MARDI Res.Bull., (1983) 11,3: (3G377)

THE EFFECT OF DIETARY COBALT AND VITAMIN B12SUPPLEMENTATION ON THE PERFORMANCE OF INDIGENOUS SHEEP

WAN ZAHARI MOHAMED, C. DEVENDRA* AND JAAFAR ALI**

Keywords: Cobalt, Vitamin B12, Supplementation, Performance, Indigenous sheep.

RINGKASAN

Kesan penambahan unsur kobalt dan vitamin B 12 terhadap prestasi biri-biri tempatan dilapurkan.Lima belas ekor biri-biri jantan yang mempunyai purata berat badan sebanyak 18 kg bagi seekor telahdigunakan di dalam kajian ini selama 20 minggu. Pelakuan-pelakuan yang telah diberikan ialah I)memberi minum 32.3 mg larutan kobalt klorida setiap minggu, II) injeksi 150,tzg vitamin 812 (cyano-cobalamin) ke dalam otot setiap minggu, III) injeksi 600,ug vitamin B12 ke dalam otot setiap bulan, IV)pemberian satu bulet kobalt dan V) kawalan. Tidak ada perbezaan yang berkesan didapati di dalampengambilan bahan kering di antara perlakuan. Pertambahan berat badan adalah paling tinggi (P(0.05)bagi perlakuan III dan paling rendah bagi kumpulan yang diberi bulet kobalt. Pengambilan air harianbertambah tinggi bagi kumpulan-kumpulan yang diberi penambahan kobalt atau vitamin 812(P<0.05) jika dibandingkan dengan haiwan-haiwan kawalan, terutama sekali bagi haiwan-haiwan didalam perlakuan I. Tidak ada perbezaan yang berkesan di antara perlakuan bagi kepekatan mineral didalam plasma darah, parameter-parameter darah, dan imbangan N serta sebahagian dari unsur-unsurmakro. Kehazaman abu semakin bertambah (P<0.05) di dalam kumpulan-kumpulan yang diberipenambahan kobalt atau vitamin B12. Kepekatan vitamin B12 di dalam plasma darah bertambah tinggibagi kumpulan-kumpulan yang diberi penambahan. Purata kepekatan vitamin B 12 mengikut perlakuan I,II, III, IV dan V adalah masing-masing 16'/8, 1460, 1619, 1002.5 dan 424.15 pgiml. Kepekatan asidforminoglutamik (FIGLU) di dalam air kencing berubah-ubah di antara perlakuan. Injeksi vitamin B12ternyata lebih berfaedah jika dibandingkan dengan pemberian bulet kobalt atau larutan kobalt klorida.

INTRODUCTION

Cobalt (Co) deficiency in grazingruminants is widespread in tropical regionsand represents an economic loss (Larrnun,1962). The severity of Co deficiency mayvary from an undetectable mild state un-accompanied by any clinical signs, to acutedeficiency. The symptoms are non-specificand often diff icult to distinguish from energyand protein malnutrit ion. The deficiencysymptoms include lack of appetite, muscularatrophy, emaciation, anaemia and pica. Lossof appet i te and lethargy. progressing roemaciation and death are common featuresin ruminants (NIILS, 1981). The loss of pro-duction attributed to Co deficiency has beenreviewed (RonEnrsoN, 1971).

The occurance of either marginal oracute Co deficiency in ruminants is believec.to be widespread in Malaysia. However, theseverity of this deficiency has never been

fu l ly invest igated. Acute Co def ic iency hashowever been reported to cause consider-able i l l - thr i f t in Kedah-Kelantan cat t le inKluang, Johore (MaNNerre er al., 1976).

Cobalt is required by the microorganisms of the rumen for the synthesis ofv i tamin B 12. I f the e lement is def ic ient in thediet , the v i tamin cannot be produced in therumen in amounts sufficient to satisfy therequirements of the protozoa that l ivesymbiotically in the rumen, nor of the animalitself (HoussER e/ al., 7916; WeN ZRHRnr,resl ).

Various methods of overcoming cobaltdeficiency have been tried. These includedirect treatment of the pasture with Co con-taining ferti l izer, oral administration orinjections of Co compounds or vitamin B12.The benefits and disadvantases of abovemethods have been reuiewel extensively(UNoenwooD. 19771

*Feed Resources and Animal Nutrition Branch, MARDI, Serdang, Selangor.* *Specialist and Reproductive Research Centre, National Family Planning Board, c/o Haematology Division, Institute

for Medical Research, Kuala Lumpur.

366

An evaluation of the relative biological

availability of Co from various sources. in

association with method of treatment in

ruminants is therefore necessary' The

objective of this paper was to study the effec-

tivlness of various methods of Co supple-

mentation on the performance of indigenous

sheep, with reference to intake, body weight

gain, digestibility, mineral and vitamin B12

plasma concentrations, including an assess-

ment of urinary forminoglutamic acid

(FIGLU) as an index of Co deficiency'

MATERIALS AND METHODS

(i) SheeP and their management

15 rams, approximately 18 months of

age and each weighing approximately 18 kg

were used in this study. The animals were

clinically normal and were randomly

assigned into five treatments according to

body weight. They were individually fed in

met;bolic cages throughout the trial period

of 18 weeks. This exludes a fortnight adapta-

tion before the start of the trial.

ChopPed NaPier grass (Pennisetum

purpureum) at the five weeks regrowth stage

*at fed to the animals ad libitum, twice daily

at 0900 and 1400 hours. Water was provided

ad libitum, and no salt lick was given'

(ii) Treatment

The animals were subjected to five

treatments as follows: -

I. 32.3 mg cobaltous chloride per week

oral drenching*

II. 150 4g vitamin Btz intramuscularinjection Per week* *

III. 600 trg vitamin Bl2 intramuscular

injection Per month

ry. Cobalt bullet Plus grinderr

V. Control - ad libitum feeding of Napier

grass onlY.

* CoCl2 6H2O, analar, Fisions Scientific

APP. Ltd', Loughborough, Fngland(doie given was equivalent to 8 -,9 9oper *"ik or 0.44mglks body weight)'

** Cyanocobalamine, M'P'F' Sdn' Bhd''

Petaling Jaya, Selangor (Containing

1,000 mcg cYanocobalamine/ml)'

r Cobalt pellet for sheep' Each con-

taining 3 g. cobalt oxide, Adelaide &

Wallaroo Fertilizer Ltd. Adelaide'Australia (administered once at the

beginning of the experiment per head) '

The animals were weighed Prior to

morning feeding at the beginning of the

experiment and every fortnight thereafter'

Af the same time, blood or plasma samples

were collected by jugular venipuncture for

the determination of vitamin B12' mineral

concentration and haematological study'

Water intake was recorded dailY.

(iii) Techniques used

Digestibility and mineral balance

studies were carried out by conventionalmethods during the last week of the

experiment. The techniques used were as

described previously (DeveNone' 1915)'

Plasma mineral concentrations were deter-

mined by emmission and atomic absorptionspectrophotometry while plasma vitamtn

812 was analysed by microbiological assay

using Lactob ac illus leichmanii (MerruEws1962). The collection of urine for FIGLUdetermination was as described bY

GawrnonNE, (1968) and was analysed by

the method of CsnNenn and BENET'r(L962). The chemical composition of the diet

is given in Table 1.

RESULTS

The average daily dry matter intake(DMI) for treatments I, II, III, IV and V

throughout the experimental period was

684 .l , 664.1 ,7 14 .9 , 694 .2 and'724 ' 9 g respec-

t ively, which is equivalent to 3.6, 3 '4, 3 '8, 3 '9

and 3.6 per cent of mean live weight'

367

TABLE 1: THE CHEMICAL COMPOSITIONPUKPUREUM) ON DRY

OF NAPIER GRASS (PENNISETUMMATTER BASIS

Constituent Yalue (%)+

Crude protein (N x 6.25)

Crude fibre

Ether extract

Ash

Nitrogen-free extract

Gross energy (MJ/kg)

P

Ca

Mg

Na

K

Co

1 1 . 0

34.0

L - O

5 . 9

46.5

17.45

0 . 1 8

0.32

0 . r 7

0.07

0.96

0.08

xCut at about five weeks of ageFertilizer regime: urea 300 kg N/ha,

TSP : 48.1 kg P/ha,MP 150.1 kg K/ha .

+Average of 10 samples throughout the experimental period.

Table 2 shows the live weight data ofthe animals. There were no significant differ-ences between treatments. Appreciableweight responses were obtained between 8thand 12th weeks. Animals in treatment III(600 pg vitamin BI2 injectionimonth)showed significantly increased body weightgain (P<0.05) throughout the experimentalperiod with the average of 3.6 kg comparedto 2 .6 ,2 .3 ,2 .0 and 0 .8 kg fo r t rea tments V ,II, I, and IV respectively. By the end of 14thweek there was no difference in live weightgain between the treatments. There was nocorrelation between DMI and body weightfor all treatments throughout the experi-mental period.

Daily water intake of treated groupsincreased significantly (P<0.01) comparedto the control group from the beginning ofthe trial up to 16th week. The average dailywater intake is highest in treatment I (437.3ml), followed by treatment IV (320 ml),

treatment V (275.2), treatment III (268.7)and I I (159.1) respect ively. D.y matterintake and water intake was correlated signi-ficantly (r : 0.519, P< 0.05) only in treat-ment I. The regression equation was Y :493.64 + 0.15 X where Y is dry matter intakeand X is water intake. The ratio of DMI ;water intake for five treatments throughoutthe experimental period was 1.3 : 1, 3.6 : 1,1.9 : 1 and 2.2 : 1, respect ively.

The average concentration of someminerals and vitamin B12 in plasma through-out the experiment is shown in Table 3.There were no significant differences in theconcentrations of Ca, P, Mg, Fe, Cu andZnbetween treatments. Regardless of the initialvalue, the repeatibility of plasma mineralvalues over time was good and within normalacceptable limits. There was a significantincrease in vitamin B12 plasma concentra-tion in treated groups compared to thecontrol (P<0.05).

368

TABLE 2: LIVE WEIGHT DATA (KG)

TreatmentAverage Average

initial finallive weight live weight

Mean Metaboliclive body

weight weight fl/Vo '7 s

)

Live weighlcnange

32.3 mg CoC12.6H2Olweek

150 pg B1 2 /week

600 pg 81 2 /month

Cobalt bullet

Control

11 .9

r 8 .5

1 7 . l

t 7 . 2

1 8 . 8

t9.9

20.7

20.6

1 8 . 0

21.4

I 8 .9c

l9.6ab

1 8.9bc

_t / .oo

20.1a

t 6 . t 4

1 6 . 5 8

16.12

l 5 . 2 8

1 6 . 8 8

2.}ab

2.3ab

3 .5a

0.8b

2.6a

*Different superscripts indicate significantly different+At the end of 20 weeks experimental period. LSD =

Table l shows the blood parameters ot

the animals. There were also no signif icant

difference in total white blood cell (RBC),haemoglobin (Hb), packed cell volume(PCV), mean corpuscular volume (MCV),mean corpuscular haemoglobin (MCH),mean corpuscular haemoglobin concentra-tion (MCHC) and platelets between treat-ments, but the values were within normall imits.

Table 5 presents the average digesti-bility coefficients of nutrients betweentreatments. Digestibility of ash in treatedgroups increased significantly compared tocontrol (P< 0.05). There was no significantdifference in N, Ca, P and Mg balancebetween the treatments (Table 6). Theaverage urinary forminoglutamic acid(FIGLU) for the five treatments were 1.9,27.0, 114.8, 101.5 and 122.8 /rglmlrespectively.

DISCUSSION

The results of present study indicatethat supplementation of Co either throughoral administration of Co salt or as vitaminB12 injection did not significantly affectappetite or dry matter intake. A response tolive weight gain of treated groups was onlydetected in the first 14 weeks after supple-mentation, especially in sheep giveninjections of vitamin B 12. As the experiment

means (P<0.05)2.09 at the 5% level.

progressed, daily gain in treatment IV(cobalt bullet) and I (cobaltous chloride)declined, which resulted in weight loss, pro-bably due to a decreased uptake of metaboli-zable energy. Therefore, bullet therapy anddirect drenching of cobaltous chlorideappear to be inferior in promoting bodyweight gain as compared to frequentin ject ions of v i tamin B12.

The lower intake of dry matter isprobably the main reason in causingincreased water intake in treated groups. Asignificant increase in water intake in treat-ment I might be attributed partly to physiolo-gical factors, as water is largely needed tobuffer the high salinity of body fluids due toexcessive ch lor ide consumDtion.

The overall l ive weight change of thesheep from this study is small. This is due tothe sheep being adult and having past theirpeak in growth. It follows therefore that ifthe best response to l ive weight gain is to beobtained in respect of Co treatments,growing animals should be used, probablyaround six months age or after weaning ashas been shown for lambs (PHII-lrrsoN andMrrcHEl1. 1952).

Changes in the mean plasma mineralconcentration is of little value, as there is nodirect antagonistic interactions between Coand other inorganic elements especially Ca,

369

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P, Mg, Fe, Cu andZn. It was found that theinitial plasma Ca and P concentrations wereinsignificantly higher compared to the finalconcentrations, regardless of the varioustreatments. The concentration of otherminerals varied little and were well withinnormal limits. However, the mean plasmavitamin B12 concentration in treated groupswas significantly higher (P<0.01) than thecontrol group, especially in the first 14 weeksafter supplementation. Apparently, this wasnot reflected in improved live weight gain.

The average concentration of vitaminB12 in plasma for groups I, II, III, IV and Vwas 1678, 1460, 1619, 1002.5 and 424.8 pg/mlrespectively. Bullet therapy increasedplasma vitamin B12 concentration from 340pglml initially to a maximum value of 1857pg/ml at 10 weeks after administration. Thenthe level started to decline as the experimentprogressed. This explains the ineffectivenessof bullet therapy in maintaining high vitaminB12 concentration for a longer period as hasbeen suggested (CoNNor-v and PooLE.1967). The concentration of plasma vitaminB 12 in the study is far higher than the valueobtained in cattle as reported (WnN ZaHentMosAur,o and Dr,vENoRR, 1982).CsRLnERs and MACI,r]ERSON (1980)indicated that bullet therapy raised plasmavitamin B12 of calves from 150-250 ng/lonly, while WHITELAw and Russr,l (1980)obtained a large increase of vitamin B 12 con-centration following similar treatment tosheep where values higher than 1000 ng/lhave been maintained. The normal plasmavitamin Bl2 concentration is between400-800 pgiml and decreased to 80- 100 pg/ml in deficient animals (MACzuERSoN et al..1973). Poor body weight gain in bullettherapy groups is therefore understandable.The present results support the finding ofALEXANDEr. et al., (1916) who also reportedno response in live weight gain after bulletadministration with similar serum Bl2 fortreated and control animals.

None of the blood parameters assistedin the interpretation of Co or vitamin B12status of sheep. The values recorded arewithin

'normal range. Indirectly, they

indicate that all animals, particularly thecontrol were not anaemic. MACPHERSON e/al., (1976) indicated that Hb and PCV (%)concentrations were between 11.6 - 12.I gl100 ml and 36.0 - 38.6% in normal animalsand fel l to 7.7 - 9.8 9/100 ml and 22.8 -

31.7t|c in deficient animals respectively.

The carbonate, chloride, sulfate andnitrate salts are considered satisfactorydietary sources of Co. However, in thisstudy, it was clear that oral drenching ofcobaltous chloride was inferior in promotingbody weight gain as compared to othermethods. This is probably due to the dietaryCo salt which when administered orally ispoorly absorbed and is excreted mainly infaeces (UNoEnwooD, 1977). Furthermore,only a small proportion of total dietary Cois converted into true vitamin B12 whichcould be ut i l ized by ruminant t issues.

Intramuscular injections of vitaminBl2 appears to be an effective means of theincreasing plasma B12 concentration, thusreducing the dependence of the animals onmicrobial B12 synthesis in the rumen. It wasobserved in this study that an in jection of 600,ug vitamin B12lmonth was more effective inpromoting body weight gain than a dose of150 pg vitamin B12lweek, with a non-significant increase in the plasma concentra-tions of Ca, P, Mg, Fe, Cu, Zn as well asvitamin B12. Supplementation of Co orvitamin B12 did not significantly increase thedigestibilities of dry matter, organic matter,crude protein, ether extract and nitrogen-free extract compared to the control (Tables).

The response to Co or vitamin B12supplementation is however not universaland primarily dependent on the extent ofdeficiency, 812 body stores and age of theanimals. The more severe the deficiency, thebetter will be the response. In this study, thecobalt content in the grass was marginal (*0.08% DM), and it is to be expected there-fore that the animals may not obtain enoughdietary Co. Indications that Co had no effecton live weight gain of the animals has beenreported (Llovo D,c.vrps, 1980).

374

The elevated excretion of urinarymethylmalonic acid (MMA) and formino-glutamic acid (FIGLU) appears to be asensitive indicator of BI2 deficiency(GawrnonNE, 1968; MrLr-RR ancLoRnNrz, 1970). As increased urinaryFIGLU concentration is associated withincomplete metabolism of histidine toglutamate, certain workers loaded histidinebefore urine collection (CHeNARTN e/ a/.,1962) whereas others did not (GewrHonNe,1968). In this study, histidine was notadministered and it was found that there wasconsiderable variation in urinary FIGLUconcegtration between treatments. Theaverage urinary FIGLU concentration fortreatments I, II, III, IV and V was 1.9,27.0,114,8, 101.5 and 122.8 pglm| respectively,which is equivalent to 1.0, 12.1, 56.2, 58.3and 51.0 pr molesikg. body weightlday.These value are far higher than L3 pr moles/kg-body weight/day as suggested for 812adequacy (GewruonNe, 1968). RussEt- eral., (7975) indicated that FIGLU urinaryconcentration in vitamin BI2 deficientanimals was 12 pglml. The discrepancy of theresults might be attributed to histidine whichalso needs to be fed to experimental animals.The sensitivity of FIGLU as an index may beinfluenced by changes in the demand forhistidine for tissue protein synthesis (Mlll-s,1981). These results suggest that in thedesign of future studies concerning Codeficiency, histidine balance is essential.

In terms of practical application, thevalue of the individual methods merit somediscussion. Vitamin Bl2 intramuscular

injection has limited value to its high cost ascompared to other techniques. However, theresponse to weekly as opposed to monthlyinjections needs to be throughly investi-gated. Oral dosing of Co salt might besatisfactory provided that the doses are givenregularly, but this is laborious, tedious andcostly. Bullet therapy, having the advantageof avoiding frequent handling of the animalsmight be less valuable for pen feeding condi-tions, eventhough this method has been wellestablished under various circumstances.These considerations suggest that vitaminB12 injections are probably the best methodpresently. However, while this is constrainedby cost, it is possible that the value of theresponse may be higher, in which case this isjustified. Further investigations arenecessary to elucidate these and otheraspects concerning Co deficiency.

ACKNOWLEDGEMENTS

The authors are grateful to PuanJamiah bte Salleh, Encik Aris bin Alias, CikMahayani bte Meor Shafie and EncikDzulkiplee bin Ahmad for technicalassistance. Thanks are also due to Dr. Khalidbin Hassan, Head, Haematology Division,Institute for Medical Research, KualaLumpur and his staff for the determinationsof vitamin B12, blood haematological studyand analysis of urinary forminoglutamicacid. We are also indebted to the head andstaff of Central Analytical Services, MARDIand to Encik Shukri bin Othman forstatistical analysis.

SUMMARY

The effect of dietary cobalt and vitamin B12 supplementation on the performance of indigenoussheep is reported. Fifteen rams, approximately 18 kg each were used for an experimental period of 20weeks. The treatments were: I) oral drenching of 32.3 mg CoClr.6FIrOlweek, II) intramuscular injectionof 150pg vitamin 812 (cyanocobalamine)/week, III) intramuscular injection of 600prg vitamin B12lmonths, IV) cobalt bullet and V) control. No significant differences in dry matter intake were foundbetween treafinents. Animals in treatment III showed significantly higher body weight gain (P<0.05),whereas the Cobalt bullet group exhibited poor response to the treatment. Daily water intake of treatedgroups was significantly increased (P<0.01) compared to the control, especially in treatment I. Nostatistical differences were found between treatrnents in plasma mineral concentrations, blood parametersand balance of N and some macro minerals. The digestibility of ash increased significantly (P< 0.05) intreated groups. Plasma vitamin 812 concentration was significantly increased in treated groups with theaverage of 1678,1,460,1619,1C[.2.5 and424.8 pg/ml for treatment I, II, III, IV and V respectively. Urinary

375

forminoglutamic acid (FIGLU) concentration varied greatly between treatments. Frequent injections ofvitamin B12 appears to be more beneficial than either bullet therapy and direct drenching ofcobaltouschloride.

REFERENCES

ALEXANDER, G,I., HARRY, J.M., LEE, J.H.and SruBBS, W.C. (1967). Studies oncopper and cobalt therapy of cattle inCentral Coastal Queensland. Aust. J.Agric. Res., 18: 169-181.

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Accelted for publication on I I th May , 1983 .

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