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DIAGNOSIS AND THERAPEUTIC MANAGEMENT OF KETOSIS IN BUFFALOES

THESIS

Submitted to the

Nanaji Deshmukh Veterinary Science University

Jabalpur

In partial fulfillment of the requirement for

the Degree of

MASTER OF VETERINARY SCIENCE

In

VETERINARY MEDICINE

by

Priya Waliya I.D. No. VP/JB/64/2010

Department of Veterinary Medicine College of Veterinary Science and Animal Husbandry

Nanaji Deshmukh Veterinary Science University, Jabalpur (M.P.)

2017

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CERTIFICATE – I

This is to certify that the thesis entitled “DIAGNOSIS AND

THERAPEUTIC MANAGEMENT OF KETOSIS IN BUFFALOES” submitted

in partial fulfillment of the requirements for the degree of MASTER OF

VETERINARY SCIENCE in VETERINARY MEDICINE of Nanaji Deshmukh

Veterinary Science University, Jabalpur is a record of the bonafide research

work carried out by PRIYA WALIYA under my guidance and supervision. The

subject of the thesis has been approved by the Student’s Advisory Committee

and the Director Instructions.

No part of the thesis has been submitted/published for any other

degree or diploma program. All the assistance and help received during the

course of the investigation has been acknowledged by her.

Place: Jabalpur Dr. Devendra Kumar Gupta

Date: / /2017 Chairman of the Advisory Committee

THESIS APPROVED BY THE STUDENT’S ADVISORY COMMITTEE

Chairman : Dr. Devendra Kumar Gupta ………..…………………………

Member : Dr. Arun Mourya ………………………………….

Member : Dr. R.P.S. Baghel ………………………………….

Member : Dr. (Mrs.) Varsha Sharma ………………………………….

Head of the Department

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CERTIFICATE – II

This is to certify that the thesis entitled “DIAGNOSIS AND

THERAPEUTIC MANAGEMENT OF KETOSIS IN BUFFALOES” submitted

by PRIYA WALIYA to the Nanaji Deshmukh Veterinary Science University,

Jabalpur in partial fulfillment of the requirements for the degree of MASTER

OF VETERINARY SCIENCE in the department of VETERINARY MEDICINE

after evaluation by the External Examiner and an oral examination, is hereby

approved by the External Examiner and Student’s Advisory Committee.

Place: Jabalpur External Examiner

Date: / /2017

MEMBERS OF THE ADVISORY COMMITTEE

Chairman : Dr. Devendra Kumar Gupta ………..…………………………

Member : Dr. Arun Mourya ……………….…………………

Member : Dr. R.P.S. Baghel ………………………………….

Member : Dr. (Mrs.) Varsha Sharma ………………………………….

Head of the Department: Dr. P.C. Shukla ………………………………….

Director Instructions : Dr. S.K. Joshi ………………………………….

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1. INTRODUCTION

India ranks first in milk production and buffalo population in

world. Total buffalo population in India is 511.05 million (Livestock Census,

2012). Buffalo milk contributes 55% of the total milk production in India. There

are many factors which reduces the milk production. During the transition

periods high milk yielders are always at verge of higher risk, having

production disorders. Production diseases are mainly man made problems,

which occupy the most key place among the diseases of dairy animals as it

directly or indirectly affect the economy of dairy farmers and ultimately dairy

farmers suffer from huge financial losses due to drastic decrease in milk

production. Among that, ketosis is very common and unaddressed, clinical

condition.

Ketosis is a common multifactorial disorder of energy

metabolism that is characterised by elevated concentrations of ketone bodies

in blood (ketonemia), urine (ketouria) and milk (ketolactia). The disease

mainly occurs in early lactation when body reserves are used to support

lactation (Constable et al., 2017). During the early post partum period, milk

production increases dramatically, while energy intake may not be adequate

to sustain the higher production level. This result in negative energy balance

and buffalo metabolize fat to meet their energy needs. It is clinically

characterized by decrease in feed intake, weight loss, drop in milk yield,

acetone/sweet smell in breath, depression and occasionally nervous signs.

Senthilkumar et al. (2015) conducted a survey on economic losses due to

ketosis in dairy farms and they extrapolated that the total economic loss in

Tamil Nadu state due to ketosis would be rupees 21.33 and 2.64 crores in

cows and buffaloes, respectively. About 90% of the ketosis cases are

diagnosed within the first two months after calving and prevalence of ketosis

are peaked in the 2nd or 3rd weak after calving (Geishauser et al., 2000).

A variety of cow side tests are available for monitoring of ketosis

in dairy herd. Various thresholds of beta hydroxybutyric acid (BHBA),

acetoacetate and acetone in blood, milk and urine have been used to define

ketosis. Blood BHBA concentration is currently the gold standard test than the

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acetone or acetoacetate (Tyopponen and Kauppinen, 1980) but this test is

costly compared with other cowside tests. BHBA in blood ranged from 1000 to

1400 µmol/L. Cows with more than 1600 µmol/L BHBA produced 1.8 kg less

milk per day. Cows with more than 1800 µmol/L BHBA produces 3 kg less

milk per day, and with > 2000 µmol/L, produces 4 kg less milk per day

(Duffield, 1997).

The treatment of ketosis requires constant monitoring of blood

glucose and ketone bodies level to achieve desired results. Sometime the

response is only transient if the treatment is discontinued and recurrence can

take place. There are enormous possibilities for ketosis therapy in the

literature. These includes, Dextrose 50%, continuous dextrose infusion, xylitol,

vitamin B12, glutamine, corticosteroids, propylene glycol, glycerol, calcium

propionate, ketamalt, insulin, chloral hydrate, niacin, rumen protected choline,

glucagon, etc. among these, the most rational and currently being used are

dextrose 50%, corticosteroids, propylene glycol and insulin. The proposed

findings will be a step forward for landless, marginal dairy owners in India in

uplifting their economic output through effective and compliant therapeutic

interventions.

Keeping the above facts in view, the present study was under

taken with the following objectives;

OBJECTIVES

1. To study the occurrence of ketosis in lactating buffaloes in Jabalpur

region.

2. To compare the efficacy of different therapeutic protocols for ketosis in

lactating buffaloes.

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2. REVIEW OF LITERATURE

In a dairy farming, production diseases are of great concern to

dairy producers worldwide. Ketosis is a common metabolic disorder in high

yielders. Ketosis or acetonemia is an increase of "ketone bodies" (acetone,

acetoacetate and beta hydroxybutyric acid, subsequently referred to as

ketones) in blood until they eventually begin to spill over into urine and milk.

Ketosis in dairy cattle occurs due to a period of negative energy balance

(NEB) that occurs almost universally at the beginning of lactation.

The detailed review of literature related to various aspects of

ketosis in buffaloes is presented in the following sub-headings;

1. Epidemiology

2. Clinical Parameters and Symptoms

3. Biochemical Parameters

4. Therapeutic Measures

2.1 EPIDEMIOLOGY

2.1.1 Occurrence

Rautmare and Anantwar (1993) tested 600 recently calved

buffaloes for clinical ketosis, out of these, 17 (2.83%) were found positive and

reported highest occurrence of clinical ketosis in 8-9 years old buffaloes

(58.82%) in the fourth lactation (41.17%). The disease occurred with greater

frequency during 0-1 month (35.29%) post partum.

Duffield (1997) studied 1333 dairy cows in various stages of

lactation and parity and found highest prevalence of ketosis for cows in early

lactation [(<65 days in milk (DIM)] was 14.1%. However, prevalence for mid

lactation (65-149 DIM), late (>149 DIM) and dry cows were 5.3%, 3.2% and

1.6% respectively.

Gupta (2012) conducted an epidemiological and therapeutic

study of subclinical ketosis in 200 lactating buffaloes of Jabalpur region of

Madhya Pradesh and found higher incidence of subclinical ketosis at 20th day

postpartum and in 5th parity.

Kumar et al. (2015) screened 145 buffaloes, suspected for

ketosis and recorded overall occurrence 16.55% and they found most of the

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cases in the third lactation, within the 2 months after parturition in the age

group of 3-9 years (average approximately 6 years). Only one animal was in

advance pregnancy with characteristic clinical signs and no variability in vital

parameters.

Vanholder et al. (2015) reported that risk for developing

subclinical ketosis was higher in the second parity and older cows compared

with heifers, whereas for clinical ketosis only, parity ≥3 cows had a higher risk.

Biswal et al. (2016) reported the prevalence rate of ketosis

36.7% (1014/2760); entailing 27.2% in clinical ketosis and 9.6% in subclinical

ketosis in Orisha state. The age-wise prevalence rate was found to be the

highest (40.8%) in the age group of 5.5-6.5 years (4th calver). The season

wise prevalence rate in 5th calver was recorded to be the highest (38.6%) in

summer season as compared to other seasons. The prevalence of ketosis

was observed to be the highest at 56.7% on the first stage of lactation at the

1st month after 2 weeks.

Sharma and Kumar (2016) studied 294 post parturient cows and

reported 10.20% prevalence of ketosis among the suspected clinical cases.

Maximum cases of ketosis were recorded during 1-2 months post-partum, 8-9

years of age and at fourth lactation.

2.1.2 CLINICAL PARAMETERS AND SYMPTOMS

Dohoo and Martin (1984) recorded the cases of cows having

subclinical ketosis, were at increased risk of subsequently developing clinical

ketosis.

Lean et al. (1991) described decrease milk yield, central

nervous excitement, typical weight loss, partial anorexia and selective intake

of forage in preference to concentrates as the major clinical signs of bovine

ketosis.

Venkateshwarlu et al. (1993) selected 313 lactating buffaloes

among three organised dairy farms. These animals were subjected to the

Ross modified Rothera’s test and 46 (14.69%) animals were positive. Clinical

examination of these animals viz. temperature, pulse, respiration and ruminal

movements were recorded. This examination did not reveal any significant

increase in the body temperature, pulse, respiratory rate and ruminal

movement.

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Mir and Malik (2003) emphasized that the clinical signs like

sudden drop in milk yield, selective feeding, wasting and depression were of

diagnostic significance because they were manifested by more than 50%

ketotic animals.

Drackley and Dann (2005) reported that decrease in dry matter

intake around parturition, increase demands for glucose and insufficient

propionate production during the early postpartum period could explain the

occurrence of ketosis at post-partum period.

Teli and Ali (2007) recorded lactation stage wise drop in milk

yield in clinically ketotic buffaloes. The average drop in daily milk yield was

recorded to be 3.52±0.16 litres (36.70%) and the recovery after treatment

could restore only 1.73±0.15 litres (25.30%) milk per day.

Dar et al. (2014) conducted study on 64 ketotic dairy cows of 4-

13 years of age and reported almost normal rectal temperature (101-103ºF),

slightly elevated pulse (60-80/min) and respiratory (30-40/min) rates.

Kumar et al. (2015) reported ketotic buffaloes exhibited clinical

signs such as selective anorexia (refusal to feed on concentrate diet), drastic

reduction in mean value of milk yield (64.4±5.35%), ketotic odour from urine,

breath, and milk and rapid loss of body condition. All the clinical vital

parameters in ketotic buffaloes i.e. body, temperature, heart rate, respiration

rate, and rumen movements were within normal range.

Bali et al. (2016) studied 18 clinical cases of ketosis in buffaloes

and found variable degrees of frequency of occurrences of symptoms with

sudden decline in milk yield being present in 100 percent of the cases. This

was followed by selective feeding (79.14%), wasting (49.91%), depression

(36.67%), complete anorexia (29.37%), acetone smelling breath (19.35%), dry

mucous coated faeces (12.90%), signs of central nervous system involvement

(6%). Rumen motility was as low as 1.77±0.11 against 2.17±0.19 per two

minutes in mild cases.

Vaja et al. (2016) studied a case of 7 years old Gir female cattle

and found normal ranges of rectal temperature (101.3ºF), slightly elevated

pulse (62/min) and respiratory (29/min) rates.

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Constable et al. (2017) opined that many bovines might be in

negative energy balance and ketonuric but may not show clinical symptoms

recognizable by the farmers.

2.3 BIOCHEMICAL PARAMETERS

Annison and Linzell (1963) stated that in a heavy milking

animals 60% to 80% of the blood glucose is utilized by the mammary glands

in the production of milk causing the animals to slip into negative energy

balance.

Emery et al. (1964) reported that Rothera’s test could detect

ketone bodies upto 2 mg % in milk.

Kronfeld (1972) opined that the nitroprusside test gave a purple

colour with acetoacetic acid and acetone but not with beta-hydroxy butyric

acid (BHBA). He further added that the test was sensitive to acetoacetate

(1mg %), acetone (2 mg %) and pyruvate (20 mg %).

Schultz (1974) observed that Rothera’s test on milk and urine

was useful for diagnosis of ketosis. Milk test gave more accurate picture and

became positive when blood ketone reached 10-15 mg%.

Caple (1977) investigated 205 ketotic cows and found blood

acetoacetate concentration in healthy and affected cows were <0.02 and

0.05-1.5 mmol/L respectively. Whereas blood BHBA concentration were 0.4 to

0.8 and 0.8 to10 in healthy and in affected cows, respectively.

Brockman (1979) reported that during early lactation plasma

insulin concentration was lower than at any time during lactation in cows, with

highest blood concentration of ketone bodies and lowest blood glucose. He

further noticed not only insulin concentration was low but also insulin

responsiveness was also impaired.

Anantwar and Singh (1993) found that Rothera test was

sensitive to acetone in urine at 60, 50 and 30 mg% when read 5, 10 and 15

minutes respectively; in the milk at 50, 30 and 20 mg% when read at 7, 10

and 15 minutes, respectively. Acetoacetic acid was sensitive to Rothera’s test

when the content was 50 mg% or more when read at 5, 10 and 15 minutes,

and in milk at 40,30 and 10 mg% when read at 5, 7 and 15 minutes,

respectively. They further added that BHBA did not react to Rothera’s test.

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Venkateshwarlu et al. (1993) selected 313 lactating buffaloes

among three organised dairy farms. These animals were subjected to the

Ross modified Rothera’s test and 46 (14.69%) animals were found positive.

Anantwar and Singh (1995) reported mean value of blood

glucose in clinically ketotic buffaloes (37.89±1.12 mg/dl). The mean blood

glucose in subclinical cases was 62.14±3.13 mg/dl. The mean glucose in

healthy buffaloes was 73.94±3.08 mg/dl.

Gutzwiller (1998) determined BHBA content in milk samples

from healthy, ketotic and high yielding cows using new dipstick semi

quantitative method. BHBA content was also calculated by using an analyzer

in milk samples as well as blood samples drawn at the time of milk sample

collection. He concluded that the dipstick test was suitable for diagnosing

clinical ketosis and for monitoring the energy status of high yielding dairy

cows.

Geishauser et al. (2000) maintained the statement that the semi

quantitative tests detected primarily acetoacetate and to a lesser degree

acetone, without detection of BHBA. The “ketolac BHBA” strip at 50 and 100

µmol of BHBA per litre of milk showed sensitivity of 92 and 72% respectively.

This test was more sensitive for detection of subclinical ketosis than any of

the Rothera’s test. The Rothera’s tests were found to be highly specific

whereas, the “ketolac BHBA” strip at 200, 500 and 1000 µmol of BHBA per

litre of milk with more than 97% specificity.

Ambore et al. (2001) studied biochemical alterations in clinical

ketosis in buffaloes. The study revealed significant decrease in blood glucose

(47.8±0.76 mg/dl) as compared to healthy control group (61.04±0.35 mg/dl).

Mandali et al. (2002) recorded significant decrease in blood

glucose level (40.93±0.85 mg%) and increase in blood ketone bodies (15.43±

0.33 mg%) in clinically ketotic buffaloes as compared with normal buffaloes

58.25±1.32 and 3.29±0.20, respectively.

Sivaraman et al. (2003) found no significant changes at 3 stages

neither in glucose (38.40±2.00, 32.67±1.09 and 36.07±1.09) protein between

30 days prior, at ketosis and 30 days after ketosis stages in crossbred Jersey

cows.

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Oetzel (2004) suggested that the most commonly used cut point

for subclinical ketosis is ≥ 1400 µmol/l of blood BHBA using 10% as the alarm

level for herd based subclinical ketosis testing (minimum 12 cows). Clinical

ketosis generally involves much higher levels of BHBA (3000 µmol /l or more).

Elizabeth (2006) reported that pH of milk and urine decreased

significantly (p<0.05) in clinical as well as in sub clinical ketotic cows.

Teli and Ali (2007) conducted study on 24 clinically ketotic

buffaloes and found that the mean glucose level before treatment was

estimated to 37.68±0.84 mg/dl, whereas insulin concentration estimated to a

mean of 15.82±0.35 µu/ ml. At clinical recovery, elevated values to a mean of

59.00±1.31 mg/dl and 24.37±1.51 µu/ml respectively were estimated. Thus

resulting in a corresponding improvement in the values of both parameters.

Safdar (2015) studied fundamental research in relation to

minimizing adverse effects on metabolic disorders found that most milk

ketone tests measure acetone and acetoacetate through a chemical reaction

with nitroprusside which causes a color change from white to either pink or

purple. These tests in general are poorly sensitive (< 40%) but highly specific

(>90%) in milk.

Constable et al. (2017) reported that blood glucose reduced

from normal levels of 50mg/dl to 20-40mg/dl in ketotic cows.

2.4 THERAPEUTIC MEASURES

Ruckebusch and Noveu (1959) administered long acting insulin

plus corticosteriod in cases of bovine ketosis. They found that the symptoms

of ketosis disappeared within three to four days following parentral

administration of insulin and corticosteroid.

Baird and Heitzman (1969) correlated milk yield and blood level

of glucose after administration of dexamethasone @10 mg/kg intramuscularly.

They found considerable rise in blood glucose concentration in non-pregnant

lactating cows on the first 3 days following treatment but a decline in milk yield

was recorded.

Hamada et al. (1982) reported altered blood glucose level in

spontaneously ketotic cows before and four hours after administration of

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glucose. They found that after 4 hours of intravenous administration of

glucose (250 g), BHBA in plasma decreased significantly.

Rings (1985) reported loss of glucose in urine after infusion of

concentrated glucose solution in cattle. He suggested that moderate rate of

infusion with insulin provided rapid utilization of glucose with speedy recovery.

Benedixen et al. (1987) treated cases of clinical ketosis and

starvation induced ketosis in cows by administration of 25% glucose, glucose

plus insulin, triamcinolone acetonide and reported high efficacy of glucose

plus insulin therapy.

Swain and Tripathy (1987) observed that the blood glucose and

calcium levels varied from 20.5-31.5 mg/dl and 6.8-8.9 mg/dl respectively

indicating hypoglycemia and hypocalcaemia in clinical cases of bovine

ketosis.

Wierda et al. (1987) studied the effects of glucocorticoids

(Dexamedium and Voreen) on milk yield and biochemical parameters in

healthy and ketotic buffaloes. They observed that both the compounds

induced and increased blood glucose concentration and temporarily

decreased the milk yield. The biochemical analysis of blood samples revealed

that after treatment, some animals responded adequately, some inadequately

where in disease relapsed after recovery.

Singh and Kasaralikar (1990) treated clinical bubaline ketosis

with infusion of glucose 20% with a dose rate of 0.5 g/kg body weight (I/v) and

triamcinolone acetonide. Triamcinolone acetonide required 2-3 treatments for

clinical response, whereas, only single treatment was required when glucose

plus insulin was used.

Nisbet and Martin (1991) reported that the higher propionic acid

production is the cause of the increased glucose levels in Saccharomyces

cerevisiae-fed cows.

Chugh et al. (1992) treated the nervous form of ketosis with

20% glucose 500 ml slow I/v and dexamethasone 16 mg I/m daily for 4 days

and found it very effective. All metabolites came in normal range within ten

days, after treatment.

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Montana et al. (1993) reported that treatment with

dexamethasone @ 7.5-10.0 mg/kg body weight intravenously for three

consecutive days was effective against bovine ketosis by aiding

gluconeogenesis in cows.

Sakai et al. (1993) studied simultaneous use of glucose and

insulin in ketotic dairy cows and reported that subcutaneous injection of

200 IU of slow release insulin (SRI) with concomitant infusion of dextrose

enhanced the effectiveness of the treatment for ketosis in cows and shortened

the recovery period. When this treatment compared with infusion of dextrose

alone for four days, resulted in recovery of appetite (88%), urinary pH

recovered (from 6.0 at the initial examination to 7.8). Blood glucose

concentration was increased significantly from 35.2 ± 3.7 mg/dl to 50.5 ± 6.1

mg/dl, blood ketone body concentration was decreased significantly from

120.5 ± 28.0 µg/ml to 22.1±13.1 µg/ml.

Rao and Balakishan (1998) observed that treatment with 20%

dextrose (500 ml I/v), insulin @ 0.5 IU/kg body weight and triamcinolone

acetonide @ 0.05 mg/kg body weight for two days, superior in causing

recovery in ketotic cows.

Gin et al. (1999) reported that on digestibility of starch by alpha-

amylase consequently tannic acid increases the glucose level in blood.

Venkateshwarlu and Choudhuri (2000) studied the therapeutic

efficacy of various regimens in subclinical ketosis in cows and concluded that

parenteral therapy with 540 ml of 25% dextrose for two days was most

efficacious with respect to early recovery time and restoration of milk yield in

comparison to different combinations of sodium propionate, nicotinic acid,

methionine, propylene glycol and jaggery.

Baishya et al. (2002) found earliest restoration of milk yield with

the treatment combination of Fructodex (10% fructose 500 ml I/v for 2 days)

and Vetalog [triamcinoline acetonide (6 mg) @1 ml I/m] followed by dextrose

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(10% 500ml I/v) plus Dexona (dexamethasone sodium phosphate 2-2.5 ml

I/m), in sub clinical ketotic Jersey cows.

Mir and Malik (2003) got least mean recovery time with no

relapses in oral glucose therapy (500 g glucose solution after premedication

with 30 g of sodium bicarbonate) in comparison to parenteral glucose therapy.

Zialitis et al. (2007) described that ten cows with sub clinical

ketosis were treated for 10 days with daily doses of 250 g of propylene glycol

which was fed with the concentrated mixture and ten cows were treated 7

days with daily doses of 12 g nicotinic acid (niacin). Cows following treatment

in both group displayed a negative blood hydroxybutyrate test, reduced milk

fat and hypoglycaemia. After treatment with niacin amount of milk lactose

increased.

Stanislaw and Przemyslaw (2009) recorded supplementation of

Saccharomyces cerevisiae increases serum glucose level and milk production

in animals.

Tufani et al. (2011) conducted study on 40 ketotic cows and

compared efficacy of different therapeutic regimen. T1, T2, T3 and T4 received

1 liter of 25% glucose I/v followed by 500 ml I/v once daily for next 2 days.

Vitamin B Complex (B1, B6 and B12) was added in group T2, T3 and T4 @ 10

ml daily with drip. In group T3 nandrolone (50 mg I/m as single dose) and in T4

dexamethasone (5 ml I/m as single dose) were also given. The mean

recovery time (days) was recorded highest in group T4 (1.7±0.26) followed by

T3 (2.1±0.23), T2 (2.6±0.16) and T1 (3.3±0.26). Frequency of relapse was

highest in group T1 (40%) in comparison to T2 (5%), T3 (5%) and T4 (5%).

Therefore, hormonal therapy with dexamethasone or nandrolone in

association parenteral glucose and B-complex (B1, B2 and B3) gave excellent

recovery rate and very less evidence of relapse.

Gupta (2012) conducted epidemiological and therapeutic studies

of subclinical ketosis in lactating buffaloes of Jabalpur region of Madhya

Pradesh. Propylene glycol was found to be more effective when compared to

the bolus of niacin, tannic acid and jaggery for the treatment of subclinical

ketosis.

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Chaudhary et al. (2013) evaluated the efficacy of oral and

parentral therapies on hypocalcaemia and hypoglycaemia in recently

parturited buffaloes. They gave the combinations to the different treatment

groups i.e. oral calcium 100 ml + jaggery 500 g + sodium acid phosphate

60 g, calcium borogluconate 300 ml I/v + sodium acid phosphate 10% I/v +

glucose 25% 500 ml I/v. Oral and parental therapies restored all the

biochemical parameters close to normalcy within 30th day of parturition.

Dar et al. (2014) conducted study on 64 ketotic dairy cows to

compare the efficacy of different therapeutic protocols for ketosis in dairy

cows and found that glucose administration either parenterally or orally in

combination with gluconeogenic precursors fortified with B-complex vitamins,

insulin, sodium bicarbonate and isoflupredone acetate gave excellent

recovery rate.

Bakr et al. (2015) studied the effect of Sacchromyces cerevisiae

supplementation on health and performance of dairy cows during transition

and early lactation period on 16 healthy cows and found that it improves their

health, milk production parameters and reduces the risk of ketosis, milk fever,

retained placenta, displaced abomasums and suppressed immune functions.

Senturk et al. (2015) determined the effect of tannin on of

negative energy balance metabolism in dairy cattle and found lower BHBA

level in tested group in comparison to healthy control group, used to prevent

negative energy balance in the ruminants.

Nigam (2016) compared different combinations of glucogenic

substances and found nicotinic acid and propylene glycol were the most

efficacious treatment for subclinical ketosis in cattle.

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3. MATERIAL AND METHODS

LOCATION AND PLACE OF WORK

The proposed work was conducted in the Department of

Veterinary Medicine, College of Veterinary Science and Animal Husbandry,

Nanaji Deshmukh Veterinary Science University, Jabalpur (M.P.), from the

period October 2016 to April 2017.

3.1 EPIDEMIOLOGY STUDY

3.1.1 Buffaloes:

A total of 159 lactating buffaloes were taken for the

epidemiological study (preferably upto 1 month of calving, 3rd to 7th parity),

suspected for ketosis from organised sectors and unorganised sectors i.e.

villages (Table 01 and Figure 01).

Table 01: Sources of buffaloes screened for ketosis

S.

No.

SECTORS No. of suspected

buffaloes

1 Organised

Livestock farm adhartal, Ashok dairy, Agrawal

dairy, Rajnath dairy, Waderiya dairy, Nandlal

dairy, Chaubey dairy, Jaipratap dairy, Maalguzar

dairy, Ajju dairy and Darshan dairy

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2 Unorganised - Villages

Padwar kala, Hathana, Chati, Kiwalari, Suhagi,

Urdua khurd, Khiriya kala, Bagheli, Sarasma,

Khiriya simra, Jatma, Nuniya kala.

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Total 159

3.1.2 Selection of buffaloes

Detail information about the suspected buffaloes (Plate 01) were

collected, viz. anamnesis, age, stage of lactation, managemental practices,

feeding standard, physical condition, normal eating or inappetence and milk

yield for selection of buffalo.

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,,3.2 CLINICAL PARAMETERS AND SYMPTOMS

The following clinical parameters were recorded to screen the

ketosis in lactating buffaloes.

3.2.1 Rectal temperature

The rectal temperature (OF) was recorded with the help of

clinical thermometer. After dropping the mercury level upto the neck level, the

thermometer was inserted into rectum and kept in contact with rectal mucous

membrane for 2 minutes.

3.2.2 Pulse rate

Pulse rate was recorded by palpating the middle coccygeal

artery of the animal. The sensitive part of the figure was employed for

counting the pulse. It was recorded for at least 30 seconds and then

converted into per minute.

3.2.3 Respiratory rate

Respiratory rate (breath/min) was recorded by observing the

movement of the ribs and sternum for at least 30 seconds and then converted

into per minutes.

3.3 BIOCHEMICAL PARAMETERS

For epidemiological study urine samples were collected from all

suspected clinical cases of buffaloes for ketosis. However, for therapeutic

study blood, urine and milk samples were collected from 18 ketotic buffaloes

on 0 (pre-treatment), 5th and 10th day (post-treatment).

3.3.1 Urine

About 10 ml of mid stream urine sample was collected at early

morning in clean containers (Plate 02) for detection of urine ketone body

(Plate 3a and 3b).

3.3.1.1 Urine biochemical parameters

Multidiagnostic strips/dipsticks were used to record following

parameters in the sample:

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Urobilinogen

Protein

Ketone

Bilirubin

Glucose

A urine test strip comprised upto 10 different chemical pads or

reagents which react (change in color) when immersed in and then removed

from urine sample. The test was read after 1-2 minutes after dipping (Plate

04).

3.3.1.2 pH - The pH of freshly collected urine samples were taken by dipping

digital pH meter for seconds till the reading stabilized (Plate 05).

3.3.2 Blood

Approximately 3.0 ml blood was collected aseptically from ear

vein (Plate 06) in vaccutainer containing sodium fluoride as anticoagulant for

blood glucose and ketone body estimation.

3.3.2.1 Quantitative estimation of blood glucose.

A glucometer (Accu Chek Active blood glucose meter kit,

Mannheim, Germany) was used for determining the approximate

concentration of glucose (mg/dl) in the blood. A small drop of blood was

placed on a disposable test strip that the meter read within seconds (Plate

07).

3.3.2.2 Quantitative estimation of blood BHBA.

To analyze blood BHBA (mmol/L) ketone bodies, FreeStyle

optium H glucose ketone meter (Abbott, U.K.) was used. A small drop of

blood was instilled on a disposable ketone test strip of the meter and after few

seconds results were recorded (Plate 08).

3.3.3 Milk

For collection of milk samples, first udder was washed with

1.0 % potassium permanganate solution and then wiped by clean towel. First

few drops of milk were discarded and then 5ml milk was collected in clean

containers.

19

3.3.3.1 pH - The pH of freshly collected milk samples were taken by dipping

digital pH meter for seconds till the reading stabilized (Plate 09).

3.3.3.2 Ketone bodies - For qualitative assessment of ketone bodies in the

milk was done by using Rothera’s reagent (Plate 10).

Rothera’s reaction

Negative –

Slight levander +

Deep levander ++

Beet red or purple +++

Deep beet red or opaque (strongly positive) ++++

3.3.3.3 Milk yield - It was recorded in litres per day basis in the morning and

evening from the individual animal.

3.4 THERAPEUTIC STUDY

On the basis of clinical signs, estimation of ketone bodies, blood

glucose in different biosamples and milk yield, 18 buffaloes confirmed for

clinical ketosis were randomly divided into 3 groups named T1, T2 and T3 with

6 buffaloes in each group. However 6 healthy buffaloes were taken as healthy

control group (T4).

Table 02: Therapeutic regimen for ketosis in buffaloes

Group No. of buffaloes

Therapeutic regimen Dose and route

Duration

T1 6 Dextrose 25% +

insulin (short acting )

1 lit (I/v) +

@0.5 IU/Kg B.wt (s/c)

3 days

T2 6 Dextrose 25% +

Dexamethasone sodium phosphate

1 lit (I/v) +

40 mg (i/v)

3 days

T3 6 Antiketotic supplement* 100 g twice a day

7 days

T4 6 Healthy control

*Antiketotic supplement (100g) - Saccharomyces cerevisiae-15g Niacin – 5g, Tannic acid - 5g, Jaggery - 75g.

20

Therapeutic response study

The response of treatment was judged by the estimation of

ketone bodies, blood glucose and milk yield of individual buffalo under

treatment on day 5th and 10th of post treatment.

Statistical Analysis

To know the effect of treatment, the data were analyzed using

analysis of variance with two way classification and Duncan’s Multiple Range

Test (Snedecor and Cochran, 1994).

21

4. RESULTS

Amongst the production diseases in livestock, ketosis

contributes a major share particularly in high producing dairy animals. It is a

production disease with high intensity of prolonged morbidity causing

substantial loss in dairy industries. Because of the economic consequences, it

is imperative to diagnose ketosis in dairy animals, particularly during early

lactation for treatment in advance and prevention of further losses. The

studies on ketosis and their relation to blood glucose level, ketone bodies, pH

and milk yield etc., along with clinical manifestations are the important

diagnostic tools for assessing the disease condition and its treatment

achieving maximum productive efficiency of the animals. The present

investigation was carried out in ketosis in lactating buffaloes and the

observations of the study are presented as under.

4.1 EPIDEMIOLOGY

4.1.1 Overall occurrence of ketosis in lactating buffaloes

An epidemiological study was conducted on buffaloes to know the

occurrence of ketosis in lactating buffaloes during the period of October, 2016

to April, 2017 in and around Jabalpur. The study was aimed on 159 buffaloes,

suspected for ketosis. The suspected buffaloes were screened by dipstick

used for urine ketone body estimation. On the basis of clinical and

biochemical parameters 30 buffaloes were found positive and an 18.6 %

overall occurrence was recorded (Table 03, Figure 01).

Table 03: Overall occurrence of ketosis in lactating buffaloes

Screened buffaloes Ketotic buffaloes Occurrence (%)

159 30 18.60

4.1.2. Sector wise occurrence of ketosis in lactating buffaloes

A total of 159 buffaloes belonging to various organised and

unorganised sectors, suspected for ketosis were screened. Higher occurrence

(24.32%) was recorded in the buffaloes belonging to unorganised sectors as

compare to the buffaloes belonging to organised sector (14.11%). The results

are shown in table 04 and figure 02.

22

Table 04: Sector wise occurrence of ketosis in lactating buffaloes

Sector Buffaloes

screened

Buffaloes

positive

Occurrence (%)

Organized 85 12 14.11

Unorganized 74 18 24.32

4.1.3 Parity wise occurrence of ketosis in lactating buffaloes

Parity wise occurrence of ketosis in buffaloes has been

presented in table 05 and figure 03. To know the parity wise occurrence of

ketosis in buffaloes wide range of parities (i.e. 1st to 10th parity), were taken

and categorized in 3 categories. Among that the maximum cases of ketosis

(21/64) were recorded in the 4th-6th parity and 32.80% occurrence was

reported. However, the lower occurrence (12.70%) of ketosis was recorded in

the buffaloes of 1st-3rd parity and minimum (3.12%) in the buffaloes of 7th and

above parity.

Table 05: Parity wise occurrence of ketosis in lactating buffaloes

Parity Buffaloes

screened

Buffaloes

positive

Occurrence (%)

1st - 3th 63 08 12.70

4th - 6th 64 21 32.80

7th and above 32 01 3.12

4.1.4 Lactation stage wise occurrence of ketosis in lactating buffaloes

Lactation stage wise occurrence of ketosis in lactating buffaloes

is shown in table 06 and figure 04. Highest occurrence (43.75%) of ketosis

was found in the buffaloes of early lactation stage followed by mid lactation

stage (10.75%) and least in the buffaloes of late lactation.

23

Table 06: Lactation stage wise occurrence of ketosis in lactating buffaloes

Lactation stage Buffaloes screened

Buffaloes positive

Occurrence (%)

Early (0-2 month) 48 21 43.75

Mid (3-4 month) 65 7 10.75

Late (5-6 month) 34 2 5.88

4.2 CLINICAL SYMPTOMS AND PARAMETERS

4.2.1 Clinical symptoms recorded in ketotic buffaloes

Various clinical symptoms observed during the study period in

30 ketotic buffaloes were summarized in table 07 and figure 05. All the ketotic

buffaloes exhibited the symptom of sudden and unexpected drop in milk yield.

Although the some ketotic buffaloes showed either singly or in combination of

symptoms i.e selective feeding, wasting appearance, sweetish breath smell

and nervous signs. However, the categorization was made on the basis of

predominance of symptoms. Among the ketotic buffaloes, predominantly

selective feeding in 18 buffaloes, wasting appearance in 15 buffaloes,

sweetish breath smell in 8 buffaloes and nervous signs in 1 buffalo was

observed.

Table 07: Clinical symptoms recorded in ketotic buffaloes

Clinical symptoms Number of buffaloes

(%)

Sudden and unexpected drop in milk yield 30 100

Selective feeding 18 60

Wasting/woody appearance 15 50

Sweetish breath smell 8 26.60

Nervous signs 1 03.33

24

4.2.2 Effect of ketosis on milk yield of lactating buffaloes

Among 30 ketotic buffaloes, 18 (60%) buffaloes showed 10-20%

drop in milk yield. However, 23.4% ketotic buffaloes had 21-30% drop in milk

yield and 16.6% ketotic buffaloes had 31-40% drop in milk yield. The results

are shown in table 08 and figure 06.

Table 08: Effect of ketosis on milk yield of lactating buffaloes

Drop in milk yield (%) Number of buffaloes Percent of ketotic buffaloes

10-20 18 60.0

21-30 7 23.4

31-40 5 16.6

4.2.3 Rectal temperature

The mean values of rectal temperature in the buffaloes of

control, T1, T2 and T3 groups were recorded on 0, 5th and 10th day of post

treatment. The mean values of rectal temperature in all the groups were in the

physiological range, there were no significant variation observed as compared

to control group in pre and post treatment days. The results are presented in

table 09 and figure 07.

Table 09: Mean values of temperature (ºF) in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 101.7±0.17 101.5±0.20 101.5±0.16

T1 101.5±0.22 101.1±0.17 101.2±0.18

T2 101.6±0.12 100.9±0.19 100.9±0.21

T3 101.5±0.15 101.7±0.12 101.5±0.16

25

4.2.4 Pulse rate

The mean values of pulse rate in the buffaloes are recorded in

table 10 and figure 08. On 0, 5th and 10th day of post treatment, there were no

significant observations in pulse rate in different treatment group at different

interval.

Table 10: Mean values of pulse rate (pulse/min) in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 62.67±0.33 61.50±0.43 62.17±0.48

T1 62.83±0.17 62.50±0.22 63.00±0.26

T2 61.67±0.42 61.83±0.48 62.33±0.42

T3 62.50±0.43 61.33±0.49 61.50±0.43

4.2.5 Respiration rate

The mean values of respiratory rate on day 0 pre treatment in

the buffaloes of control, T1, T2 and T3 groups were recorded. No significant

variation were observed in relation to the mean values of control group, on

day 5th and 10th of post treatment (Table 11 and Figure 09).

Table 11: Mean values of respiration rate (breaths/min) in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 15.33±0.21 16.67±0.42 15.67±0.33

T1 15.83±0.31 17.33±0.33 16.33±0.61

T2 16.67±0.33 17.00±0.45 16.83±0.48

T3 17.17±0.17 16.50±0.43 16.17±0.48

26

4.2.6 Blood glucose

Blood glucose level of buffaloes of different groups under

treatment are presented in the table 12 and figure 10. The mean values of

blood glucose level of T1, T2 and T3 groups were 36.67 ±1.15, 36.17±1.08 and

40.17±0.48 respectively on day 0 (pre treatment) and were significantly lower

than the mean value of control group i.e. 58.17±0.83. On day 5th of post

treatment there were significantly increased in mean values of respective

groups in comparison to their pre treatment values. Further, on day 10th of the

treatment there were slight decrease in the mean values in group T1 and T2,

although there were slight increase in blood glucose level in group T3.

Moreover, at the end of treatment, the blood glucose level in the buffaloes of

all the treatment groups were in physiological range.

Table 12: Mean values of blood glucose (mg/dl) in buffaloes before and after treatment

Group

Interval (days)

0 5th 10th

Control 58.17aA±0.83 57.83aA±1.96 59.83aA±0.91

T1 36.67bB ±1.15 62.50aA±0.62 58.00aA±1.06

T2 36.17bB±1.08 60.17aA±1.01 57.00aA±1.63

T3 40.17bB±0.48 50.67bA±0.80 53.33bA±0.84

Mean values between treatments (lower case) and between intervals (upper case) with different superscript differed significantly (p<0.05)

4.2.7 Beta hydroxy butyric acid (BHBA)

The mean values of BHBA (mmol/L) in all treatment groups

were significantly higher in comparison to the mean values of control group

before treatment (day 0). Consequently on day 5th of the treatment there were

significant reduction in mean BHBA values i.e. 0.77±0.11 and 0.95±0.08 in

groups T1 and T2, respectively. Although, there was decrease in mean BHBA

value 1.7±0.06 in group T3 on 5th day of treatment but it was not significant

when compared with 0 day value. On day 10th of the treatment the mean

27

BHBA values came into physiological range i.e. 0.57±0.07, 0.60±0.08 and

0.82±0.06 in groups T1, T2 and T3 respectively. However, on day 10th in group

T3 the mean value of BHBA were significantly lower when compared the

values of day 0 and 5th (Table 13 and Figure 11).

Table 13: Mean values of BHBA (mmol/L) in buffaloes before and after treatment

Group

Interval (days)

0 5th 10th

Control 0.75bA±0.07 0.67bA±0.08 0.55aA±0.07

T1 2.15aA±0.20 0.77bB±0.11 0.57aB±0.07

T2 2.20aA±0.09 0.95bB±0.08 0.60aB±0.08

T3 2.37aA±0.11 1.70aA±0.06 0.82aB±0.06

Mean values between treatments (lower case) and between intervals (upper case) with different superscript differed significantly (p<0.05)

4.2.8 Urine pH

The mean values of urine pH of control, T1, T2 and T3 groups

were statistically analyzed pre and post treatment. The mean values of urine

pH were significantly lower in all the treatment groups i.e. 6.60±0.09,

6.65±0.06 and 6.63±0.09 in T1, T2 and T3 groups respectively when compared

to control group i.e. 8.21±0.09 before the treatment. There were significant

increased in urine pH of buffaloes of groups T1 and T2 i.e. 8.17±0.10 and

8.13±0.10 respectively, although there were marginal increased in the urine

pH value in group T3 on day 5th post treatment but it was non significant.

Further, on the 10th day of post treatment, mean values of urine pH of

buffaloes of group T3 showed the significant increase when compared to the

values of 0 and 5th day of post treatment (Table 14 and Figure 12).

28

Table 14: Mean values of urine pH in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 8.21aA±0.09 8.27aA±0.10 8.20aA±0.06

T1 6.60bC±0.09 8.17aA±0.10 7.20bB±0.06

T2 6.65bC±0.06 8.13aA±0.10 7.10bB±0.05

T3 6.63bB±0.09 6.97bB±0.10 7.40bA±0.22

Mean values between treatments (lower case) and between intervals (upper case) with different superscript differed significantly (p<0.05)

4.2.9 Urine ketone bodies (acetoacetate)

The presented table 15 and figure 13 shows the mean values of

urine ketone bodies. The mean urine ketone bodies were measured as

120.00±17.89, 120.00±17.89 and 106.67±16.87 mg/dl in groups T1, T2 and T3,

respectively on day 0 pre treatment. These were significantly higher in

comparison to the buffaloes of control group i.e 12.17±1.25 mg/dl before

treatment. On day 5th of the treatment, there were significant decrease in the

mean values and came down as 28.50±9.54, 30.00±4.66 and 32.50±11.16

mg/dl in T1, T2 and T3 groups, respectively. Whereas, on day 10th of the

treatment, decrease in the trend of mean values of all treatment groups were

recorded.

Table 15: Mean values of urine ketone bodies (mg/dl) in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 12.17bA±1.25 14.00aA±1.77 13.33aA±1.26

T1 120.00aA±17.89 28.50aB±9.54 16.17aB±1.17

T2 120.00aA±17.89 30.00aB±4.66 18.00aB±4.76

T3 106.67aA±16.87 32.50aB±11.16 24.17aB±11.29

Mean values between treatments (lower case) and between intervals (upper case) with

different superscript differed significantly (p<0.05).

29

4.2.10 Urine biochemical parameters

Pre and post treatment urine biochemical parameters were

analysed by multidiagnostic dipstick. However, urobilinogen, protein and

bilirubin could not be detected by multidiagnostic dipstick.

4.2.11 Milk pH

The milk samples for pH were analyzed statistically pre and post

treatment, are presented in table 16 and figure 14. There were significant

decrease in the milk pH i.e. 6.43±0.02, 6.50± 0.00 and 6.42±0.03 in of T1, T2

and T3 groups of ketotic buffaloes on day 0 pre treatment and when compared

with the mean value (6.67±0.03) of control group. Later on day 5th of post

treatment, there were significant increase in the mean values of milk pH as

6.71±0.03, 6.65±0.03 and 6.51±0.03 in T1, T2 and T3 groups, respectively.

However, on day 10th of post treatment, there were slight variation in mean

values of milk pH in all treatment groups which were under the normal

physiological range and did not show significant difference when compared to

values of 5th day of treatment.

Table 16: Mean values of milk pH in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 6.67aA±0.03 6.70aA±0.03 6.71aA±0.02

T1 6.43bB±0.02 6.71aA±0.03 6.63bA±0.03

T2 6.50bB± 0.00 6.65aA±0.03 6.60bA±0.04

T3 6.42bB±0.03 6.51cA±0.03 6.55bA±0.02

Mean values between treatments (lower case) and between intervals (upper case) with

different superscript differed significantly (p<0.05).

4.2.12 Milk Yield

The parameter of milk yield were analysed statistically and are

shown in table 17 and figure 15. The mean values of milk yield in control

group on day 0, 5th and 10th was 8.53±0.76, 8.22±0.76 and 8.47±0.74

litre/day. However, the milk yield in ketotic buffaloes under treatment on day 0

30

were significantly decreased as 6.28±0.42, 6.33±0.56 and 5.51±0.41 in T1, T2

and T3 groups, respectively when compared with milk yield of buffaloes of

control group. On day 5th of the treatment there were significantly increase in

milk yield in the buffaloes of group T1 and slight improvement in milk yield in

the buffaloes of group T3 while there was decrease in milk yield in the

buffaloes of group T2 when compared with the milk yield of day 0. Further, on

10th day treatment increasing trend of milk yield was noticed in all the

treatment groups but it was less when compared with the buffaloes of control

group. The improvement in milk yield was markedly observed in the buffaloes

of group T1 followed by buffaloes of group T2. In terms of recovery in milk yield

on 10th day of treatment, the buffaloes of group T1 seems to be better.

Table 17: Mean values of milk yield (litre/day) in buffaloes before and after treatment

Group Interval (days)

0 5th 10th

Control 8.53aA±0.76 8.22aA±0.76 8.47aA±0.74

T1 6.28bA±0.42 6.83abA±0.42 7.40abA±0.31

T2 6.33bA±0.56 5.33bB±0.64 6.55bcA±0.56

T3 5.51bA±0.41 5.58 bA±0.39 5.63cA±0.38

Mean values between treatments (lower case) and between intervals (upper case) with different superscript differed significantly (p<0.05).

Response to therapy was evaluated on the basis of clinical

parameters, estimation of ketone bodies and blood glucose in the different

biosamples, milk yield on day 5th and 10th of the post treatment the buffaloes

of groupT1 (received Dextrose 25% + insulin short acting) showed better

recovery by all the means.

Therefore, combination of Dextrose 25 % and Insulin was found

to be most effective followed by combination of Dextrose 25% and

Dexamethasone sodium phosphate and least being the antiketotic

supplement (Saccharomyces cerevisiae, niacin, tannic acid and jaggery) for

the treatment of clinical ketosis.

31

5. DISCUSSION

The recent farming techniques are aimed to ensure high milk

yields at the minimum cost, imposing severe stress on the metabolism of dairy

animals. Production disorders in milch animals are of great concern to the

growth of dairy industry as they seriously jeopardize productive and

reproductive capabilities of animals. Ketosis is a pretentious metabolic

disorder of heavily lactating dairy animals having detrimental effects on the

milk production. Buffaloes contribute the major chunk of milk production in

India and are very precious dairy animals because of organoleptic preference

of milk. The magnitude of ketosis has very little been studied in buffaloes in

Madhya Pradesh. Hence, in the present study, results of occurrence clinical

parameters, biochemical parameters and various therapeutic protocols for

ketosis have been discussed.

5.1 EPIDEMIOLOGY

5.1.1 Overall occurrence of ketosis in lactating buffaloes

Ketosis of lactating animals has been identified since 19th

century in both ancient and modern dairy management system, with varied

level of incidence worldwide (Lean et al., 1991). In the present investigation a

total of 159 buffaloes, suspected for ketosis were screened during the period

of October, 2016 to April, 2017 in and around Jabalpur. Of these, 30 buffaloes

were found positive and an 18.6 % overall occurrence was recorded. The

results of the study correlate well with the findings of various workers (Kumar

et al., 2015; Biswal et al., 2016 and Sharma and Kumar, 2016). However,

Rautmare and Anantwar (1993) reported comparatively less occurrence

(2.83%) of clinical ketosis in recently calved buffaloes. Earlier reports have

also documented varying rates of incidence of ketosis in buffaloes in India

(Rautmare et al., 1989; Ambore et al., 2001; Mandali et al., 2002).

Regional and country difference in dairy husbandry plays an

important role in ketosis prevalence. Differences in study design and

methodology may also account for differences in prevalence estimated. Such

32

type of wide variation in prevalence might be attributed to agroclimatic

condition, climate change, breed susceptibly, stage of calving , feeding habit,

production potential and managemental practices followed by the dairy

owners.

5.1.2 Sector wise occurrence of ketosis in lactating buffaloes

A total of 159 buffaloes belonging to various organised and

unorganised sectors, suspected for ketosis were screened. Higher occurrence

(24.32%) was recorded in the buffaloes belonging to unorganised sectors as

compare to the buffaloes belonging to organised sector (14.11%).

Our investigation was confined to small and marginal dairy

farmers in the milk shed areas of Jabalpur who were deprived of sophisticated

managemental system. The lactating buffaloes in the milk shed areas were

managed in natural village condition on feeding inadequate quantity of rice

bran, wheat bran, broken pulses, oil cakes, common salt, mineral mixtures,

paddy straw. The buffalo sheds were mostly thatched roofs with kutcha floors

and had poor sanitary conditions.

5.1.3 Parity wise occurrence of ketosis in lactating buffaloes

It was noted that parity of buffalo influenced the occurrence of

ketosis. Buffaloes of 4th-6th parity were found to be most susceptible (32.80%),

followed by 1st-3rd parity (12.70%). Whereas, comparatively less occurrence

(3.12%) was reported in the buffaloes of 7th and above parity. The results are

in agreement with the findings of Biswal et al. (2016) who found the highest

prevalence (40.8%) in 4th calver. In similar pattern Sharma and Kumar (2016)

recorded the maximum cases of ketosis in 8-9 years of age at 4th lactation.

However, Rautmare and Anantwar (1993) reported comparatively higher

occurrence (58.80%) of ketosis in 8-9 years old buffaloes.

This pattern of occurrence of ketosis might have cropped up due

to the fact that in the prime stage of productive life, the young buffaloes were

accomplished with increased body activity and physiological tolerance against

adverse conditions and had the greater power of adjustment between input

33

and output physiologically. In advancement of aging process, different

physiological processes became gradually slower. The prevalence increased

gradually with the increased calving status upto 4th calving period and then

decline. It might be elucidated that the buffaloes of very first stage of calving

were not accomplished with the highest lactation potential as the udder

development was still to attain its fulfilment with required amount of secretory

tissues for synthesis of milk.

5.1.4 Lactation stage wise occurrence of ketosis in lactating buffaloes

In the present study, highest occurrence (43.75%) of the ketosis

was recorded during early lactation after calving followed by mid lactation

(10.75%) and late lactation (5.88%). These observations are in accordance

with the findings of Anantwar and Singh (1993), Rautmare and Anantwar

(1993), Kumar et al. (2015), Biswal et al. (2016) and Sharma and Kumar

(2016).

To satisfy the requirements of milk production, the animal can

draw on two sources of nutrients namely; feed intake and body reserves.

During early lactation, the energy intake is insufficient to meet the energy

output in milk and the animal is in a negative energy balance. In conventional

farming, this is considered to be a normal metabolic situation in high yielding

dairy buffaloes. Buffaloes in early lactation are therefore, in a vulnerable

situation and any stress that causes a reduction in feed intake may stand

more chances for clinical ketosis.

5.2 CLINICAL PARAMETERS AND SYMPTOMS

The results of the present study revealed ketotic buffaloes had

slight variations in clinical parameters (temperature, pulse and respiration) but

within the normal physiological range. The similar findings were observed by

various scientists Venkateshwarlu et al. (1993), Teli and Ali (2007), Kumar et

al. (2015) and Bali et al. (2016). On the contrary, Dar et al. (2014) and Vaja et

al. (2016) reported slight elevated pulse and respiration rate.

34

Varying degrees of frequency of occurrence of symptoms of

ketosis were recorded in the present study. Topping the order was the sign of

sudden and unexpected decline in milk yield being repeated in 100 % of the

cases. This was followed by selective feeding (60%), wasting (50%), sweetish

breath smell (26.60%) and signs of central nervous system involvement

(03.33%) of the cases of ketosis. The pattern of signs observed in this study

was similar to clinical profile described by other workers Baird and Heitzman.

(1969), Schultz (1974), Singh and Kasaralikar (1988), Lean et al. (1991), Mir

and Malik (2003) and Constable et al. (2017). At the onset of lactation, even

towards the end of pregnancy period, energy consumption of dairy animals is

increased. If this energy is not compensated, it is inevitable occurrence of

ketosis

The variable pattern of drop in milk yield was noticed in present

investigation. Among 30 ketotic buffaloes, 18 buffaloes showed 10-20% drop

in milk yield, 07 had 21-30% drop in milk yield and 05 ketotic buffaloes had

31-40% drop in milk yield. Decline of 25-60 per cent in milk production in

bovine clinical ketosis has been recorded by Dohoo et al. (1984), Swain and

Tripathy (1987), Mir and Malik (2003), Teli and Ali (2007) and Kumar et al.

(2015).

The possible reason for the decreased milk production could be

reduced capacity of the animal to supply the lactogenic precursors to

mammary gland than the capacity of gland to produce due to homeorhetic

drive for production (Lean et al., 1991). Moreover, elevated blood ketones

also result in decreased milk production (Anderson and Lundstorm, 1985).

5.3 BIOCHEMICAL PARAMETERS

5.3.1 Blood glucose

In the present study, the mean blood glucose levels in the pre-

treated ketotic buffaloes were reduced ranging from 36 to 40 mg/dl which

were significantly increased and came to normal physiological range in

clinically recovered cases after instituting the therapy. Decrease in blood

glucose level in ketosis has also been documented by Kronfeld (1972), Chugh

35

et al. (1992) in cows and Anantwar and Singh (1993), Ambore et al. (2001),

Mandali et al. (2002), Teli and Ali (2007) in lactating buffaloes.

In ruminants, glucose is synthesized from propionic acid and

fulfils the requirement of glucose. Normal ruminants have low blood glucose

levels and slight decrease is enough to push them into hypoglycemic state

(Doxy, 1983). Glucose is the principal substrate of lactose and 60-80 per cent

of blood glucose is utilized by the mammary glands for milk production

(Annison and Linzell, 1963). A heavily milking animal may lose over 1,000 g of

lactose per day in its milk (Doxy, 1983) and the relative demand of glucose

during early lactation exceeds the available source leading to negative

carbohydrate balance (Grohn et al., 1983). In absence of sufficient energy

intake for high producing dairy animal, requirement of glucose increases

which causes mobilization of body fat, fat accumulation in the liver and the

rate of ketone body production and contributes to result in ketosis (Benedixen

et al., 1987). Severity of the clinical syndrome in ketosis is proportional to the

degree of hypoglycemia which is the principal metabolic disturbance in ketosis

(Constable et al., 2017).

5.3.2 Ketone Bodies

The increased BHBA levels were measured in ketotic buffaloes

above 2 mmol/L. However, the significant decrease in BHBA concentration

was recorded in all treatment groups on different intervals of treatment. The

findings shows resemblance with the observations of Caple (1977) who

measured BHBA concentrations as 0.4 to 0.8 and 0.8 to10 mmol/L in normal

and affected cows, respectively.

The mean values of urine ketone were significantly higher in

comparison to the buffaloes of control group i.e. 12.17±1.25 before treatment

and were significantly dropped at clinical recovery after treatment. The

findings corroborate the findings of Caple (1977) and Geishauser et al.

(2000).

Among different test options to measure ketone bodies in blood,

milk and urine, the concentration of BHBA represents the gold standard for

36

ketosis detection (Oetzel, 2004). Blood BHBA is an indicator of in appropriate

oxidation of non esterified fatty acids in liver (LeBlance, 2010). It is used as

early marker for detection of ketosis in ruminants (Duffield, 1997).

Ketone bodies are one of the important metabolic end products

of free fatty acid metabolism. Blood, milk and urine contains ketone bodies

and can be used for a cowside diagnostic. Total ketone bodies concentrations

are reported four times higher in urine than blood (Scultz, 1974). The

concentration of BHBA in milk is about 1/8 of the concentration in blood.

Whereas, concentration of acetoacete in milk is 40 to 45% of that of the blood

(Elizabeth, 2006).

5.3.3 pH

The mean values of urine pH were significantly lower in all the

treatment groups i.e. 6.60±0.09, 6.65±0.06 and 6.63±0.09 in T1, T2 and T3

groups, respectively when compared to control group i.e. 8.21±0.09 before

the treatment. There was significant increased in urine pH during the

treatment. Similar profile of urine pH in ketosis has been documented by

Sakai et al. (1993) and Elizabeth (2006).

The mean values of milk pH were significantly lower in all ketotic

buffaloes before treatment. There was restoration of normal milk pH after

employing the therapy. Similar profiles of milk pH in ketosis have been

documented by Elizabeth (2006). In reference to slight fall in milk pH

suggested that there might be increase in ketone bodies concentration in

blood and milk resulted in acidosis and may lead to drop in milk pH.

The reduction in milk and urine alkalinity was due to the

presence of ketone bodies which increases the acidity. After the treatment

increase in pH was reported this might be due to reduction in ketone bodies in

urine.

37

5.3.4 Milk Yield

The mean values of milk yield in ketotic buffaloes under

treatment on day 0 were significantly decreased as 6.28±0.42, 6.33±0.56 and

5.51±0.41 in T1, T2 and T3 groups, respectively when compared with milk yield

of buffaloes of control group. Increasing trend of milk yield was noticed in all

the treatment groups but it was less when compared with the buffaloes of

control group. The present findings are in the agreement with Teli and Ali

(2007) who recorded the recovery after treatment could restore only 25.30%

milk per day.

The possible reason for the decreased milk production could be

reduced capacity of the animal to supply the lactogenic precursors to

mammary gland than the capacity of gland to produce due to homeorhetic

drive for production (Lean et al., 1991). Moreover, elevated blood ketones

also result in decreased milk production (Anderson and Lundstorm, 1985).

Restoration in milk yield in different treatment groups might be due to

decrease in the concentration of ketone bodies which have negative effect on

milk production.

5.4 THERAPEUTIC STUDY

Baird and Heitzman (1969) has emphasized the hypoglycemic

theory in which hypoglycaemia is the driving force in the syndrome and

causes ketonemia. In the present study, the blood glucose level and ketone

body concentration were used to diagnosed healthy and ketotic buffaloes. The

intension of ketosis therapy is the support of gluconeogenesis via glucogenic

precursors and the reduction of lipolysis to improve the voluntary feed intake

(Allen and Piantoni, 2013).

Group T1 (Dextrose 25% plus Insulin)

The ketotic buffaloes of this group were treated with dextrose

25% along with insulin (short acting) and had significant increase in blood

glucose concentration (62.50±0.62 mg/dl) on 5th day post treatment, decrease

in ketone body concentration in blood and urine. Whereas, increase in pH of

38

urine and milk and improvement in milk yield (6.28±0.42 on 0 day and came

to 7.40±0.31 on day 10th post treatment) were observed. These findings are in

corroboration with the findings of Rings (1985), Singh and Kasarlikar (1988),

Anantwar and Singh (1993), Sakai et al. (1993) and Anantwar et al. (1995).

The concentrated glucose solution given intravenously is

excreted through urine when administered alone resulting in fall in blood

glucose level within two hours of administration (Schultz, 1968). Adjunction of

insulin is beneficial because it promotes uptake of glucose by cells of

peripheral tissues and accelerates carbohydrate metabolism (Sakai et al.,

1993). Insulin facilitates cellular uptake of glucose, suppresses fatty acid

metabolism and stimulates hepatic gluconeogenesis. Insulin should be

administered in conjunction with either glucose or a glucocorticoids and may

be of particular value in early onset cases of ketosis that are unresponsive to

glucose or corticosteroid therapy (Constable et al., 2017).

Group T2 (Dextrose 25% plus dexamethasone)

The buffaloes of this group were treated with dextrose 25%

along with dexamethasone sodium phosphate and had significant increase in

blood glucose concentration (60.17±1.01 mg/dl) on 5th day post treatment,

decrease in ketone body concentration in blood and urine. Whereas, increase

in pH of urine and milk, and improvement in milk yield were observed. Typical

trend of milk yield was noticed in this group. In comparison to 0 day

(6.33±0.56 lit/day). There was significance decrease in milk yield on 5th day

(5.33±0.64 lit/day) which increases significant on 10th day (6.55±0.56) of post

treatment.

Glucocorticoids have been used in the treatment of ketosis

because of their ability to produce hyperglycemia as a result of changes in

glucose. Baird and Heitzman (1969) and Wierda et al. (1987) stated that after

administration of dexamethasone, the blood glucose level rises but decline in

milk yield was recorded, similar findings were observed in present study.

Chuge et al. (1992) and Montana et al. (1993) found dexamethasone effective

against ketosis by aiding gluconeogenesis in animals within 3-4 days,

39

although all the metabolites came in the normal range within ten days. The

efficiency of glucocorticoids in the bovine ketosis has been demonstrated in

both experimental and field cases. Hyperglycemia occurs within 24 hours of

glucocorticoid administration and appears to result from a repartitioning of

glucose in the body rather than from gluconeogenesis (Constable et al.,

2017).

Group T3 (Antiketotic supplement)

The buffaloes of this group received antiketotic supplement

(Saccharomyces cerevisiae, niacin, tannic acid and jaggery) @ 100 g twice

daily for 7 days. Significant increase in blood glucose concentration was

noticed on 5th day post treatment. There was gradual increase in milk yield but

it was less when compared with group T1 and T2 on day 10th of post treatment.

The results are in partial agreement with findings of Abd El- Tawab, (2007),

Kawas et al. (2007) and Stanislaw and Przemyslaw (2009) who have obtain

variable pattern of recovery of ketosis and milk yield.

Saccharomyces cerevisiae feeding increases the production of

acetate, propionate and total VFA in dairy animals. It is worth mentioning that

propionate is the major precursor for gluconeogenesis and thus increment of

rumen propionate production results in an increase of hepatic glucose

production (Bakr et al., 2015). The primary function of niacin is in fatty acid

oxidation and glucose synthesis. Jaggery is the source of glucose and tannic

acid bypass it from the rumen and absorbed to increase blood glucose level

due to this animal cope up from negative energy balance and thus ketone

bodies level reduces in the blood.

The poor recovery in comparison to groups T1 and T2 could be

attributed to the fact that antiketotic supplement is not sufficient to maintain

consistent blood glucose level and to restablize disturbed body metabolism in

the ketotic buffaloes as faster as required. However, antiketotic

supplementation could be of more value in the treatment of subclinical

ketosis.

Comparative study of all therapeutic regimens indicated that

restoration of clinical symptoms, biochemical parameters and milk yield was

40

highest in the buffaloes of group T1 followed by buffaloes of group T2 and

least in the buffaloes of group T3. Thus, the results of the present study

indicated that the dextrose 25% + Insulin could be of potential therapeutic

value for the treatment of ketosis in buffaloes followed by dextrose 25% +

dexamethasone as compared to antiketotic supplement (Saccharomyces

cerevisiae, niacin, tannic acid and jaggery).

41

6. SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK

6.1 SUMMARY

Ketosis is a common multifactorial disorder of energy

metabolism that is characterised by elevated concentrations of ketone bodies

in ketonemia, ketouria and ketolactia. The disease mainly occurs in early

lactation when body reserves are used to support lactation. During the early

post partum period, milk production increases dramatically, while energy

intake may not be adequate to sustain the higher production level. This result

in negative energy balance and buffalo metabolize fat to meet their energy

needs. It is clinically characterized by decrease in feed intake, weight loss,

drop in milk yield, acetone/sweet smell in breath, depression and occasionally

nervous signs. Because of the economic consequences, it is imperative to

diagnose ketosis in dairy animals, particularly during early lactation for

treatment in advance and prevention of further losses.

The present work was carried out to study the diagnosis and

therapeutic management of ketosis in buffaloes. For epidemiological study, a

total of 159 lactating buffaloes were screened for ketosis from various

organized and unorganized dairy farms in and around Jabalpur. The

information pertaining to anamnesis of buffaloes like, age, stage of lactation,

managemental practices followed, feeding standards adopted, physical

condition, normal eating or inappetance and milk yield were collected. For

therapeutic study, 18 ketotic buffaloes were randomly divided into 3 groups

viz. T1, T2 and T3, each comprised of 6 buffaloes. Buffaloes of group T1

received dextrose 25% and short acting insulin, T2 received dextrose 25%

and dexamethasone sodium phosphate while T3 received anti-ketotic

supplement (Saccharomyces cerevisiae, niacin, tannic acid and jaggery) and

six apparently healthy buffaloes were grouped as T4 as healthy control. Blood,

urine and milk samples were collected and various parameters (i.e. blood

glucose and ketone bodies, urine pH) were analysed pre and post treatment.

The milk production was also recorded. Based on these parameters

performance, therapeutic efficacy of different treatments was decided.

42

The overall occurrence of the ketosis in buffaloes was recorded

as 18.60% (30/159). Higher occurrence of ketosis was recorded in the

unorganized sector 24.32% in comparison to organized sector. Highest

occurrence of ketosis was found in buffaloes of 4-6 parity (32.80%) and in

their early stage of lactation. On the basis of history sudden drop in milk

production was reported almost in all the ketotoic buffaloes. However,

10-20 % milk yield drop was noticed in majority of ketotic buffaloes. Whereas,

no significant variation was observed in the ketotic buffaloes of all treatment

groups at different intervals in rectal temperature, respiration and pulse rates.

The mean values of ketone bodies in blood and urine were found significantly

(p<0.05) higher in all the ketotic buffaloes before treatment but after treatment

it decreased significantly on 5th and 10th day of post treatment and reached to

normal physiological range. Significant decrease in the mean values of blood

glucose, milk yield, milk and urine pH were observed which increased

significantly on 5th and 10th day of post treatment.

On the basis of restoration in clinical signs, milk yield and

biochemical parameters (ketone bodies, blood glucose level) treatment with

dextrose 25% + Insulin was found to be most effective for ketosis followed by

dextrose 25% and dexamethasone sodium phosphate and least with

antiketotic supplement (Saccharomyces cerevisiae, niacin, tannic acid and

jaggery).

43

6.2 Conclusion

Overall occurrence of ketosis was reported as 18.6% in lactating

buffaloes in Jabalpur region.

Highest occurrence of ketosis was reported in buffaloes in 4 to 6 parity

in early lactation stage.

On the basis of restoration in clinical signs, milk yield and biochemical

parameters; treatment with dextrose 25% + Insulin was found to be

most effective for ketosis followed by dextrose 25% and

dexamethasone sodium phosphate and it was found least effective

with antiketotic supplements (Saccharomyces cerevisiae, niacin, tannic

acid and jaggery).

44

6.3 Suggestions for further work

Considering the widespread occurrence of bovine ketosis at

global scale and economic losses incurred by the farmers, the early and

accurate diagnosis is essential for the prevention of the disease. The present

study was planned looking into the limited time period and feasibility of work.

However, future studies are warranted in present topic in the following areas:

An extensive survey in the larger part of the state to find out the

occurrence of ketosis in buffaloes is needed.

Simple and sensitive diagnostic kit needs to be evolved for the

diagnosis of disease well before manifestation of clinical signs of

ketosis.

More studies on therapeutic aspects are needed taking other

glucogenic agents alone or in combination.

45

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