogram. Gentamicin, Enrofloxacin, Norfloxacin and Kanamycin were found most effective drugs amongst the antibiotics tested in vitro, moderate sensitivity was observed to Cepharidine and Streptomycin, and the least sensitivity was observed to Penicillin and Cloxacillin. Key words: Mastitis, bacteriology, antibiogram, in vitro, Streptococcus; Staphylococcus; Escherichia Coli; Gentamycin; Chloramphenicol; Streptomycin; Penicillin; Cepharidine; Cloxacillin; Cattle.

INTRODUCTION

Mastitis can be defined as an inflammatory condition of the udder

irrespective of the cause, either infection or trauma and is generally thought the most economically important disease in dairy animals. Mastitis is a multietiological complex disease which is characterized by physical, chemical and microbiological changes in the milk and pathological changes in the glandular tissue of the udder (Radostits et al., 1995). It is a multifactorial and the most costly disease of the dairy industry throughout the world (DeGraves & Fetrow, 1993) that affects both quality and quantity of milk (Arshad, 1999). It has been indicated that there is a positive associations between SCC, mastitis, low quality of management and low hygienic level in farms (Koivula et. al., 2005). Bovine mastitis is a global problem as it adversely affects animal health, quality and quantity of milk and so economics of milk production and every country including developed ones suffer huge financial losses (Sharma et al., 2004). Major pathogens i.e. streptococci spp., S. aureus and coliforms can be controlled

A. A. Anjum ET AL BIOLOGIA (PAKISTAN)

118

by udder washing and disinfection (Sori et al., 2005). Minor and environmental pathogens can be 70% treated by dry period antibiotic therapy (Jones, 2006). Apart from its economic importance, mastitis also carries public health risk, especially for the drug residues in milk and passage of pathogenic organisms to humans. After Foot and Mouth disease it is the most challenging disease in dairy animals in Pakistan. According to Ratafia (1987), worldwide annual losses caused by this disease amounted to nearly 35 billion US dollars.

The losses due to mastitis might be higher in Pakistan because the mastitis prevention practices like teat dipping and dry period antibiotic therapy are not in practice (Arshad, 1999). The study conducted in North Western Frontier Province (NWFP) of Pakistan has indicated that mastitis is an important cause of premature culling in local born and imported Holstein-Friesian cattle; accounting for 22.5% of all culling during a 10 years period (Samiullah et al., 2000). Mastitis is caused by many bacteria, which include the coliform group (specifically Escherichia coli, Enterobacter, Klebsiella species), Streptococci, Staphylococci, Corynebacteria, Pasteurella, Mycoplasma, Leptospira, Yersinia, Mycobaceteria, Pseudomonas, Serratia, and other organisms like fungi, yeasts and virus (Boynukara et al., 2008; Ericsson et al., 2009).

This study was conducted to detect the mastitis in cattle by various indirect laboratory diagnostic tests, to isolate the prevailing causal bacterial organisms from mastitis positive milk and to carry out in vitro sensitivity tests of the isolates to the commonly used antibiotics.


Collection of milk samples The samples were collected from dairy cattle, suspected for mastitis from 20 small herds and 30 private commercial dairy farms in district Hafiz Abad. A total of 158 milk samples from all the sites (78 from small herds & 80 from private commercial dairy farms) were collected aseptically from lactating animals suspected for mastitis. After proper sanitization of teat orifice with 70% ethyl alcohol,10-20ml of milk samples from the teats were collected aseptically after discarding first few streams, in sterile polyethylene screw caped wide mouth vials (Buswell 1995). The milk samples were kept in an ice box and carried to the Laboratoy of Microbiology Department, University of Veterinary And Animal Sciences, Lahore, Pakistan, where the milk samples were kept at 4-8

0C in

refrigerator for further laboratory investigation. Testing of milk samples for mastitis All the milk samples were subjected to the California Mastitis Test (CMT) according to Schneider and Japer (1964). Isolation and identification of bacteria A total of fifty two milk samples positive for mastitis were subjected to isolation and identification of bacteria on the basis of morphological, cultural and biochemical characteristics (Boynukara et al., 2008). Susceptibility of individual bacterial isolates to commonly used antibiotics was determined by disc diffusion method (Baur et al., 1996). The positive samples collected from the field were subjected to cultural examination using standard laboratory procedures of inoculating the samples in Nutrient agar, Blood agar, MacConkey’s agar and

Vol. 56 (1&2) Bacteriology of mastitic milk of cattle 119

Staph 110 agar. The inoculated plates were incubated aerobically at 37°C for 24-48 hours. The bacterial isolates were identified on the basis of their cultural, morphological characteristics and biochemical reactions (Boynukara et al., 2008). The isolated colonies were studied for the staining character, morphology and hemolytic activity. All the isolates were tested in vitro for their sensitivity to commonly used antibiotics in veterinary practice.

The antibiotic discs were placed five minutes later selectively with the help of an antibiotic disc dispenser in a clockwise direction. The charged plates were incubated overnight at 37

ºC by keeping the plates in an inverted position

(Baur et al., 1966). For determining the sensitivity of each antibiotic for therapeutic purpose, the diameters of zone of inhibition including the disc were measured in millimeters (mm). The organisms were considered sensitive to a particular drug when no growth could be seen in an area of 7mm diameter around the antibiotic discs. Drug sensitivity The area of zone of inhibition with the diameter measured in mm and the quantum of sensitivity was interpreted as follows.

+++ >12mm Highly sensitive

++ 10-12mm Moderately sensitive

+ 7-10mm Relatively sensitive

0 < 7mm Resistant


Out of 158 milk samples from different farms, 52 were found positive for mastitis. The overall occurrence of mastitis was 32.91%. These findings agree well with those of Khan and Muhammad 2005), who recorded 23.59 to 34.5% mastitis cases in milking cows and buffaloes. But our findings differ from Ikhwan et al., (1989), who reported overall occurrence of mastitis in buffaloes, cattle, sheep and goats as 9.53%. The test used in their study was White Side Test which was not very sensitive in detecting mastitis positive cases (Iqbal et al., 2003; Ali et al., 2010). Different antibiotics used in the study and their concentrations were Chloramphenicol 30µg, Ciprofloxacin 5µg, Cloxacillin 5µg, Clorotetracycline 30µg, Doxycycline hydrochloride 30µg, Erythromycin 15µg, Gentamicin 10µg, Kanamycin 30µg, Lincomycin 10µg, Norfloxacin 10µg. In this study, out of the total 52 mastitis positive milk samples, bacterial growth was obtained from 37 (71.153%), while the remaining 15 (28.846%) samples yielded no growth. The causes of non-growth in milk samples may be due to premedication of the animals with antibiotics, non-bacterial causing factor and the type of media that did not support the growth of whole range of bacteria associated with mastitis (Arshad et al.,1998). The 37 milk samples of cow yielded 54 isolates, containing Staphylococcus aureus 25(46.29%), Streptococcus agalactiae 16(29.62%), Escherichia coli 6(11.11%), Bacillus spp. 3(5%), Corynebacterium 2(3.7%), Pseudomonas and mixed growth 2(3.7%) (Table: 1). Similar results were observed by Memon et al. (1999) that the major pathogenic organism in mastitis was Staphylococcus aureus (38%), followed by


120

Streptococcus uberus (13%), Escherichia coli (11%) and Klebsiella pneumoniae (11%) in cross cows and buffaloes belonging to Faisalabad-Pakistan. Bhalerao et al., (2000) noted the major pathogenic organism as Staphylococcus aureus (54.55%), followed by Streptococci (36.36%), Escherichia coli (4.55%) and Klebsiella (2.27%) in bovines in India. The contaminated udder is an indication of sub-standard dairy farming. E. coli is the commonest environmental contaminants, which is closely associated with hygiene. It becomes pathogenic whenever the hygienic conditions of the animal or environment become poor. Moreover it also indicates the relatively poor quality of milk, related with substandard hygiene of farm management.

Table 1: Relative occurrence of various bacterial species isolated from mastitic milk samples

Bacteria No. of Cattle %age

Staphylococci 25 46.29

Streptococci 16 29.62

E. coli 6 11.11

Bacillus 3 5

Corynebacterium 2 3.7

Pseudomonas 2 3.7

TABLE 2: Antibiogram of Mastitis Associated Isolates

Name of Antibiotics No. of isolates sensitive % sensitivity

Chloramphenicol 57 24.2

Cloxacillin 22 9.3

Cepharidine 23 9.8

Amoxicillin 38 16.1

Enrofloxacin 92 39

Gentamicin 93 39.4

Norfloxacin 83 35.2

Kanamycin 80 33.9

Flumequin 46 19.5

Oxytetracycline 35 14.8

Penicillin 19 8.1

Streptomycin 29 12.3

Table 2 indicates the antibiotic sensitivity of the isolates. In this trial 12 available antibiotics were used. It is evident from this table that gentamicin, enrofloxacin, norfloxacin and kanamycin were the most effective drugs amongst the 12 antibiotics tested in vitro. The responsiveness of host immune system was a key factor to the invading microorganisms for the severity and control of infection (Bannerman et al., 2009). The infection of the udder by a pathogen is a complex pathological process which specific for each pathogen, Swanson et al., (2009).

Vol. 56 (1&2) Bacteriology of mastitic milk of cattle 121

REFERENCES

Ali, Z., Muhammad G., Ahmad T., Khan R., Naz S., Anwar H., Farooqi, F. A., Manzoor M. N. & Usama A. R., 2010. Prevalence of caprine sub-clinical mastitis, its etiological agents and their sensitivity to antibiotics in indigenous breeds of Kohat, Pakistan. Pakistan J. Life Soc. Sci., 8: 63-67.

Arshad, M., Qamar F.K., Afzal H. & Sidique M., 1998. Epidemiological studies of bovine mastitis in district Gujrat. Proc. Intern. Seminar on Microbial Disease of Livestock and Poultry, CVS, Lahore, pp: 23.

Arshad, G. M. 1999. A population based active disease surveillance and drug trails of mastitis in cattle and buffaloes of District Sargodha. MSc Thesis, Deptt: Vet. Clinical Medicine and Surgery, Univ. Agri., Faisalabad, Pakistan.

Bannerman D.D., Rinaldi M., Vinyard B.T., & Laihia J., 2009. Effects of intramammary infusion of cis-urocanic acid on mastitis-associated inflammation and tissue injury in dairy cows. Am. J. Vet. Res., 70: 373-382.

Baur, A.W., Kieby W.M.M., Shrenis J.C. & Turck M., 1996. Antibiotic susceptibility testing by a standardized single disc diffusion method. Am. J. Clin, Pathol., 45;453-496.

Bhalerao, D. P., Jagadish, S., Keskar, D. V., Dangore, A. D. & Sharma, L. K., 2000. Antibiogram and treatment of bovine subclinical mastitis. Indian Vet. J., 77: 244-246.

Boynukara B, Gulhan, T., Alisarli, M., Gurturk, K., & Solmaz, H., 2008. Classical enterotoxigenic characteristics of S. aureus strains isolated from bovine subclinical mastitis in Van, Turkey. Int. J. Food Microbiol., 125: 209-211.

Buswell, J., 1995. Simple mastitis bacteriology for the practice. In; Practice,pp;426-432.

DeGraves, F J & Fetrow, J., 1993. Economics of mastitis and mastitis control. Veterinary Clinics of North America: Food Animal Practice, 9, 421-434.

Ericsson U.H., Lindberg, A., Persson, W.K., Ekman, T., Artursson, K., Nilsson-Ost, M., & Bengtsson, B., 2009. Microbial aetiology of acute clinical mastitis and agent-specific risk factors. Vet. Microbiol., 137: 90-97.

Ikhwan, K., Shah, M., Anwar, M., Rehman, S., Hafeez, M., & Qureshi, M. B., 1989. To detect the evidence of mastitis in cows and buffaloes and to study the anti-biogram (in vitro) of the causative organisms. Proc: 2nd Inter Provincial Seminar on Animal Health and Production. Organized by Pakistan. Vet. Med. Assoc., Peshawar, pp: 27-32.

Iqbal, M., Amjed, M., & Khan, M., 2003. Comparative study of various laboratory diagnostic tests of sub-clinical mastitis in dairy cows and buffaloes. MSc Thesis, NWFP Agri. Univ., Peshawar, Pakistan.

Jones, G. M., 2006. Understanding the basics of mastitis. Virginia Cooperative Extension, Publication No. 404-233. Virginia State University, USA, pp: 1-7.

Khan A.Z. & Muhammad, G., 2005. Quarter wise comparative prevalence of mastitis in buffaloes and crossbred cows. Pakistan Vet. J., 25(1): 9-12.


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Koivula M, Mantysaari, E. A., Negussie, E., & Serenius, T., 2005. Genetic and phenotypic relationships among milk yield and somatic cell count before and after clinical mastitis. J. Dairy Sci., 88: 827-833.

Memon, M.I., Mirbahar, K.B., Memon, M.R., Akhtar, N., Soomoro, S.A., & Dewani, P., 1999. A study on the etiology of subclinical mastitis in buffaloes. J. Agri. Agri. Engg., 15: 34- 36.

Muhammad, G., Athar, M., Shakoor, A., Khan, M.Z., Fazal-ur-Rehman & Ahmad, M. T., 1995. Surf field mastitis test: An inexpensive new tool for evaluation of wholesomeness of fresh milk. Pakistan. J. Food Sci., 3(3-4): 91-93.

Murphy, J. M. & Hanson, J. J. A., 1941. Modified White Side Test for the detection of chronic bovine mastitis. Cornell Vet., 32: 439.

Radostits, O. M., Blood, D. C., & Gay, C. C., 1995. Veterinary Medicine, 8th Ed.

ELBS- Bailliere Tindall, London. Ratafia, M., 1987. Worldwide opportunities in genetically engineered vaccines.

Bio-Technology (New York), 5: 1154. Sammiullah, M., Syed, U.D., Arif, M., & Khan, M., 2000. Frequency and causes

of culling and mortality in Holstein Friesian cattle in NWFP (Pakistan). J. Anim. Hlth. Prod., 20: 22-24.

Schneider, R., & Japer, D.E., 1964. Standardization of California Mastitis Test. Amer. J. Vet. Res., 25: 1635.

Sharma, Neelesh, Maiti, S.K., & Singh, R., 2004. Mastitis in dairy animals-A global problem. Dairy Planner, 1; 40-41.

Sori, H., Zerihum, A., & Abdicho, S., 2005. Dairy cattle mastitis in and around Sebeta, Ethiopia. Int. J. Appl. Res. Vet. Med., 3: 332-338.

Swanson K.M., Stelwagen, K., Dobson, J., & Henderson, H.V., 2009. Transcriptome profiling of Streptococcus uberis induced mastitis reveals fundamental differences between immune gene expression in the mammary gland and in a primary cell culture model. J. Dairy Sci., 92: 117-129.


PK ISSN 0006-3096

Effect of acute fluoride intoxication on some hematological changes in chicken (Gallus

domesticus).

KHALID MAHMOOD ANJUM, ATIF YAQUB, NAUREEN MUMTAZ, UZMA SAYYED, MUHAMMAD ASAD & SHAHID ALI KHAN

Department of Wildlife & Ecology, University of Veterinary And Animal Sciences, Lahore (KMA, MA, SAK).Department of Zoology, GC University, Lahore (AY, US).

Department of Zoology, University of Education, Bank Road Lahore (NM)

ABSTRACT

A very small amount of fluoride is beneficial for human; whereas overdosing cause problems like neural disorders, memory loss and deficit in intelligence in human as well as other mammals. Present study was designed to look into the hematological changes by acute fluoride intoxication in Gallus domesticus (domestic chicken). The birds

were divided into four treatment groups: group A acted as control and three groups as B, C & D injected with NaF at 10, 20 and 30 µg/g of body weight, respectively. The birds were sacrificed and blood was collected for hematological studies. It was observed that there was significant decrease (P < 0.05) in Total Leukocyte Count (TLC), Total Erythrocyte Count (TEC) and hemoglobin content in groups C and D. The results showed that birds were anemic after the exposure to acute dose of NaF. It was also observed hat bones were deformed leading to lesser production of bone marrow, a site for blood formation, which is the reason for severe decrease in hematological values.

Key words: NaF, Hematology, Chicken, Acute Intoxication, Bone Deformation.

INTRODUCTION

Any combination of elements containing fluorine ion is fluoride. In high concentrations, high concentrations of fluoride salts are toxic to skin or eye. The lethal dose of most common salt of fluoride i.e. sodium fluoride (NaF) for most adult humans is 5 to 10 g (Gosselin et al., 1984 and Baselet, 2008). The fluorine was not produced commercially before the Second World War, the use of fluorine in the processing of uranium ores promoted its manufacture (International Programme on Chemical Safety, 2002).

After the absorption of NaF from intestine, a big part of the fluoride is rapidly distributed to skeletal system and small proportion in the teeth. In skeletal system it promotes the development of bone marrow; whereas, in teeth gives protection from caries (Schmidt & Leuschke, 1990). Studies have shown that high levels of fluoride are accumulated in the brain causing neurologic disorders, motor disruption, intelligence deficit and learning disabilities in human (Li et al., 1995). Fluoride overdose in rabbits caused corneal epithelial defects and necrotic area in the conjunctive of the rabbits. Furthermore, lameness was observed in ungulates due to fluoride accumulation in their skeleton. Fluoride accumulation also creates premature mortalities and dental lesions in wild animals (Phyllis, 1995).

K. M. Anjum ET AL BIOLOGIA (PAKISTAN) 124

Excessive fluoride contamination in drinking water is common in developing countries like India, China, Uzbekistan, Ethiopia and Pakistan. Many minerals contain soluble fluoride, when the ground water passes through such fluoride bearing rock formations; the water becomes contaminated (Abida et al., 2007).

In Pakistan, during year 2000, bone deformity disease was reported in districts; Kalanwala and Kasur of Punjab. The investigations showed that disease was due to high levels of fluoride in the drinking water affecting extremities of legs, vertebral column and the skull bones. The present study was aimed to evaluate the effects of acute fluoride intoxication on different blood parameters as in domestic chicken, Gallus domesticus. In the affected area the poultry birds were also given drinking tap water containing the high levels of fluoride. The study was focused to determine the risks of NaF intoxication in poultry as model organism and validation of results obtained on other organisms.


Eighty domestic chickens were used in the study. One day old chicks were purchased from local market. Before starting the experiment, birds were raised and acclimatized for a period of two weeks in Animal House at GC University, Lahore. After two weeks animals were divided into four groups of 20 birds each. Group A: This group was kept as control, fed on normal feed and was not exposed to any toxicant. Five chicks were sacrificed each day and the hematological values were used as a reference to investigate the NaF intoxication effects in the treatment groups. Group B: This group received normal feed and each bird was injected with NaF 10 µg/g body weight. First day all the chicks were injected. On second day, five chicks were sacrificed for hematological studies. Remaining 15 chicks were again exposed to the same dose. Five chicks were sacrificed each after one, two, three and four days with the exposure of 10, 20, 30 & 40 µg /g body weight of NaF, respectively. Group C: This group was given normal feed and each bird was injected with NaF 20 µg/g body weight. First day all the chicks were injected. On second day, five chicks were sacrifices for hematological and serological studies. Remaining 15 chicks were again exposed to the same dose. Five chicks were sacrificed each after one, two, three and four days with the exposure of 20, 40, 60 & 80 µg /g body weight of NaF, respectively. Group D: This group was given normal feed & was injected with 30 µg/g body weight of NaF. First day all the chicks were injected. On second day, five chicks were sacrificed for hematological and Serological studies. Remaining 15 chicks were again exposed to the same dose. Five chicks were sacrificed each after one, two, three and four days with the exposure of 30, 60, 90 & 120 µg /g body weight of NaF, respectively.

Hematological studies were carried out by using improved nebular Hemacytometer (Bright-Line™). The quantitative data are represented as mean + S.D (Standard Deviation). Statistical package for social sciences (SPSS-13)

Vol. 56 (1&2) Effect of Acute Fluoride Intoxication

125

software was used for statistical analysis. Hypothesis testing methods included the one way analysis of variance (ANOVA). P values less than 0.05 were considered to indicate the statistical significance. (Sokal & Rohlf, 1995; Kumar et al., 2006).


The erythrocytes of the birds are nucleated and oval-shaped. The colour of individual cell is yellowish green due to presence of hemoglobin content. The red blood cell count per millimeter of the mature chicken varies from 2.74 × 10

9 to

3.2 × 109 (Susheela & Jain, 1983). In present study, erythrocyte count in control

group was recorded as 2.76 × 109 + 0.01. In the Group B, RBC counts after first

dose was 2.74 × 109 + 0.02. The erythrocyte count decreased after second, third

and fourth dosing to 2.73 × 109 + 0.02, 2.71 × 10

9 + 0.03, and 2.72 × 10

9 + 0.32,

respectively (Table 1). In Group C, there was drastic decrease in RBC count after first, second, and third dose to 2.73× 10

9 + 0.02, 2.72× 10

9 + 0.25 and 2.71× 10

9

+ 0.02 respectively (Table 1). The birds exposed to four doses of NaF showed severe anemia and their RBC count decreased to 2.68× 10

9 + 0.03 (Table 1). In

Group D, the condition was too severe especially with the birds that received four doses of toxicant. The RBC values recorded were: 2.73× 10

9 + 0.02, 2.69× 10

9 +

0.03, 2.66× 109 + 0.04 and 2.63× 10

9 + 0.03, after receiving one, two, three and

four doses of the toxicant, respectively as shown in Table 1.

Table 1: Effect of NaF Intoxication on Total Erythrocyte Count (TEC)

*indicates the significant results (P < 0.05)

The erythrocyte count may vary due to the hemolysis of R.B.Cs or destruction of erythropoietic organs. Anjum & Shakoori (1993) also reported anemia induced by fluoride intoxication.

In birds the average leukocyte count is 13.5× 106/ml. Different studies

have shown that Total Leukocyte Count (TLC) reduced when the individual birds were exposed to different intoxicants. Al. Khel et al., (1998) observed the reduction in leukocyte after the exposure of Oreochromis niloticus to different sub-lethal concentrations of Manganese.

In the present study the TLC values decreased. In the control group TLC was recorded as 13.62+ 0.24 × 10

6. In group B, the TLC was recorded almost

same as in the control group indicating that the birds were able to cope with dosage of fluoride intoxication; whereas, the birds exposed to four doses of NaF showed the 13.04 + 0.25 × 10

6. In group C, reduction in TLC was more

pronounced especially in birds receiving three and four doses of toxicant. Total

Dose received

TEC. in group A (× 10

9)

TEC. in group B (× 10

9)

TEC. in group C (× 10

9)

TEC. in group D (× 10

9)

1 2.76 + 0.01 2.74 + 0.02 2.73 + 0.02 2.73 + 0.02 2 2.76 + 0.01 2.73 + 0.02 2.72 + 0.25* 2.69 + 0.03* 3 2.76 + 0.01 2.71 + 0.03* 2.71 + 0.02* 2.66 + 0.04* 4 2.76 + 0.01 2.72 + 0.32* 2.68 + 0.03* 2.63 + 0.03*

K. M. Anjum ET AL BIOLOGIA (PAKISTAN) 126

leukocytic count were as; 13.04+ 0.25 × 106 and 13.02+ 0.18 × 10

6, respectively

(Table 2). The TLC values were adversely reduced in group D, 13.14+ 0.26 × 106,

12.90+ 0.14 × 106, 12.85+ 0.24 × 10

6 and 12.78+ 0.14 × 10

6 were the TLC

values receiving one, two, three and four doses of NaF. Table 2: Effect of NaF Intoxication on Total Leukocyte Count (× 10

6)

*indicates the significant results (P < 0.05) The same findings were also reported by Hashmi (1999), during mercury

intoxication in Labeo rohita. The leucopoiesis was impaired by fluoride in the present study.

Hemoglobin is an important protein which supplies the oxygen to the body of animals. The concentration of Hemoglobin in chicken varies from 7- 8.9 (g/dl). The avian hemoglobin contains the four heme units just like the mammals (Ali, 1990). The hemoglobin concentration in group A (control) was 7.96+ 0.20. In group B, there was a decrease in hemoglobin in response to dose of the toxicant. Following were the hemoglobin concentrations; 7.84+ 0.26, 7.74+ 0.21, 7.48+ 0.32 and 7.38+ 0.25 recorded after one, two, three and four doses (Table 3). In Group C, the birds showed remarkable decrease in hemoglobin concentration as the leukocytes counts were 7.50+ 0.30, 7.44+ 0.30, 7.36+ 0.18 and 7.12+ 0.28 after the one, two, three and four doses respectively. In group D, there was a drastic decrease in hemoglobin concentration, the birds after first dose of the toxicant showed 7.46+ 0.30 of hemoglobin while the birds that received four doses in this group had 6.68 + 0.26 (g/dl) of hemoglobin.

Table 3: Effect of NaF Intoxication on Haemoglobin Concentration. (g/dl).

*indicates the significant results (P < 0.05).

The possible reasons for the decrease in hemoglobin concentration were low level of RBC production and hemolysis. The bones of these chicks were deformed due to fluoride intoxication resulting in decrease of bone marrow production which is a site for RBC production, leading to anemia and low hemoglobin concentration.

Dose received

TLC. in group A (× 10

6)

TLC. in group B (× 10

6)

TLC. in group C (× 10

6)

TLC. in group D (× 10

6)

1 13.62+ 0.24 13.60+ 0.30 13.26+ 0.33* 13.14+ 0.26* 2 13.62+ 0.24 13.54+ 0.24 13.20+ 0.30* 12.90+ 0.14* 3 13.62+ 0.24 13.34+ 0.37* 13.04+ 0.25* 12.85+ 0.24* 4 13.62+ 0.24 13.04+ 0.25* 13.02+ 0.18* 12.78+ 0.14*

Dose received

Hb Conc. in group A (g/dl)

Hb Conc. in group B (g/dl)

Hb. Conc. in group C (g/dl)

Hb Conc. in group D (g/dl)

1 7.96+ 0.20 7.84+ 0.26 7.50+ 0.30* 7.46+ 0.30* 2 7.96+ 0.20 7.74+ 0.21* 7.44+ 0.30* 7.38+ 0.23* 3 7.96+ 0.20 7.48+ 0.32* 7.36+ 0.18* 7.12+ 0.28* 4 7.96+ 0.20 7.38+ 0.25* 7.12+ 0.28* 6. 68 + 0.26*

Vol. 56 (1&2) Effect of Acute Fluoride Intoxication

127

REFERENCES

Abida, F., Harue, M., Minoru, K., Muhammad, N., & Nousheen, F., 2007 Distribution of highly arsenic and fluoride contaminated groundwater from east Punjab, Pakistan, and the controlling role of anthropogenic pollutants in the natural hydrological cycle. Geochem. J., 41: 213- 234.

Al-Akel, A. S., Shamsi, M. J., Al-Kahem, H. F., & Ahmad, Z., 1998. Effect of cadmium on chichild fish, Oreochromis niloticus; Behavioral & Physiologic response. J. Uni. Kuwait (Sci.), 15: 341-346.

Ali, M., 1990. Internal Medicine, 2nd

edition. Ilmi Kitab Khana, Lahore, Pakistan. Anjum, F. & Shakoori, A. R., 1993. Effect of cadmium on the survival, total body

weight, relative liver weight and the liver function enzymes of promethazine- pretreated and phenobarbitone pretreated rabbits. Pak. J. Zool., 25:177-185.

Baselt, R. C., 2008. Disposition of toxic drugs and chemicals in man, 2nd

edition, Foster City (CA): Biomed. Publ. pp. 636–640.

Gosselin, R. E., Smith, R. P., & Hodge, H. C., 1984. Clinical toxicology of commercial products, 5

th edition. 428 East Preston Street, Baltimore, MD

21202: Williams & Wilkins. pp. III-185–93. Hashmi, F., 1999. Hematological & Kidney changes in Labeo rohita following

murcury intoxication. M.Sc. Thesis, Submitted to Zoology Department, Government College, Lahore, Pakistan.

International Programme on Chemical Safety (IPCS), 2002. Environmental health criteria 227 (Fluoride). Geneva: International Programme on Chemical Safety, WHO., pp. 100.

Kumar, G. P. S., Arulselvan, P., Kumar, D. S., & Subramanian, S. P., 2006. Anti-Diabetic activity of fruits of Terminalia chebula on Streptozotocin induced Diabetic rats, J Heal. Sc., 52: 283-291.

Li, X. S., Zhi, J.L. & Gao, R.O., 1995. Effect of Fluoride Exposure on Intelligence in Children. Fluor., 28: 189-192.

Phyllis, J. M. 1995. Neurotoxicity of sodium fluoride in rats. Neurotox. & Terat., 17:169-177.

Schmidt, C.W. & Leuschke, W., 1990. Fluoride content of deciduous teeth after regular intake of black tea. Dtsch Stomatol., 40: 441-442.

Sokal, R. R. & Rohfl, F. J. 1995. Biometry: The principles and practice of statistics in biological research. 3

rd edition. W. H. Freeman, New York.

Susheela, A. K. & Jain, S. K., 1983. Fluoride-induced haematological changes in rabbits. Bull. Environ. Contam. & Toxi., 30: 388-93.


PK ISSN 0006 – 3096

Clerodendrum splendens G. Don., of Family Verbenaceae:

An addition to the flora of Pakistan

ZAHEER-UD-DIN KHAN & MUHAMMAD AJAIB


ABSTRACT

In the Botanic Garden GC University, Lahore, as well as in parks and gardens

throughout Punjab Province a woody climber introduced long ago was as Clerodendrum splendens G. Don (Bleeding Heart Vine) of family Verbenaceae. The plant voucher specimen was preserved in Dr. Sultan Ahmad Herbarium, GCU Lahore, Pakistan for reference. This climber has not been reported in Flora of Pakistan, therefore the present report is an addition into it.

Key words: Clerodendrum splendens, Flora of Pakistan, Bleeding Heart Vine

INTRODUCTION

The genus Clerodendrum of family Verbenaceae was first described by Linnaeus during 1953 in Genera Plantarum (Jafri & Ghafoor 1974). Clerodendrum splendens G. Don is the native to Western Africa and is widely distributed in west and central Africa from Senegal to Angola and Dem. Rep. Congo. It is cultivated as an ornamental climber elsewhere in tropics and sub-tropics and recorded as naturalized in Dem. Rep. Congo and Angola (Fernandes 2005).

Clerodendrum splendens G. Don commonly called Bleeding Heart Vine, Flaming Glorybower, Pagoda Flower) is found growing well in Lahore, the Capital of Punjab and the popular city of Pakistan, situated along the left bank of river Ravi and is greatly expanding due to profuse urbanization, high population growth rate and emigration.

Climatically Lahore falls in subtropical arid type in which summers are really hot and the winters are mild cold (Anonymous, 2009).

Clerodendrum splendens G. Don was introduced as an ornamental climber in Pakistan and it adapted well in this climate. This species has not been reported by Parker (1956), Chaudhary (1969), Stewart (1972) and Jafri & Ghafoor (1974) in Flora of Pakistan. Description to the species after Fernandes (2005):

Clerodendrum splendens G. Don in Edinburgh New Philos. J. 11: 349 (1824). —J.G. Baker in F.T.A. 5: 300 & 517 (1900). —R. Good in J. Bot. 68, Suppl. 2 (Gamopet.): 141 (1930). —Hutchinson & Dalziel, F.W.T.A. 2: 275 (1931). —Thomas in Bot. Jahrb. Syst. 68: 56 (1936). —Huber in F.W.T.A., ed. 2, 2: 444 (1963). —H. Moldenke & A. Moldenke in Rev. Fl. Ceylon 4: 435 (1983). —Verdcourt in F.T.E.A., Verbenaceae: 87 (1992). —R. Fernandes in C.F.A., Verbenaceae. Type from Sierra Leone.

Z. KHAN & M. AJAIB BIOLOGIA (PAKISTAN)

130

An evergreen densely branched climber or sarmentose vine or running shrub up to 5m high with minutely puberulous branchlets; main branches long and flexuose. Leaves upto 18cm long and 12 cm broad, ovate-oblong to broadly ovate to suborbicular or lanceolate, slightly acuminate, usually rounded at the base, entire with undulate margins, glabrous, glacous, dark green above and paler or greyish-green below, nerves prominent; petiole upto 2.7 cm long. Flowers scarlet in many-flowered, axillary and terminal corymbose cymes; peduncles stout, upto 9 cm long; pedicels 0.3–1.3cm long; Calyx, sepal lobed, less than 1cm long, purplish; corolla tube upto 2cm long, ; stamens exerted upto 3cm; drupes, ovoid, black when mature, glabrous, 1.5 cm long (Pl. 1). Syn.: Clerodendrum aurantium G. Don, C. botryodes, C. gilettii De Wild. & T. Durand C. splendens var. gilettii (De Wild. & T. Durand) B. Thomas, Sphinanthus splendens (G. Don) Hiern.

Vern. Bleeding Heart Vine, Flaming Glorybower, Pagoda Flower. Fl.Per. Feb.-April. Voucher No. SAH. 780.

Ethnobotany: According to Jiofack et al. (2009) the plant is used to treat yellow fever and panacea of witchcraft.

Plate 1: Clerodendrum splendens G. Don in natural habitat

REFERENCES

Anonymous, 2009. Annual rainfall, relative humidity and temperature of Lahore 2004-2009. Pakistan Metrological Department Jail Road Lahore, Pakistan.

Vol. 56 (1&2) Clerodendrum splendens G. Don

131

Chaudhary, S.A., 1969. Flora in Layallpur and adjacent Canal Colony. W. Pak. Agricultural University Lyallpur.

Fernandes, R., 2005. Flora Zambesiaca. Lamiaceae. Vol. 8, part 7.

Jafri, S.M.H. & Ghafoor, A., 1974. Flora of West Pakista. No. 77. Verbenaceae. E. Nasir and S.I.Ali (eds.) Department of Botany, University of Karachi, Karachi.

Jiofack, T., Ayissi, I., Fokunang, C., Guedje, N., & Kemeuze, V., 2009. Ethnobotany and Phytomedicine of the upper Nyong Valley Forest in Cameroon. African J. Pharmacy and Pharmacology, 3(4). 144-150.

Parker, R.N., 1956. A Forest Flora for the Punjab with Hazara and Delhi, Ed. 3. Superintendent, Govt. printing press Lahore.

Stewart, R.R., 1972. An Annotated Catalogue of the Vascular Plants of West Pakistan and Kashmir. Flora of West Pakistan E. Nasir and S.I. Ali,

(eds.) Fakhri Printing Press, Karachi.


PK ISSN 0006 – 3096

Melochia corchorifolia L. of family Sterculiaceae: An

addition to the flora of Pakistan

MUHAMMAD AJAIB & ZAHEER-UD-DIN KHAN


ABSTRACT

During the field survey of District Kotli, Azad Jammu & Kashmir as a part

of PhD studies a naturalized herbaceous weed found growing in corn (Zea mays L.) fields was identified as Melochia corchorifolia L. (Chocolate weed) of family Sterculiaceae. The inhabitants of the area reported that the weed was growing for more than 50 years and has ethnobotancal significance. The voucher plant specimen was deposited in Dr. Sultan Ahmad Herbarium, GCU Lahore, Pakistan. The present report on this weed is an addition to the Flora of Pakistan, as it was not reported previously.

Key words: Melochia corchorifolia, Chocolate weed, Family Sterculiaceae

District Kotli, AJK

INTRODUCTION

The genus Melochia of family Verbenaceae was first described by Linnaeus during 1953 in Genera Plantarum (Abedin & Ghafoor 1976). Melochia corchorifolia L. is the native to Americas but naturalised in tropical Africa, Madagascar, south, south east and east Asia, Malesia, New Guinea, northern Australia and some Pacific islands (Goldberg 1967). The chromosomal count no. is 2n = 36 (Matoba & Uchiyama 2009).

District Kotli, Azad Jammu & Kashmir is divided into Kotli, Sehnsa, Khuiratta, Fatehpur and Nakiyal Tehsils. The area is rich in plant diversity and represents sub-tropical Pine vegetation and Melochia corchorifolia is found in and near corn fields of plains in throughout the District. The District is bounded on the eastern side by Occupied Kashmir, Western side by Rawalpindi (Pakistan), Southern side by Mirpur and Northern side by District Poonch its population is 0.558 million, according to census 1998. Its area is 1862sq.km. The

investigated area lies between longitude 73 6′ to 74

7′ East and latitude 33

20′

to 33 40′ North (Topo sheet No. 43G /15). The annual rainfall of District Kotli is

1227.91 mm, maximum during July to August, i.e. 306.93 mm and 256.53 mm, respectively, while low during winter. Thus average monthly rainfall is 102.32 mm. Humidity is low during the day time as compared to night. January, February, August and September are more humid months than May and June (Ajaib et.al., 2010).


134

DESCRIPTION OF THE SPECIES Melochia corchorifolia L. (Syn.: Melochia affinis Wall.) A coarse, erect or decumbent herb or subshrub upto 1m high with fistular stem. Leaves ovate-oblong, petiolate, pubescent with stellate hairs beneath, serrate, acute, upto 8cm long, upto 4cm broad, 5-nerved at base. Flowers white, pink or yellowish in congested terminal clusters, bracteate; bracts ciliate, linear; calyx pale green, united; corolla usually white or pink, petals spathulate; stamen 5-10, isomerous with perianth; anthers yellow, bilocular, dehiscing via longitudinal slits; carpel 5, syncarpous, isomerous with perianth; pistil 5-celled; ovary superior, pubescent 5-locular; ovules 2 in each locule; style 5, free; stigma 5, united at the base; placentation axile; capsule upto 6mm long, dehiscent, septicidal, black or purplish 5mm in diameter; seeds black (Pl. 1).

Fairly common in corn fields during rainy season. Fl. Per. July-September. Vern. Dhamni, Bundava, Chocolate weed. Voucher No. SAH 1113. Melochia corchorifolia L. is looking natural and adapted well in this climate. This species has not been reported by Parker (1956), Chaudhary (1969), Stewart (1972) and Abedin & Ghafoor (1976) in Flora of Pakistan. Ethnobotany. It is used as a fodder for cattle. Leaves are sometime used as vegetable. Fruit powder is used for ear problems, anthelmintic, dysentery, abdominal swellings and snake bites.

a b

Plate 1. Melochia corchorifolia L. a. Plant in natural habitat b. Flowers and Fruit

Vol. 56 (1&2) Melochia corchorifolia L. 135

REFERENCES

Abedin, S. & Ghafoor, A., 1976. Flora of West Pakistan. No. 99. Sterculiaceae. E. Nasir and S.I.Ali (eds.) Department of Botany, University of Karachi, Karachi.

Ajaib, M., Khan, Z., Khan, N. & Wahab, M., 2010. Ethnobotanical Studies on useful Shrubs of District Kotli, Azad Jammu & Kashmir, Pakistan. Pak. J. Bot., 42(3):1407-1415.

Chaudhary, S.A., 1969. Flora in Layallpur and adjacent Canal Colony. W. Pak. Agricultural University Lyallpur.

Goldberg, A., 1967. Genus Melochia (Sterculiaceae), Contr. U.S. Nat. Herb, 34(5): 191-363. Matoba, H. & Uchiyama, H., 2009. Chromosome Counts of Aquatic and Wetland

Plants Growing in Acid Sulfate Soil from the Central Plain,Thailand. The J. Japanese Bot., 84(5): 313-320.

Parker, R.N., 1956. A Forest Flora for the Punjab with Hazara and Delhi, Ed. 3. Superintendent, Govt. printing press Lahore.

Stewart, R.R., 1972. An Annotated Catalogue of the Vascular Plants of West Pakistan and Kashmir. Flora of West Pakistan E. Nasir and S.I. Ali, (eds.) Fakhri Printing Press, Karachi.

http://www.abebooks.co.uk/book-search/author/ABEDIN,-S-GHAFOOR,-A

http://www.botanicus.org/page/399293


Bacteriological quality of bottled water brands in Karachi,

Pakistan

AMNA KHATOON & ZAID AHMED PIRZADA

Department of Microbiology, University of Karachi, Karachi-75270, Pakistan

ABSTRACT

In the present study, bacteriological quality of 187 different mineral water bottle brands marketed in Karachi was analyzed from September 2009 to June 2010. All the collected samples were analyzed for the presence of coliform bacteria, fecal coliform bacteria, E. coli, fecal entrococci, P. aeruginosa and total viable plate count (TVPC). Alarmingly, 67 (36%) of these samples did not comply with Pakistan and WHO standard guidelines for drinking water. The majority of the specimens (39 samples) were contaminated with higher TVPC and P. aeruginosa, while many were contaminated with both coliform (20 samples) and fecal coliform bacteria (11 samples). Total heterotrophic bacterial number in 49 samples was higher than even 200 cfu/mL. The presence of coliform bacteria in drinking water suggests the possible presence of pathogenic enteric microorganisms thus unsafe for drinking. The data presented here clearly raise the concerns regarding the quality of drinking mineral water and highlights the danger posed to the public health. Key words: Bottled, water, coliform, bacteria, contamination

INTRODUCTION

Water is an essential requirement of all life forms. Satisfactory supply of

clean, safe and hygienic water is imperative for health. Drinking unsafe and unhygienic water can cause high prevalence of waterborne diseases like diarrhoea, typhoid and cholera (Oyedeji et al., 2010, Fawell & Nieuwenhuijsen, 2003). As a preventive measure, consumption of bottled water has increased in recent years in developing countries and elsewhere. Sale of bottled water has gone to more than 35 billion US dollars (Raj, 2005) and by an average of 12% increase per year the world over (El-Salam et al., 2008). People from all over the world drink about 13x10

10 liters of bottled water annually (Samadi et al., 2009). In

Pakistan the consumption of bottled water increased from 5.9% to 9.5% during 2003-2007 (Samadi et al., 2009).

In general people are concerned about the poor quality of tap water that is why they have switched over to bottled water perceiving it to be clean and safe. Although, people consider bottled water safe but it can be also be contaminated with chemical and biological agents (Khaniki et al., 2010). Presence of coliform bacteria, E coli or Pseudomonas in bottled water can pose a great threat to the public health. Infants, young children, debilitated and immuno-compromised people are at high risk of waterborne diseases, even at lower infective doses (WHO, 2005).

According to world health organization (WHO) recommendations, potable water should have <20 cfu/mL heterotrophic bacterial count with

138 A. KHATOON & A. Z. PIRZADA BIOLOGIA (PAKISTAN)

complete absence of coliform bacteria, fecal coliforms, E. coli, enterococci and Pseudomonas aeruginosa. Although, coliform organisms may not always be considered as indicator of fecal contamination but their presence in drinking water suggests the potential presence of pathogenic enteric microorganisms such as Salmonella spp., Shigella spp. and Vibrio cholera. Pseudomonas aeruginosa is an opportunistic pathogen causing a variety of infections like urinary and respiratory tract infections, particularly in patients who are severely immuno-compromised (Zamberlan da Silva et al., 2008). Presence of P. aeruginosa is a great risk to human health because Pseudomonas species are multi drug resistant organisms having the ability to transfer its resistance genes to other bacteria in the human body.

The main objectives of the current study are to analyze the bacteriological quality and bacterial contamination of bottled mineral water from Karachi markets and to check their compliance with the standards.


Sample collection: A total of 187 different local brands of commercially available bottled water were collected randomly from supermarkets and various shops of Karachi from September 2009 to June 2010. Bacteriological analysis of all the samples was conducted within 2 hours of collection. Sample analysis: All the collected water samples were analyzed for the following parameters according to WHO and Pakistan standard guidelines and as described previously (Liguori et al., 2010): Total viable plate count: Total viable plate count (TVPC) was performed by pour plate technique. Bottled water samples were mixed in R2A media (Merck) and incubated at 37

oC for 48 hours and at 22

oC for 72 hours for thermophilic and

psychrophilic bacteria respectively. After incubation colonies were counted with the help of colony counter. Coliform bacteria: Membrane filter technique was performed to isolate the coliform bacteria. Briefly, 250mL of each sample was filtered through 0.45 µm pore size cellulose nitrate membrane filter (Sartorius) and placed on Tergitol 7 TTC agar (Merck) and plates were incubated at 37

oC for 18-24 hours. Plates

were checked for the presence of coliform bacteria by routine diagnostic tests like TSI, Citrate, SIM. Escherichia coli: E. coli was also detected from the same plates that were used for the isolation of coliform bacteria. Identification of E coli was done by routine diagnostic tests like TSI, Citrate, SIM. Fecal coliforms: 250 mL of each sample was filtered through 0.45 µm cellulose nitrate membrane filter. Filter membrane was placed on MFC agar (Merck) & plates were incubated at 44.5

oC for 18-24 hours. After incubation plates were

observed for dark blue colored colonies. Fecal coliform bacteria were confirmed by the gas formation in EC broth (Merck) incubated at 44.5

oC for 18-24 hours.

Fecal enterococci: 250 ml of each sample was filtered through 0.45 µm cellulose nitrate membrane filter which was then placed on Slanetz-Bartley agar (Merck) and plates were incubated at 37

oC for 48 hours. After incubation plates

were observed for typical colonies. Isolated colonies were confirmed as fecal enterococci by bile esculin azide test.

Vol. 56 (1&2) Bacteriological quality of bottled water 139

Pseudomonas aeruginosa: Similarly, 250 ml of each sample was filtered through 0.45 µm cellulose nitrate membrane filter and then membrane was placed on Pseudomonas CN agar (Citrimide-Nalidixic agar; Merck) and plates were incubated at 37

oC for 24-48 hours. Identification of P. aeruginosa was done

by routine diagnostic tests like TSI, Citrate, SIM and oxidase test.


Karachi city is a highly urbanized area of Pakistan, where approximately 200 brands of bottled water are marketed. To the best of our knowledge, very few studies have been conducted to analyze the quality of bottled mineral water from Pakistan. Therefore this study was undertaken to analyze the bacteriological quality of bottled water from Karachi markets and to check their compliance with the standards. According to the guidelines of national and international recommendations, all the specimens were checked for the following parameters: TVPC at 22

oC or 37

oC, presence of coliform bacteria, fecal

coliforms, E. coli, fecal enterococci and P. aeruginosa. In the present study the bacteriological examination of 187 different

bottled mineral water samples was carried out. Alarmingly, 67 (36%; figure 1) of these samples did not comply with the standard guidelines of Pakistan and WHO for drinking water. In these specimens TVPC at 37

oC was found higher than the

recommended range which is <20 cfu/mL (Figure 2). TVPC was found even higher than 200 cfu/mL in 49 (26%) mineral water samples (Table 1). TVPC count shows the presence of heterotrophic bacteria in the water samples, which indicates the bacterial pollution of drinking water. Although heterotrophic bacteria are themselves non pathogenic but there are chances that the higher heterotrophic bacterial count is associated with the presence of coliform bacteria or other pathogens, as indicated in our results. Out of 67 non-complied samples only 8 failed due to higher TVPC count parameter alone, while 39 samples failed because TVPC was associated with P. aeruginosa. Whereas, rests of the samples failed because higher TVPC was associated with P. aeruginosa, coliform and E coli in different combinations as indicated in figure 2.

Shockingly, 20 (11%; figure 2) mineral water bottles were contaminated with coliform bacteria. Although all the analyzed bottled samples were negative for fecal coliform and fecal enterococci but 11 (6%; figure 2) of these were found positive for E coli, an indicator of fecal contamination. The presence of coliforms and E coli in bottled water samples not only indicates the potential presence of pathogenic enteric microorganisms but also questions the efficiency and integrity of production system. It is therefore recommended to monitor the complete water processing system to avoid major public health problems.

Yet another point of concern was the presence of P. aeruginosa in 45 (24%; figure 2) mineral water samples. In this study, the main reason for the non-compliance of the majority of the mineral water samples to standards was that these were contaminated with P. aeruginosa along with higher TVPC (Figure 3). One of the main reasons for P aeruginosa isolation in high percentage was that it can survive longer in low nutrient environment by slowing down its metabolic activity and its resistance against disinfectants commonly used for the treatment of water (Mena & Gerba, 2009). Presence of P. aeruginosa in mineral water is


considered as an indicator of the quality of water. In a recent study, majority of Pseudomonas spp. isolated from bottled mineral water were found to be multi drug resistant (Daood, 2009), which can cause significant health hazards to the consumers.

Different studies have shown that bottled drinking water is not always safe (Venieri et al., 2006, Zeenat et al., 2009). In India 40% of the bottled drinking water samples were failed in TVPC parameter and most of the isolated were highly resistant to many antibiotics (Jeena et al., 2006). In a study conducted in Brazil, 20 liters mineral water bottles were found contaminated with coliform bacteria, fecal coliform and P. aeruginosa (Zamberlan da Silva et al., 2008). Many outbreaks have also been resulted due to contaminated bottled water (Reynolds et al., 2008). CDC reported in 1994 that Cholera out break in US was associated with bottled water (El-Salam et al., 2008). Similarly, bottled water was reported to be one of the vehicles for Vibrio cholera transmission in Portugal (Addo et al., 2009). According to a previous study in Pakistan the majority of the mineral water samples were contaminated with P. aeruginosa and E. coli (Taj & Baqai, 2007).

Contaminated mineral water poses great health risk especially when it is used by elderly persons, infants, hospitalized patients and immuno-compromised ones (Leclerc et al., 2002). There can be many reasons for the bacterial contamination like improper disinfection, infiltration of contaminated water, leakage points. Furthermore, the improper storage of the products provides favorable conditions for the bacteria to grow up to harmful levels. Therefore, a regular checking of mineral water bottles is necessary to prevent the spread of diseases and to keep vigilance on quality of water available in the market. In addition, studies about the chemical analysis of these brands are equally important. The data presented here clearly raise the concerns regarding the quality of drinking mineral water; danger posed to public health and highlights the importance of a continued surveillance system to minimize the chances of contamination in the mineral water.

Table 1: Total viable plate count (TVPC) denoted in cfu/mL in

mineral water samples.

TVPC range (cfu/mL) Number of samples Percentage

<1 75 40%

15-20 45 24%

20-200 18 10%

200-300 25 13%

>300 24 13%


Compliance with Pakistan and WHO

standards

64%

36%

Compliance with standard

Does not compliance withstandard

Fig. 1: Total number of samples having compliance and non-

compliance with Pakistan and WHO standards.

Compliance with the basic parameters

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Fail 35.82% 35.29% 24.06% 10.69% 5.88% 0% 0.00%

Pass 64.10% 64.70% 75.93% 89.30% 94.12% 100% 100.00%

T.V.P.C at 37o C T.V.P.C at 22o CPseudomonas

aeruginosaColiform E.coli Fecal Coliform

Fecal

Enterococci

Fig. 2: Pass and fail ratio of all the parameters used to analyze the quality

of mineral water samples.

Total no of Samples 187 100 %

Complied with the standards 120 64 %

Not complied with the standards 67 36 %


Profile of samples fail in different parameters

Coliform,

E.coli,

P. aeruginosa

and TVPC (02)

Coliform,

P. aeruginosa

and TVPC (04)

Coliform,

E.coli and

TVPC (09)

P. aeruginosa

and TVPC (39)

Coliform and

TVPC (05)

TVPC (08)

Fig. 3: Profile of samples failing in different parameters.

REFERENCES

Addo, K.K., Mensah, G. I., Donkor, B., Bonsu, C. & Akyeh, M. L., 2009.

Bacteriological quality of bottled water sold on the Ghanaian market. African journal of food agriculture nutrition and development, 9: 1378-1387.

Daood, N., 2009. Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water Consumed in Syria. Damascus university journal for basic sciences, 25: 21-42.

El-Salam, A.M.M., Al-Ghitany, E.M. & Kassem, M.M., 2008. Quality of Bottled Water Brands in Egypt Part II: Biological Water Examination. J. Egypt Public Health Assoc., 83: 468-486.

Fawell, J. & Nieuwenhuijsen, M.J., 2003. Contaminants in drinking water. Br Med Bull 68: 199-208.

Jeena, M.I., Deepa, P., Mujeeb, R.K.M., Shanthi R.T. & Hatha, A.A., 2006. Risk assessment of heterotrophic bacteria from bottled drinking water sold in Indian markets. Int. J. Hyg. Environ. Health, 209: 191-196.

Khaniki, G.R.J., Zarei, A., Kamkar, A., Fazlzaddehdavil, M., Ghaderpoori, M. & Zarei, A., 2010. Bacteriological evaluation of bottled water from domestic brands in Tehran markets, Iran. World Applied Sciences Journal, 8: 274-278.

Leclerc, H., Schwartzbrod, L. & Dei-Cas, E., 2002. Microbial agents associated with waterborne diseases. Crit. Rev. Microbiol., 28: 371-409.


Liguori, G., Cavallotti, I., Arnese, A., Amiranda, C., Anastasi, D. & Angelillo, I.F., 2010. Microbiological quality of drinking water from dispensers in Italy. BMC Microbiol., 10: 19.

Mena, K. D. & Gerba, C.P., 2009. Risk assessment of Pseudomonas aeruginosa in water. Rev. Environ. Contam. Toxicol., 201: 71-115.

Oyedeji, O., Olutiola, P.O. & Moninuola, M.A., 2010. Microbiological quality of packaged drinking water brands marketed in Ibadan metropolis and Ile-Ife city in South Western Nigeria. African Journal of Microbiology Research, 4: 96-102.

Raj, S. D., 2005. Bottled water: how safe is it? Water Environ. Res., 77: 3013-3018.

Reynolds, K. A., Mena, K.D. & Gerba, C.P., 2008. Risk of waterborne illness via drinking water in the United States. Rev. Environ. Contam. Toxicol., 192: 117-158.

Samadi, M.T., Rahmani, A.R., Sedehi M. & Sonboli, N., 2009. Evaluation of Chemical Quality in 17 Brands of Iranian Bottled Drinking Waters. J. Res. Health Sci. 9: 25-31.

Taj, A. & Baqai, R., 2007. Antimicrobial effects of Alum and Sulphur on bacteria isolated from Mineral and Hospital water. Infectious Diseases Journal, 16: 10-13.

Venieri, D., Vantarakis, A., Komninou G. & Papapetropoulou, M., 2006. Microbiological evaluation of bottled non-carbonated ("still") water from domestic brands in Greece. Int. J. Food Microbiol., 107: 68-72.

WHO, 2005. Guidelines for drinking water quality. Health criteria and other supporting information. 2 Geneva

Zamberlan da Silva, M.E., Santana, R.G., Guilhermetti, M., Filho, I.C., Endo, E.H., Ueda-Nakamura, T., Nakamura, C.V. & Dias Filho, B.P., 2008. Comparison of the bacteriological quality of tap water and bottled mineral water. Int. J. Hyg. Environ. Health, 211: 504-509.

Zeenat, A., Hatha, A.A., Viola, L. & Vipra, K., 2009. Bacteriological quality and risk assessment of the imported and domestic bottled mineral water sold in Fiji. J. Water Health, 7: 642-649.

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