Tropical Animal Health and Production, 36 (2004) 473^482 # 2004 Kluwer Academic Publishers. Printed in the Netherlands

Serum Vitamin B12 Levels in Omani Goats A. Al-Zadjali, E.H. Johnson* and A. Srikandakumar Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al-Khod 123, Muscat, Sultanate of Oman *Correspondence: E-mail: [email protected] Al-Zadjali, A., Johnson, E.H. and Srikandakumar, A., 2004. Serum vitamin B12 levels in Omani goats. Tropical Animal Health and Production, 36(5), 473^482 ABSTRACT Blood and faecal samples were studied from 200 Omani goats of both sexes and various age groups, within ¢ve di¡erent regions in Oman. Survey questionnaires were undertaken on each goat. The serum vitamin B12 values ranged from 105 to 42000 pg/ml, with a mean value of 723.1 pg/ml and a 95% con¢dence interval of 61^1507 pg/ml. Kids in the age group 1^3 months showed signi¢cantly lower levels of serum vitamin B12 than the older animals. Varying levels of serum vitamin B12 were found in di¡erent regions of Oman and on farms within the same region. Goats maintained on commercial feed showed higher levels of serum vitamin B12 than those maintained on pasture or fed on household leftover food, as commonly practised in Oman. Coccidial counts were highest in animals with lower levels of serum vitamin B12. The results of this study indicate that there is a wide range of serum vitamin B12 levels in Omani goats and suggests that younger animals, with lower levels of serum vitamin B12, might be more susceptible to vitamin B12 de¢ciencies than older animals. Keywords: cobalt, goats, Oman, vitamin B12

INTRODUCTION Cobalt is an essential dietary trace element in ruminants, in which it is synthesized to vitamin B12 by rumen microorganisms. Sheep and cows feeding on forages containing less than 0.07 mg/kg (ppm) dry matter (DM) of cobalt (Fell, 1981) are likely to develop de¢ciencies resulting from a reduction in the number of rumen microorganisms (Gall et al., 1949; Marston and Lee, 1952; Marston and Smith, 1952) and subsequently in the synthesis of vitamin B12 (Walker and Elliot, 1972; Bigger et al., 1976). Clinically, sheep and cows are reported to develop inappetance, poor production, weight loss, photosensitivity and anaemia (Ulvund and Pestalozzi, 1990). Immunode¢ciency (Vellema et al., 1996), disturbances in reproductive performance (Fisher and MacPherson, 1991) and increased susceptibility to certain parasite infections (MacPherson et al., 1987; Ferguson et al., 1989; Paterson and MacPherson, 1990) have also been described. In addition, sheep develop a condition commonly referred to as white liver disease (Sutherland et al., 1979; Ulvund and Pestalozzi, 1990). Goats, however, have been described as being more resistant than sheep to low dietary cobalt levels (Clark et al., 1986; Mburu et al., 1993). The reason for this apparent lack of sensitivity has not been 473

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adequately addressed. In Oman, hepatic lipidosis has been described as the most commonly occurring liver pathology in goats (Johnson et al., 1999a). This condition was found to be associated with low levels of cobalt in the livers of a¡ected animals (Johnson et al., 1999b). Prior to this study there were only two reports of hepatic lipidosis associated with cobalt de¢ciency, which were described in a small number of Angora or Angora cross goats in New Zealand. These animals were also described as su¡ering from ill-thrift (Pearson et al., 1987; Black et al., 1988). In the ¢eld, goats in Oman with chronic weight loss but no other apparent clinical symptoms have responded to the intraruminal implantation of cobalt pellets or vitamin B12 injections with improved weight gain, suggesting that vitamin B12 de¢ciency might be a disorder of clinical signi¢cance (unpublished observations). As a consequence of the rarely reported occurrence of clinical manifestations of cobalt de¢ciencies in goats, few studies have addressed the determinations of normal serum levels of vitamin B12 in this species. Consequently, it is often assumed that normal serum vitamin B12 levels reported for sheep can be extrapolated for goats. However, it has been suggested that normal values for sheep may be inappropriate for goats (Mburu et al., 1993). Owing to the scarcity of information relating to normal serum vitamin B12 levels in goats, and because of the widespread occurrence of caprine hepatic lipidosis associated with cobalt de¢ciency in Oman, the present study was undertaken to establish baseline normal reference values. These values will eventually be used to assist in the clinical and laboratory characterization of cobalt de¢ciencies in Omani goats. MATERIALS AND METHODS Geographic location and animals Two hundred goats of both sexes and from various age groups (1 months to 11 years) were used in this study. Goats were from various northern and eastern regions of the country, including Batinah (n = 74), Muscat (n = 67), Sharqiya (n = 20), Dhahira (n = 19) and Dakhiliya (n = 20). Survey questionnaire A survey questionnaire was used to obtain information from the owners regarding the deworming history and type of feed given to their animals. In addition, this survey also addressed issues relating to sex, age, breed and mineral supplementation.

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Vitamin B12 A microparticle enzyme immunoassay (MEIA) (IMx B12; Abbott Laboratories Diagnostics Division, Abbott Park, IL, USA) was used to determine vitamin B12 levels. The assay is based on the competitive binding of serum vitamin B12 and a standard B12 alkaline phosphatase conjugate to an intrinsic factor coated matrix. The procedure outlined by the manufacturer was followed. Analysis was performed on serum samples obtained from each individual goat. The inter-assay and intra-assay coe¤cients of variations were 6.6% and 5.7%, respectively. Faecal sample analysis Rectal faecal samples were taken and kept in an icebox until they were analysed for egg counts by the morphological identi¢cation of eggs using a standard McMaster £otation technique under a light microscope. Statistical analysis Data were analysed using the Statistical Analysis System (SAS, 1993). The analysis of variance procedure was used to determine the e¡ect of age, feed type, region, farm and sex on vitamin B12 levels in goats. Correlation analysis was used to determine the relationship between vitamin B12 and faecal egg counts. Means were used to compare the e¡ect of region, sex, age group and serum chemistry on vitamin B12 level in goats using Duncan's multiple range test. In all cases the a level was equal to 0.05. RESULTS Serum vitamin B12 levels The serum vitamin B12 levels in Omani goats ranged from 105 to 42000 pg/ml, with a mean value of 723.1 pg/ml. The 95% con¢dence interval was 61^1507 pg/ml. Relationship between age and serum vitamin B12 levels Kids in the 1^3 months age group had mean serum vitamin B12 level of 458+101 pg/ ml, which was signi¢cantly (p50.05) lower than the values for goats in the 4^5 months (621+59.2 pg/ml), 6^11 months (847+61 pg/ml), 1^2 years (731+67.5 pg/ml), 42^4 years (737+87.4 pg/ml), and 44^11 years (710+47.3 pg/ml) age groups (Figure 1).

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Figure 1. Mean serum vitamin B12 levels (pg/ml) in six di¡erent age groups

E¡ect of feed on serum vitamin B12 levels All goats were free grazing and were most commonly supplemented with household leftover food. However, some goats were given commercially available concentrate feed. Goats maintained on pasture and/or given additional feed supplements (leftovers) had signi¢cantly lower (p50.05) mean serum vitamin B12 level (676.1+21.8 pg/ ml) than those fed commercial feed (938.1+86.2 pg/ml). Commercial feed was given only by some farmers in the Muscat, Batinah and Sharqiya regions. Relationship between regions and farms on serum vitamin B12 levels The mean serum vitamin B12 level of goats in Dakhiliya region (509.1+56.7 pg/ml) was signi¢cantly lower (p50.05) than the mean values of goats in Muscat (751.7+58 pg/ml), Batinah (770.6+41 pg/ml) and Sharqiya (808.5+81.6 pg/ml) regions. The mean serum vitamin B12 level of goats in Dhahira region (572.8+56.8 pg/ml) was signi¢cantly lower (p50.05) than the mean values of goats in Sharqiya region (808.5+81.6 pg/ml) (Figure 2).

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Figure 2. Mean serum vitamin B12 levels (pg/ml) in ¢ve di¡erent regions in Oman

Relationship between sex and serum vitamin B12 levels There was no signi¢cant e¡ect of sex on the mean serum vitamin B12 levels of male and female goats. Faecal egg counts From the survey results, none of the farmers routinely dewormed their animals. Faecal egg counts were generally high in all goats. No relationship was observed between the serum vitamin B12 levels and faecal egg counts. However, goats with low levels of serum vitamin B12 had signi¢cantly higher coccidial counts (Figure 3). DISCUSSION In the present study, the serum vitamin B12 levels of 200 goats from ¢ve regions of Oman ranged from 105 to 42000 pg/ml with a mean value of 723.1 pg/ml. The normal range for adult goats maintained at the Sultan Qaboos University Agricultural

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Figure 3. Log relationship between mean serum vitamin B12 levels (pg/ml) in six di¡erent age groups versus coccidial oocyst counts (oocysts/g) (&, serum vitamin B12 levels; ^, coccidial oocyst counts)

Experiment Station (AES) is 940^1131 pg/ml. These goats, however, receive a standard diet consisting of hay ad libitum and a commercially prepared concentrate ration containing 0.16 ppm of cobalt. In addition, these animals had access to mineral blocks containing cobalt. The results from the present study are more likely to be representative of the serum vitamin B12 status of goats in Oman within the ¢ve regions studied. The study also takes into account the variability associated with age, general feeding practices and regional e¡ects. There is scarcity of information available on normal serum values of vitamin B12 in goats. Mburu and colleagues (1993) reported a range of 200^250 pg/ml, whereas Mgongo and colleagues (1981) reported normal values to be 400 pg/ml in East African

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short-horned goats. It is di¤cult to compare these values. In the present study a microparticle enzyme immunoassay was employed, whereas in the other studies radioimmunoassays and competitive binding assays were used. These assays are likely to di¡er signi¢cantly in their respective sensitivities. In addition, previous studies looked at serum vitamin B12 levels of adult animals, whereas in the present study goats were surveyed ranging from kids to 1 month of age to adults over 11 years of age. There are also likely genetic di¡erences between breeds of the same species. Indeed, we have observed di¡erent plasma copper levels for di¡erent breeds of goats but of the same age and receiving identical diets (Osman et al., 2000). The present study showed that serum vitamin B12 levels were signi¢cantly (p50.05) lower in the animals in the 1^3 month age group than in older animals. This is supported by the ¢ndings of Findlay (1972), who found that adult sheep tend to have higher serum vitamin B12 levels than young sheep. Colostrum provides the only source of vitamin B12 to the newborn during the ¢rst 24 h (Ramos et al., 1994) to 48 h (Halpin and Caple, 1982) after birth, and thereafter vitamin B12 levels begin to decline over a period of days. In addition, neonates born to dams with insu¤cient levels of vitamin B12 in their colostrum will be expected to exhibit much lower levels of serum vitamin B12 levels. Robertson (1971) suggested that the need of young animals for vitamin B12 is greater than that of adults because of their higher metabolic rate. Young goats also have underdeveloped rumens in which the microbial synthesis of vitamin B12 may not be at optimum levels. Although the cobalt levels in the feeds provided to the goats in the present study were not determined, it is clear that the dietary intake of cobalt played a signi¢cant role in the serum vitamin B12 values observed. Indeed, there was a signi¢cant di¡erence in vitamin B12 levels observed in goats that were maintained on diets that included commercial feeds in Muscat, Batinah and Sharqiya regions. The commercial feeds in Oman are all supplemented with cobalt. The varying levels of serum vitamin B12 found in goats in the study in di¡erent regions of Oman may be due to di¡erent levels of alkalinity of the soils. The majority of soils in Oman are alkaline, which is generally associated with low levels of cobalt (unpublished observations). Animals grazing on plants from these soils would conceivably receive inadequate amounts of cobalt, as grasses are not generally rich in cobalt compared to legumes and many other plants (Robertson, 1971; Smith and Sherman, 1994). In Oman, grasses and shrubs are the main sources of nutrients for grazing goats. It cannot be ruled out, however, that some animals may exhibit low levels of serum vitamin B12 as a response to secondary minerals in their diet that may inhibit the intestinal absorption of cobalt. Similarly to the ¢nding of Ulvund (1990) in lambs, the present study showed no signi¢cant di¡erence between males and females. However, Vellema and colleagues (1997) showed sex-related di¡erences in weight gains and survival rates from the age of 1 month until 5 months in sheep, where both the non-supplemented ewe and ram lambs showed weight loss. It was postulated that the sex di¡erences might be due to the fact that rams are more active and grow faster than females and therefore need to consume more feed to meet their nutritive requirements.

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It is generally appreciated that infections with coccidia occur most commonly in young goats between 3 weeks and 5 months of age (Smith and Sherman, 1994). Although it can be argued that this coincides with the period during which young animals have not fully developed an immunological repertoire capable of limiting the development of this parasite, it cannot be ruled out that there might be a causal relationship between low levels of serum vitamin B12 and susceptibility to infection. Such a relationship has been observed by MacPherson and colleagues (1987) and by Paterson and MacPherson (1990), who reported that cobalt-de¢cient calves had signi¢cantly higher worm egg counts than cobalt-supplemented calves. Similar results were reported by Ferguson and colleagues (1989) in lambs with Ostertagia circumcincta infections. The results of the present study indicate that there is a broad range of serum vitamin B12 levels in Omani goats. The lower values observed in kids 1^3 months of age suggest that this might be a very critical period during the development of the young animal. Indeed, we have observed under experimental conditions that low dietary intake of cobalt in animals of this age group results in severe clinical manifestations compared to those in older animals (unpublished observations). Further studies are underway to determine the exact dietary requirements of cobalt for Omani goats and to assess the clinical e¡ects of sub-optimal levels of cobalt on the health, growth and the immune system of these animals. REFERENCES Bigger, G.W., Elliott, J.M. and Rickard, T., 1976. Estimation of pseudovitamin B12 factor A and factor B in sheep. Journal of Animal Science, 42, 1077^1081 Black, H., Hutton, J.B., Sutherland, R.J. and James, M.P., 1988. White liver disease in goats. New Zealand Veterinary Journal, 36, 15^17 Clark, R.G., Mantelman, L. and Verkerk, G.A., 1986. Failure to obtain a weight gain response to vitamin B12 treatment in young goats grazing pasture that was cobalt de¢cient for sheep. New Zealand Veterinary Journal, 35, 38^39 Fell, B.F., 1981. Pathological consequences of copper de¢ciency and cobalt de¢ciency. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 294, 153^169 Ferguson, E.G., Mitchell, G.B. and MacPherson, A., 1989. Cobalt de¢ciency and Ostertagia circumcincta infection in lambs. The Veterinary Record, 124, 20 Findlay, C.R., 1972. Serum vitamin B12 levels and the diagnosis of cobalt de¢ciency in sheep. The Veterinary Record, 90, 468^471 Fisher, G.E. and MacPherson, A., 1991. E¡ect of cobalt de¢ciency in the pregnant ewe on reproductive performance and lamb viability. Research in Veterinary Science, 50, 319^327 Gall, L.S., Smith, S.E., Becker, D.E., Stark, C.N. and Loosli, S.K., 1949. Rumen bacteria in cobalt de¢cient sheep. Science, 109, 468^469 Halpin, C.G. and Caple, I.W., 1982. Vitamin B12 nutrition of foetal and newborn lambs. Proceedings of the Australian Society of Animal Production, 14, 658 Johnson, E.H., Muirhead, D.E., King, G.J., Ochei, J. and Al-Busaidy, R., 1999a. An abattoir survey of caprine liver diseases in the Sultanate of Oman. Veterinary Journal, 158, 216^220 Johnson, E.H., Muirhead, D.E., Annamalai, K., King, G.J., Al-Busaidy, R. and Shahul Hameed, M., 1999b. Hepatic lipidosis associated with cobalt de¢ciency in Omani goats. Veterinary Research Communications, 23, 215^221 MacPherson, A., Gray, D., Mitchell, G.B. and Taylor, C.N., 1987. Ostertagia infection and neutrophil function in cobalt-de¢cient and cobalt supplemented cattle. British Veterinary Journal, 143, 348^353

481 Marston, H.R. and Lee, H.J., 1952. Response of cobalt-de¢cient sheep to massive doses of vitamin B12. Nature, 17, 791 Marston, H.R. and Smith, R.M., 1952. Control of cobalt de¢ciency in sheep by injection of vitamin B12. Nature, 170, 792^793 Mburu, J.N., Kamau, J.M.Z. and Badamana, M.S., 1993. Changes in serum levels of vitamin B12, feed, live weight and hematological parameters in cobalt de¢cient small East African goats. Internationale Zeitschrift fur Vitamin-und Ernahrungsforschung, 63, 135^139 Mgongo, F.O.K., Gombe, S. and Ogaa, J.S., 1981. Thyroid status in cobalt and vitamin B12 de¢ciency in goats. The Veterinary Record, 109, 51^53 Osman, N.H.I.E.D., Johnson, E.H., Al-Busaidy, R. and Suttle, N.F., 2003. The e¡ects of breed, neonatal age and pregnancy on the plasma copper status of goats in Oman. Veterinary Research Communications, 27, 219^229 Paterson, J.E. and MacPherson, A., 1990. The in£uence of a low cobalt intake on the neutrophil function and severity of Ostertagia infection in cattle. British Veterinary Journal, 146, 519^530 Pearson, A.B., 1987. White liver disease and pestivirus infection in goat kids. Surveillance, 14, 21 Ramos, J.J., Saez, T., Bueso, J.P., Sanz, M.C. and Fernandez, A., 1994. Vitamin B12 levels in ewes colostrum and milk and in lamb serum. The Veterinary Research, 25, 405^409 Robertson, W.W., 1971. Cobalt de¢ciency in ruminants. The Veterinary Record, 89, 5^12 SAS, 1993. SAS/STAT User's Guide, Volume 2, Version 6, (Statistical Analysis Systems, Cary, NC) Smith, C.S. and Sherman, D.M., 1994. Goat Medicine, (Lea and Febiger, Philadelphia) Sutherland, R.J., Cordes, D.O. and Carthes, G.C., 1979. Ovine white liver disease ^ an hepatic dysfunction associated with vitamin B12 de¢ciency. New Zealand Veterinary Journal, 27, 227^232 Ulvund, M.J., 1990. Ovine white liver disease (OWLD). Vitamin B12 and methyl malonic acid (MMA) estimations in blood. Acta Veterinaria Scandinavica, 31, 267^275 Ulvund, M.J. and Pestalozzi, M., 1990. Ovine white liver disease (OWLD) in Norway: clinical symptoms and preventative measures. Acta Veterinaria Scandinavica, 31, 53^62 Vellema, P., Rutten, V.P., Hoek, A., Moll, L. and Wentink, G.H., 1996. The e¡ect of cobalt supplementation on the immune response in vitamin B12 de¢cient Texel lambs. Veterinary Immunology and Immunopathology, 55, 151^161 Vellema, P., Moll, L., Barkema, H.W. and Schukken, Y.H., 1997. E¡ect of cobalt supplementation on serum vitamin B12 levels, weight gain and survival rate in lambs grazing cobalt-de¢cient pastures. Veterinary Quarterly, 19, 1^5 Walker, C.K. and Elliot, J.M., 1972. Lactational trends in vitamin B12 status on conventional and restricted roughage rations. Journal of Dairy Science, 55, 474^478 (Accepted: 26 March 2003) Taux de vitamine se¨rique B12 chez des che©vres Omani Re¨sume¨ ^ Des e¨chantillons de sang et fe¨caux e¨manant de 200 che©vres Omani des deux sexes et de tranches d'aªge di¡e¨rentes, provenant de cinq re¨gions di¡e¨rentes d'Oman, ont e¨te¨ e¨tudie¨s. Des questionnaires a© type d'enqueªte ont e¨te¨ entrepris sur chaque che©vre. Les valeurs de la vitamine se¨rique B12 se sont situe¨es dans la plage de 105 a© 42000 pg/ml, avec une valeur moyenne de 723.1 pg/ml et un intervalle de con¢ance de 61 a© 1507 pg/ml. Les chevreaux de la tranche d'aªge de 1 a© 3 mois ont pre¨sente¨ des taux de vitamine se¨rique B12 conside¨rablement plus bas que les animaux plus aªge¨s. Des taux variables de vitamine se¨rique B12 ont e¨te¨ retrouve¨s dans di¡e¨rentes re¨gions d'Oman et dans des fermes de la meªme re¨gion. Les che©vres maintenues sur une nourriture commerciale avaient des taux de vitamine se¨rique B12 plus e¨leve¨s que celles maintenues au pre¨ ou nourries de restes de nourriture domestique, comme c'est la pratique courante en Oman. Les nume¨rations des coccidies ont e¨te¨ les plus e¨leve¨es chez les animaux a© taux de vitamine se¨rique B12 plus bas. Les re¨sultats de cette e¨tude indiquent qu'il existe une vaste gamme de taux de vitamine se¨rique B12 chez les che©vres Omani et sugge©rent que les animaux plus jeunes, a© taux de vitamine se¨rique B12 plus bas pourraient eªtre plus pre¨dispose¨s a© des carences en vitamine B12 que les autres animaux.

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Niveles de vitamina B12 en suero en cabras Oman|¨ es Resumen ^ Se estudiaron muestras sangu|¨ neas y fecales de 200 cabras oman|¨ es de ambos sexos y distintos grupos de edad, en cinco regiones diferentes de Oma¨n. Se llevaron a cabo cuestionarios de inspeccio¨n para cada cabra. Los valores de vitamina B12 en suero variaban de 105 a 42000 pg/ml, con un valor medio de 723.1 pg/ml y un intervalo de con¢anza del 95% de 61^1507 pg/ml. Las cabritillas de 1^3 meses de edad mostraban niveles de vitamina B12 en el suero signi¢cativamente ma¨s bajos que las cabras mayores. Se encontraron distintos niveles de vitamina B12 en suero en diferentes regiones de Oma¨n y en distintas granjas de la misma regio¨n. Aquellas cabras que se sustentaban con alimentos comerciales mostraban niveles ma¨s altos de vitamina B12 en el suero, que aquellas que se sustentaban con pastos o se alimentaban de sobras dome¨sticas, como es la pra¨ctica habitual en Oma¨n. Los recuentos coccidiales resultaron ma¨s altos en animales con bajos niveles de vitamina B12 en suero. Los resultados de este estudio indican que hay una gran variedad de niveles de vitamina B12 en suero en las cabras oman|¨ es, y sugiere que los animales ma¨s jo¨venes, que tienen unos niveles ma¨s bajos de vitamina B12 en suero, podr|¨ an ser ma¨s susceptibles a de¢ciencias de vitamina B12 que los animales mayores.