T. Apichartsrungkoon, P. Pongpiachan¹, C. Khoohathapharakand K. Sanwong²

Abstract

Faecal samples were obtained from 7 female bantengs and 2 male bantengs (controls) twice a week during hot-dry season (April - May 1995), hot-wet season (August-September 1995) and cold-dry season (December 1995-January 1996). Faecal progesterone concentrations were assayed by radioi-mmunoassay (RIA) technique. Progesterone profiles were found in 4 cycling females with the baseline levels of < 60 ng/g dry faeces and maximum levels ranged from 525 to 1,380 ng/g dry faeces during hot-dry and hot-wet seasons but the ovarian activities were hardly detected in cold-dry season. In 3 pregnant bantengs, progesterone profiles fluctuated with the progesterone levels ranged from < 60 to 1,740 ng/g dry faeces. In conclusion, progesterone profiles were detected in female bantengs compared with the absent profile of the males in hot-dry and hot-wet seasons but not in cold-dry season. This may be due to seasonal dietery variations.

Banteng (Bos javanicus) is one of the most beautiful wild cattle in South East Asia. It ranges from north Burma to northern edge of the Malay Peninsula, Thailand, Cambodia, Laos, Java, Bali and Borneo. Banteng in Thailand is of the subspecies of Bos javanicus birmanicus which are also found in other countries on the Asian mainland. Banteng is bigger than native domestic cattle. The coat is golden brown or chestnut in males and bright rufous brown or fawn in females (IUCN/SSC, 1995). The markings of banteng includes white "stocking", white lips, white hair in the ears and a large white rump patch.

Today, Banteng is seriously endangered. The world population is less than 10,000 and the population in Thailand is around 500. Srikosamatara and Suteethorn (1995) reported that the number of banteng in Thailand decreased by approximately 80% during the past 25 years, from 2,300-2,500 animals in 1970 to 500 animals in 1995. Hunting, habitat degradation and diseases from domestic livestock are the major reasons for the reduction in population.

Increase in number of banteng is required urgently. A possible way is to breed more animals in zoological parks and then release them in protected natural forest. The study of banteng reproduction is important for breeding management. Progesterone determination in females is a criterion in monitoring the ovarian function. However, taking blood sample from wild animals seems to be impossible. Therefore, noninvasive methods like determining the concentration of progesterone in faeces might be an alternative. Faecal sample is easy to collect and can be done without disturbing the animals. The purpose of the present study was to monitor the ovarian function of the banteng by determination of faecal progesterone level.

Seven female bantengs (cow no. 1-7), 10 - >20 years old, at Chiangmai Zoo, were used for this study with 2 males (bull no. 1-2) as the controls. They were divided into 2 groups and kept in 2 separate restricted areas. 4 females and 2 males in one group and 3 females in another group. Their diet varied from season to season with water ad libitum.

Fresh faecal samples were collected from the ground and were stored at -20 C until analysis. Samples were taken twice a week (3-4 days interval) during hot-dry season (April-May 1995), hot-wet season (August-September 1995) and cold-dry season (December 1995-January 1996).

Faecal steroids were extracted using the technique developed by Wasser et al. (1993). Faecal sample was dried in an oven at 45 C overnight. Then 0.1 g dry faeces was mixed with 3 ml ethanol and shaken for 30 minutes. The mixture was centrifuged at 1000X G for 10 minutes. The supernatant was then used for progesterone concentration measurement by radio-immunoassay (RIA) technique.

Progesterone analysis from extracted faecal samples by RIA, based on the method described by Pongpiachan and Apichartsrungkoon (1990) was shown in figure 1. Antisera was raised in rabbit against progesterone linked with molecule of bovine serum albumin. Labeled progesterone used in the assay was [1,2,6,7 - ³H] - P₄ . The separation of bound/free fractions was accomplished by the use of charcoal solution. The 50% relative bending of the standard curves ranged from 168 to 228 ng/g dry faeces. The intra-assay and inter - assay coefficient of variation were 19.4% and 11.15%, respectively. Faecal progesterone concentration was expressed in nanogram per gram dry faeces (ng/g dry faeces).

During the period of study, cow no. 2, 3 and 4 were pregnant. The parturition dates were 26/12/95, 06/03/96 and 19/04/96, respectively. Progesterone profile of each animal was presented in figure 2. During hot-dry and hot-wet seasons, changes in progesterone levels with the baseline levels of < 60 mg/g dry faeces and maximum levels ranged from 525 to 1,380 ng/g dry faeces were found in cycling nonpregnant cows, compared to the steady basal levels of progesterone profiles in bulls. Normal oestrous cycles, ranging from 18-21 days, were also observed in cycling cows. During cold-dry season, the progesterone profiles were not clear due to the incomplete faecal sampling. However, progesterone levels tended to be lower in cold-dry season than those in hot-dry and hot-wet seasons.

In the 3 pregnant cows, progesterone levels fluctuated between

< 60 to 1,740 ng/g dry faeces. Progesterone profiles were not consistent with the pregnancy status, particularly during cold-dry season.

0.1 g dry faeces + 3 ml ethanol

¯ shake for 30 min.

centrifuge at 1000X g , 10 min.

dilute the supernatant by PBS ratio 1:20

50 ul faecal supernatant, P₄ standard sol.

add 10 ul antiserum

¯ vortex, leave for 1 hr.

add 100 ul ³H-P₄

¯ vortex, 4 íC, leave overnight

add 250 ul charcoal solution

¯ vortex, 4 íC, leave for 20 min.

centrifuge 3,000 g, 4 íC , 15 min.

separate bound form

add 1 ml Scintillation

¯ leave overnight

Beta counter

Fig. 2 Concentration of progesterone in faecal samples of female bantengs (Cow no. 1-7) and male bantengs (Bull no. 1-2) during hot-dry, hot-wet and cold-dry seasons. H-D = hot-dry, H-W = hot-wet, C-D = cold-dry. (H-D-0 = 17/04/1995, H-W-0 = 07/08/1995, C-D-0 = 4/12/1995)

Mekchay (1994), studying dairy cattle, confirmed that there was a relationship between milk and faecal progesterone concentrations with a maximum positive correlation (r = 0.739) for third degree polynominal on curvilinear regression; Y_cubic = -1.157 + 0.0083x - 2.25*10^-6x² + 2.1*10^-10x³. This was supported by Wasser et al. (1993) who reported the relationship between serum and faecal progesterone concentrations in baboons. The use of faecal progesterone concentrations for ovarian activity determination and pregnancy diagnosis were supported by Mekchay (1994), in dairy cattle; Schwarzenberger et al. (1991), in mares ; Schwarzenberger et al. (1992), in mares; Schwarzenberger et al. (1993), in okapi and Larter et al. (1994), in dairy cattle. Therefore, only faecal samples were used in this study without the collection of blood sample to avoid disturbing the animals. There were progesterone profiles showed in cycling and pregnant female bantengs compared to the absent profiles of the males. However, the progesterone concentrations were not consistent with the ovarian status in some periods of study, particularly in cold-dry season. This may due to the variation of diet that animals received in different seasons. Wasser et al. (1993) reported that faecal progestagen concentrations decreased as dietary fibre increased. They suggested that indexing faecal progestagens by cholestanone (cholesterol metabolite which positively correlated with dietary fibre) improved the correlation between serum and indexed faecal progestagens. We suggested that more study should be done to confirm the results and to adjust the progesterone value calculation.

This study was supported by the grant from Chiangmai University (in 1997). We also thank the staff of Chiangmai Zoo for the collection of samples and Mr. C. Tonginn for technical assistance.

IUCN/SSC (1995) Asian Wild Cattle, Conservation Assessment and Management Plan Workshop. p. 51-52.

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Wasser, S.K., Thomas, R., Nair, P.P., Guidry, C., Southers, J., Lucus, J., Wildt, D.E. and Monfort, S.L. (1993) Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus). J. Reprod. Fert. 97 : 569 574.