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Plasma concentrations of progesterone during oestrous cycles of ethiopian menz sheep using enzyme immunoassay
Small Ruminant Research, 3 (1990) 57-62
57
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Plasma Concentrations of Progesterone during Oestrous Cycles of Ethiopian Menz Sheep using Enzyme Immunoassay E. MUKASA-MUGERWA, ZERE EZAZ and P. VIVIANI
International Livestock Centre for A[rica (ILCA), P.O. Box 5689, Addis Ababa (Ethiopia) (Accepted 19 November 1988)
ABSTRACT Mukasa-Mugerwa, E., Zere Ezaz and Viviani, P., 1990. Plasma concentrations of progesterone during oestrous cycles of Ethiopian Menz sheep using enzyme immunoassay. Small Rumin. Res., 3: 57-62. Plasma progesterone concentrations were assessed with a commercially available ELISA technique in 20 Ethiopian Menz ewes during their oestrous cycles which averaged 17.2 _+1.0 (range 15-20 ) days (n = 36). Average progesterone levels were significantly (P < 0.0001 ) different between ewes (range 2.43-4.80 ng/ml) and days (P < 0.0001 ) of their oestrous cycles (0.32-5.84 ng/ ml ). Progesterone values were under 1.0 ng/ml from 2 days before to 4 days after oestrus. Hormone concentration rose steadily to peak at 5.0-5.6 ng/ml on days 10 to 14. This was followed by a rapid decline to 3.0 ng/ml (53% of day 14 peak value) on day 15; 0.8 ng/ml (15% of peak value) on day 16; and 0.2 ng/ml (3.7%) on the day before oestrus. It is concluded that the ELISA method can be used for progesterone determination in Ethiopian sheep, and also, that progesterone levels of under 1.0 ng/ml are indicative of either anoestrous or the follicular and early luteal phases of the oestrous cycle.
INTRODUCTION
Reproductive wastages can strain tropical sheep productivity to the extent that only 800 t of mutton and lamb are produced from the approximately 12 million sheep in Africa (FAO, 1985). There are several reports on small ruminant reproductive performance in tropical Africa (Jollans, 1960; Wilson, 1975; ILCA, 1979; Mulokwu and Umunna, 1980; Agyemang et al., 1985; Hambolu and Ojo, 1985; Hambolu et al., 1985; Mukasa-Mugerwa and Tekelye, 1988). Nevertheless, details of reproductive physiology are scanty in contrast to that of temperate breeds (Thorburn et al., 1969; McNatty et al., 1973; Sarda et al., 1973; Quirke et al., 1979). Monitoring peripheral progesterone concentration is useful in studying reproductive physiology of ewes (Bassett et al., 1969; Thorburn et al., 1969; Rob0921-4488/90/$03.50
© 1990 Elsevier Science Publishers B.V.
58
E. MUKASA-MUGERWAET AL.
ertson and Sarda, 1971 ). However, most investigations have been carried out with radioimmunoassay (RIA) techniques, even in Africa (Katongole and Gombe, 1985). Consequently, laboratories that are not equipped or qualified to handle radioisotopes have been unable to monitor progesterone values. The enzyme-linked immunosorbent assay (ELISA) technique, using plasma or serum (Nakao, 1980; Boland et al., 1985; Parker et al., 1988) or milk (Sauer et al., 1981; Foulkes et al., 1982) partly overcomes this problem (Nakao et al., 1982). This study aimed to (1) investigate progesterone levels during the oestrous cycle of indigenous sheep using the ELISA method, and (2) establish minimum (discriminatory) progesterone values that could be adopted in investigations on anoestrus, pregnancy diagnosis and prenatal reproductive wastage. MATERIALSAND METHODS
Study location and animals The study was carried out at the ILCA Debre Berhan experimental station, 120 km north of Addis Ababa, during the dry but cool months of April and May 1988. Twenty-eight Ethiopian Menz type ewes (Mukasa-Mugerwa and Tekelye, 1988), 3 to 4 years old, and averaging 31 + 2 kg body weight, were used. Ewes scored 3 to 4 in body condition (Russell et al., 1969). Scores of 0, 1, 2, 3, 4 and 5 represent animals in emaciated, poor, fair, good, very good and excellent condition, respectively. All ewes had weaned a lamb during the previous 3 months but had been kept isolated from a fertile ram. The flock was run over 4 months with a vasectomised, harnessed ram to help detect oestrus. Animals were grazed during the day and penned at night with water and mineral licks available ad libitum. Blood collection Blood samples were collected daily from 20 ewes, starting 10 days after first oestrus was observed. Samples were taken from the jugular vein into heparinised vacutainers (Becton Dickinson). Sampling was continued until ewes had exhibited two subsequent oestrous periods. Plasma was recovered by centrifugation within 20 rain and was stored frozen at - 2 0 °C until required for progesterone assay. Progesterone determination Plasma progesterone concentration was estimated by the enzyme-linked immunosorbent assay (ELISA) technique (Boland et al., 1985; Parker et al., 1988) using Ovucheck test kits from Cambridge Veterinary Sciences (U.K.). The test is based on competitive binding between unlabeUed progesterone in test samples and a known quantity of enzyme alkaline phosphatase (AP)-labelled progesterone for binding sites of a standardised amount of specific anti-proges-
PLASMA PROGESTERONE OF CYCLING ETHIOPIAN MENZ SHEEP MEASURED BY ELISA
59
terone antibody, precoated onto microtiter plates. Ten/~1 of four undiluted standards (0.5, 1.0, 5.0 and 10.0 ng/ml) and each test sample were added to the appropriate wells on the microtiter plates. As far as possible, samples from the same ewe were analysed using the same kit. Two hundred/ll of conjugate (progesterone-alkaline phosphatase) were added to each well. The plate was covered with aluminium foil and incubated at room temperature (19-22 °C) for 30 min. After this period, the plates were emptied, washed twice with cold water and tap-dried onto absorbent paper. Two hundred/zl of substrate (pnitrophenyl phosphate: prepared by dissolving 3 X 40 mg tablets of the substrate in 25 ml of substrate buffer containing 1 M diethanolamine and 0.5 mM magnesium chloride at pH 9.8) were then added to each well and the plate again incubated as described. After this period, 100/zl of stopper solution (supplied as 0.5 M dipotassium hydrogen orthophosphate and 5 mM EDTA at pH 10.0) were added to all wells and the plate was read at 405 nm, using a Biorad plate reader after blanking the instrument with air. Standard absorbancy values were plotted and the curve obtained was used to interpolate progesterone values in test samples with a programmable Hewlett Packard microcomputer. For plasma containing 5.1 ng/ml progesterone, the intra- and inter-assay coefficients of variation for kits delivered in batches has been established locally as 13.9% and 39.6%, respectively. Data were analysed by least squares (Harvey, 1976) using a model in which animals were regarded as a main effect and day of the cycle as a continuous variable. RESULTS
Mean length of oestrous cycles was 17.2+1.0 days ( n = 3 6 ) . Two cycles (5.6%) lasted 15 days, five (13.9%) were 16 days, seventeen (47.2%) lasted 17 days, ten (27.8%) were 18 days and one each were 19 and 20 days. Two animals had oestrous cycles of 31 and 32 days after manifesting silent oestrus without external signs as detected by hormone analyses. The general pattern of plasma progesterone during normal cycles is shown in Fig. 1. Average progesterone values differed significantly between ewes (P < 0.0001, range 2.43-4.80 ng/ml) and day of oestrous cycle (P < 0.0001, range 0.32-5.84 ng/ml). In general, mean values were under 1.0 ng/ml (basal levels) from 2 days before oestrus (the "follicular" phase) until 4 days after oestrus (the "early luteal" phase). Average plasma progesterone was 0.46 + 0.26 ng/ml on the day of oestrus (day 0). A "rising" phase in hormone concentration was noticeable from day 5 (1.45 + 0.52 ng/ml) to day 9 (4.69 + 0.70 ng/ml). This was followed by peak mean values of 5.0-5.6 ng/ml from day 10 to day 14 (the "plateau" phase). Thereafter, there was a rapid drop during the "declining" phase from day 15 as animals again moved into the follicular phase, with basal progesterone levels, of the next oestrous cycle. From the time progesterone
60
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ET AL.
? A
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12
14
16 0
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Fig. 1.Meandailyplasmaprogesteroneconcentrationduringthe oestrouscycleofEthiopianHighland ewes (El -- oestrus 1; E2= oestrus 2). decline started, concentration dropped to 3.0 ng/ml (53% of the day 14 peak of 5.61 ng/ml taken as 100%) within 24 h, to 0.84 ng/ml (15%) in 48 h and 0.21 ng/ml (3.7%) in 72 h, the day before oestrus. DISCUSSION The mean oestrous cycle length of 17.2 + 1.0 days obtained in this study is intermediate between estimates of 16.4-18.6 days, previously reported for tropical ewes (Narayanaswamy and Belaine, 1976; Galliard, 1979; Yenikoye et al., 1981; Yenikoye, 1983; Farid and Makarechian, 1987) and cycling temperate ewes (Hafez, 1952; Joubert, 1962; Lamond et al., 1972; Quirke, 1978; Quirke et al., 1979). Sheep in the tropics tend to breed year-round with definite peaks (Mukasa-Mugerwa and Tekelye, 1988). These peaks are modulated through variable nutrition and other environmental factors. Consequently, not all ewes may exhibit cycles of 15-20 days. Cycle lengths of 28-30 days were observed by El-Wishy et al. (1971) among some Awassi ewes while cycles of 70 days were reported by Yenikoye et al. (1982) in some Fulani ewes. Nevertheless, extended cycles may result also from improper heat detection, and some ewes may not exhibit oestrus externally. This was noticed in two (5.2%) of the 40 heats in this study through monitoring plasma progesterone levels. Except for reports of Yenikoye et al. (1981,1982) and Yenikoye (1983), data on reproductive endocrinology of African tropical sheep are very scanty. Patterns in progesterone concentrations in this study agree with previous studies and those for temperate ewes in the tropics (Martinez et al., 1980). Present average progesterone values were, however, higher than those found by Yenikoye ( 1983 ) whose peak values in the luteal phase were 1.5 ng/ml. In contrast, our values were in the range of 3.5-7.5 ng/ml peaks as obtained by Rhind et
PLASMAPROGESTERONEOF CYCLINGETHIOPIANMENZSHEEP MEASUREDBYELISA
61
al. (1985), who like Yenikoye (1983) used RIA for progesterone determination. Irrespective of differences in assay techniques, it was evident that progesterone levels were under 1.0 ng/ml in all studies from 2 days before to 4 days after oestrus. This level can therefore be adopted as the minimum (discriminatory) value to indicate anoestrus or the follicular and early luteal phase of the oestrous cycle. This level may, therefore, be used in studies of prenatal reproductive wastage, detecting silent oestrus and to assist in the better reproductive management of Ethiopian Menz sheep. ACKNOWLEDGEMENTS
Thanks are due to Mr. Tekeste Tibebu and Mr. Girma Abebe in sample collection and processing, and Mr. Solomon Zewde for assistance in data analysis. REFERENCES Agyemang, K., Negussie, A., Voorthuizen, A. and Anderson, F.M., 1985. A rapid survey of reproduction in the traditional sector of Debre Berhan, Ethiopian Highlands. In: R.T. Wilson and D. Bouzart (Editors), Proc. Conf. Small Ruminants in African Agriculture. ILCA, Addis Ababa, Ethiopia, 30 Sept.-4 Oct., pp. 175-185. Bassett, J.M., Oxborrow, T.J., Smith, I.D. and Thorburn, G.D., 1969. The concentration of progesterone in the peripheral plasma of the pregnant ewe. J. Endocrinol., 45: 449. Boland, M.P., Foulkes, J.A., MacDonnell, H.F. and Sauer, M.J., 1985. Plasma progesterone concentrations in superovulated heifers determined by enzymeimmunoassay and radioimmunoassay. Br. Vet. J., 144: 409-415. El-Wishy, A.B., EI-Sawaf, S.A. and E1-Mikkawi, F., 1971. Some aspects of reproduction in fattailed sheep in sub-tropics. I. Reproductive behaviour of local Ausimi and imported Awassi ewes. Vet. Med. J. United Arab Rep., 19: 131-155. FAO, 1985. Production Yearbook, Vol. 39. Food and Agricultural Organization, Rome, Italy. Farid, A. and Makarechian, M., 1987. Onset of oestrus and length of oestrous cycle during breeding season in five breeds of fat-tailed sheep. World Rev. Anim. Prod., 23(2): 39-45. Foulkes, J.A., Cookson, A.D. and Saner, M.J., 1982. Artificial insemination of cattle based on daily enzyme immunoassay of progesterone in whole milk. Vet. Rec., 111: 302-303. Gaillard, Y., 1979. Caracteristiques de la reproduction de la brebis Oudah (Reproductive characteristics of the Uda ewe). Rev. Elev. Med. Vet. Pays Trop., 32: 285-290. Hafez, E.S.E., 1952. Studies in the breeding season and reproduction of the ewe. J. Agric. Sci. (Camb.), 42: 189-265. Hambolu, J.O. and Ojo, S.A., 1985. Ovarian activity of Sokoto Red goats using abattoir specimens. Theriogenology, 23 (2): 273-282. Hambolu, J.O., Ojo, S.A., Jamdar, M.N. and Molokwu, E.C.I., 1985. Ovarian activity of Yankasa sheep using abattoir specimens. Theriogenology, 23 (2): 263-272. Harvey, W.R., 1976. User's guide for mixed model least-squares and maximum likelihood computer program (LSML 76). Ohio State University, Columbus, OH. ILCA, 1979. Small Ruminant Production in the Humid Tropics. ILCA Systems Study No. 3. International Livestock Centre for Africa, Addis Ababa, Ethiopia, 122 pp. Jollans, J.L., 1960. A study of West African dwarf sheep in the closed forest zone of Ashanti. West Afr. J. Biol. Appl. Chem., 3(4): 74-78. Joubert, D.M., 1962. Sex behaviour of purebred and crossbred Merino and Blackface Persian ewes. J. Reprod. Fertil., 3: 41-49.
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Katongole, C.B. and Gombe, S., 1985. A study of the reproductive hormones of indigenous goats in Uganda. In: R.T. Wilson and D. Bouzart (Editors), Proc. Conf. Small Ruminants in African Agriculture. ILCA, Addis Ababa, Ethiopia, 30 Sept.-4 Oct., pp. 2-11. Lamond, D.R., Gaddy, R.G. and Kennedy, S.W., 1972. Influence of season and nutrition on luteal plasma progesterone in Rambouillet ewes. J. Anita. Sci., 34: 626-629. Martinez, A., Herrera, J., Valencia, J. and Fernandez-Baca, S., 1980. Estudio de la actividad ovarica pos-parto mediante la determinacion de progesterona en ovejas Dorset, Suffolk y Tabasco (A study of postpartum ovarian activity by means of determining progesterone levels in Dorset, Suffolk and Tabasco ewes). Veterinaria (Mexico), 11: 127-131. McNatty, K.P., Revfeim, K.J.A. and Young, A., 1973. Peripheral plasma progesterone concentrations in sheep during the oestrous cycle. J. Endocrinol., 58: 219-225. Mukasa-Mugerwa, E. and Tekelye, B., 1988. The reproductive performance of Ethiopian Highland sheep. Anita. Reprod. Sci., 17: 95-102. Mulokwu, E.C. and Umunna, N.N., 1980. Reproductive performance of Yankasa sheep of Nigeria. Theriogenology, 14: 239-249. Nakao, T., 1980. Practical procedure for enzyme immunoassay of progesterone in bovine serum. Acta Endocrinol., 93: 223-227. Nakao, T., Sugihashi, A., Ishibashi, Y., Tosa, E., Nakagawa, Y., Yuto, H., Nomura, T., Ohe, T., Ishimi, S., Takahashi, H., Koiwa, M., Tsunoda, N. and Kawata, K., 1982. Use of milk progesterone immunoassay for early pregnancy diagnosis in cows. Theriogenology, 18 (3): 267-274. Narayanaswamy, M. and Belaine, D.S., 1976. A note on the oestrous cycle in Bannur sheep. Indian Vet. J., 53: 235-236. Parker, B.N.J., Foulkes, J.A., Jones, P.C., Dexter, I. and Stephens, H., 1988. Prediction of calving times from progesterone concentrations. Vet. Rec., 122 (4): 88-89. Quirke, J.F., 1978. Onset of puberty and oestrous activity in Galway, Finnish Landrace and Finncross ewe lambs during their first breeding season. Irish J. Agric. Res., 17: 15-23. Quirke, J.F., Hanrahan, J.P. and Gosling, J.P., 1979. Plasma progesterone levels throughout the oestrous cycle and release of LH at oestrus in sheep with different ovulation rates. J. Reprod. Fertil., 55: 37-44. Rhind, S.M., Leslie, I.D., Gunn, R.G. and Doney, J.M., 1985. Plasma FSH, LH, prolactin and progesterone profiles of Cheviot ewes with different levels of intake before and after mating, and associated effects on reproductive performance. Anim. Reprod. Sci., 8: 301-313. Robertson, H.A. and Sarda, I.R., 1971. A very early pregnancy test for animals: its application to the cow, ewe and sow. J. Endocrinol., 49: 407-419. Russell, A.J.F., Doney, J.M. and Gunn, R.G., 1969. Subjective assessment of body fat in live sheep. J. Agric. Sci., 72: 451-454. Sarda, I.R., Robertson, H.A. and Smeaton, T.C., 1973. Sequential changes in plasma progesterone in the ewe during the oestrous cycle and during pregnancy in intact and ovariectomised sheep. Can. J. Anim. Sci., 53: 25-34. Sauer, M.J., Foulkes, J.A. and Cookson, A.D., 1981. Direct enzyme immunoassay of progesterone in bovine milk. Steroids, 38: 45-53. Thorburn, G.D., Bassett, J.M. and Smith, I., 1969. Progesterone concentration in the peripheral plasma of sheep during the oestrous cycle. J. Endocrinol., 45: 459-469. Wilson, R.T., 1975. Comparative data on two populations of indigenous sheep and goats in Sudan and Ethiopia. Sudan J. Vet. Sci. Anim. Husb., 16: 1-11. Yenikoye, A., 1983. Etudes de quelques caracteristiques de reproduction et de leurs variations saisonniaires chez brebis Peulh du Niger. (Studies on seasonal variation in some reproductive characteristics in Peulh ewes in Niger.) In: Proc. IFS/ILCA Workshop on Small Ruminant Research in the Tropics, October, Addis Ababa, Ethiopia, pp. 205-229. Yenikoye, A., Andre, D., Ravault, J.P. and Mariana, J.C., 1981. Etude de quelques caracteristiques de reproduction chez la brebis Peuh, du Niger. (Some reproductive characteristics of the Fulani ewe in Niger. ) Reprod. Nutr. Dev., 21: 937-951. Yenikoye, A., Pelletier, J., Andre, D. and Mariana, J.C., 1982. Anomalies in ovarian function of Peulh ewes. Theriogenology, 17: 355-364.
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Plasma Concentrations of Progesterone during Oestrous Cycles of Ethiopian Menz Sheep using Enzyme Immunoassay E. MUKASA-MUGERWA, ZERE EZAZ and P. VIVIANI
International Livestock Centre for A[rica (ILCA), P.O. Box 5689, Addis Ababa (Ethiopia) (Accepted 19 November 1988)
ABSTRACT Mukasa-Mugerwa, E., Zere Ezaz and Viviani, P., 1990. Plasma concentrations of progesterone during oestrous cycles of Ethiopian Menz sheep using enzyme immunoassay. Small Rumin. Res., 3: 57-62. Plasma progesterone concentrations were assessed with a commercially available ELISA technique in 20 Ethiopian Menz ewes during their oestrous cycles which averaged 17.2 _+1.0 (range 15-20 ) days (n = 36). Average progesterone levels were significantly (P < 0.0001 ) different between ewes (range 2.43-4.80 ng/ml) and days (P < 0.0001 ) of their oestrous cycles (0.32-5.84 ng/ ml ). Progesterone values were under 1.0 ng/ml from 2 days before to 4 days after oestrus. Hormone concentration rose steadily to peak at 5.0-5.6 ng/ml on days 10 to 14. This was followed by a rapid decline to 3.0 ng/ml (53% of day 14 peak value) on day 15; 0.8 ng/ml (15% of peak value) on day 16; and 0.2 ng/ml (3.7%) on the day before oestrus. It is concluded that the ELISA method can be used for progesterone determination in Ethiopian sheep, and also, that progesterone levels of under 1.0 ng/ml are indicative of either anoestrous or the follicular and early luteal phases of the oestrous cycle.
INTRODUCTION
Reproductive wastages can strain tropical sheep productivity to the extent that only 800 t of mutton and lamb are produced from the approximately 12 million sheep in Africa (FAO, 1985). There are several reports on small ruminant reproductive performance in tropical Africa (Jollans, 1960; Wilson, 1975; ILCA, 1979; Mulokwu and Umunna, 1980; Agyemang et al., 1985; Hambolu and Ojo, 1985; Hambolu et al., 1985; Mukasa-Mugerwa and Tekelye, 1988). Nevertheless, details of reproductive physiology are scanty in contrast to that of temperate breeds (Thorburn et al., 1969; McNatty et al., 1973; Sarda et al., 1973; Quirke et al., 1979). Monitoring peripheral progesterone concentration is useful in studying reproductive physiology of ewes (Bassett et al., 1969; Thorburn et al., 1969; Rob0921-4488/90/$03.50
© 1990 Elsevier Science Publishers B.V.
58
E. MUKASA-MUGERWAET AL.
ertson and Sarda, 1971 ). However, most investigations have been carried out with radioimmunoassay (RIA) techniques, even in Africa (Katongole and Gombe, 1985). Consequently, laboratories that are not equipped or qualified to handle radioisotopes have been unable to monitor progesterone values. The enzyme-linked immunosorbent assay (ELISA) technique, using plasma or serum (Nakao, 1980; Boland et al., 1985; Parker et al., 1988) or milk (Sauer et al., 1981; Foulkes et al., 1982) partly overcomes this problem (Nakao et al., 1982). This study aimed to (1) investigate progesterone levels during the oestrous cycle of indigenous sheep using the ELISA method, and (2) establish minimum (discriminatory) progesterone values that could be adopted in investigations on anoestrus, pregnancy diagnosis and prenatal reproductive wastage. MATERIALSAND METHODS
Study location and animals The study was carried out at the ILCA Debre Berhan experimental station, 120 km north of Addis Ababa, during the dry but cool months of April and May 1988. Twenty-eight Ethiopian Menz type ewes (Mukasa-Mugerwa and Tekelye, 1988), 3 to 4 years old, and averaging 31 + 2 kg body weight, were used. Ewes scored 3 to 4 in body condition (Russell et al., 1969). Scores of 0, 1, 2, 3, 4 and 5 represent animals in emaciated, poor, fair, good, very good and excellent condition, respectively. All ewes had weaned a lamb during the previous 3 months but had been kept isolated from a fertile ram. The flock was run over 4 months with a vasectomised, harnessed ram to help detect oestrus. Animals were grazed during the day and penned at night with water and mineral licks available ad libitum. Blood collection Blood samples were collected daily from 20 ewes, starting 10 days after first oestrus was observed. Samples were taken from the jugular vein into heparinised vacutainers (Becton Dickinson). Sampling was continued until ewes had exhibited two subsequent oestrous periods. Plasma was recovered by centrifugation within 20 rain and was stored frozen at - 2 0 °C until required for progesterone assay. Progesterone determination Plasma progesterone concentration was estimated by the enzyme-linked immunosorbent assay (ELISA) technique (Boland et al., 1985; Parker et al., 1988) using Ovucheck test kits from Cambridge Veterinary Sciences (U.K.). The test is based on competitive binding between unlabeUed progesterone in test samples and a known quantity of enzyme alkaline phosphatase (AP)-labelled progesterone for binding sites of a standardised amount of specific anti-proges-
PLASMA PROGESTERONE OF CYCLING ETHIOPIAN MENZ SHEEP MEASURED BY ELISA
59
terone antibody, precoated onto microtiter plates. Ten/~1 of four undiluted standards (0.5, 1.0, 5.0 and 10.0 ng/ml) and each test sample were added to the appropriate wells on the microtiter plates. As far as possible, samples from the same ewe were analysed using the same kit. Two hundred/ll of conjugate (progesterone-alkaline phosphatase) were added to each well. The plate was covered with aluminium foil and incubated at room temperature (19-22 °C) for 30 min. After this period, the plates were emptied, washed twice with cold water and tap-dried onto absorbent paper. Two hundred/zl of substrate (pnitrophenyl phosphate: prepared by dissolving 3 X 40 mg tablets of the substrate in 25 ml of substrate buffer containing 1 M diethanolamine and 0.5 mM magnesium chloride at pH 9.8) were then added to each well and the plate again incubated as described. After this period, 100/zl of stopper solution (supplied as 0.5 M dipotassium hydrogen orthophosphate and 5 mM EDTA at pH 10.0) were added to all wells and the plate was read at 405 nm, using a Biorad plate reader after blanking the instrument with air. Standard absorbancy values were plotted and the curve obtained was used to interpolate progesterone values in test samples with a programmable Hewlett Packard microcomputer. For plasma containing 5.1 ng/ml progesterone, the intra- and inter-assay coefficients of variation for kits delivered in batches has been established locally as 13.9% and 39.6%, respectively. Data were analysed by least squares (Harvey, 1976) using a model in which animals were regarded as a main effect and day of the cycle as a continuous variable. RESULTS
Mean length of oestrous cycles was 17.2+1.0 days ( n = 3 6 ) . Two cycles (5.6%) lasted 15 days, five (13.9%) were 16 days, seventeen (47.2%) lasted 17 days, ten (27.8%) were 18 days and one each were 19 and 20 days. Two animals had oestrous cycles of 31 and 32 days after manifesting silent oestrus without external signs as detected by hormone analyses. The general pattern of plasma progesterone during normal cycles is shown in Fig. 1. Average progesterone values differed significantly between ewes (P < 0.0001, range 2.43-4.80 ng/ml) and day of oestrous cycle (P < 0.0001, range 0.32-5.84 ng/ml). In general, mean values were under 1.0 ng/ml (basal levels) from 2 days before oestrus (the "follicular" phase) until 4 days after oestrus (the "early luteal" phase). Average plasma progesterone was 0.46 + 0.26 ng/ml on the day of oestrus (day 0). A "rising" phase in hormone concentration was noticeable from day 5 (1.45 + 0.52 ng/ml) to day 9 (4.69 + 0.70 ng/ml). This was followed by peak mean values of 5.0-5.6 ng/ml from day 10 to day 14 (the "plateau" phase). Thereafter, there was a rapid drop during the "declining" phase from day 15 as animals again moved into the follicular phase, with basal progesterone levels, of the next oestrous cycle. From the time progesterone
60
E. MUKASA-MUGERWA
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? A
~6 ~5 o ~4 o~
b9
I0
12
14
16 0
2
4
6
8
I0
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16 0
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Doy of the oestrous cyc{e
Fig. 1.Meandailyplasmaprogesteroneconcentrationduringthe oestrouscycleofEthiopianHighland ewes (El -- oestrus 1; E2= oestrus 2). decline started, concentration dropped to 3.0 ng/ml (53% of the day 14 peak of 5.61 ng/ml taken as 100%) within 24 h, to 0.84 ng/ml (15%) in 48 h and 0.21 ng/ml (3.7%) in 72 h, the day before oestrus. DISCUSSION The mean oestrous cycle length of 17.2 + 1.0 days obtained in this study is intermediate between estimates of 16.4-18.6 days, previously reported for tropical ewes (Narayanaswamy and Belaine, 1976; Galliard, 1979; Yenikoye et al., 1981; Yenikoye, 1983; Farid and Makarechian, 1987) and cycling temperate ewes (Hafez, 1952; Joubert, 1962; Lamond et al., 1972; Quirke, 1978; Quirke et al., 1979). Sheep in the tropics tend to breed year-round with definite peaks (Mukasa-Mugerwa and Tekelye, 1988). These peaks are modulated through variable nutrition and other environmental factors. Consequently, not all ewes may exhibit cycles of 15-20 days. Cycle lengths of 28-30 days were observed by El-Wishy et al. (1971) among some Awassi ewes while cycles of 70 days were reported by Yenikoye et al. (1982) in some Fulani ewes. Nevertheless, extended cycles may result also from improper heat detection, and some ewes may not exhibit oestrus externally. This was noticed in two (5.2%) of the 40 heats in this study through monitoring plasma progesterone levels. Except for reports of Yenikoye et al. (1981,1982) and Yenikoye (1983), data on reproductive endocrinology of African tropical sheep are very scanty. Patterns in progesterone concentrations in this study agree with previous studies and those for temperate ewes in the tropics (Martinez et al., 1980). Present average progesterone values were, however, higher than those found by Yenikoye ( 1983 ) whose peak values in the luteal phase were 1.5 ng/ml. In contrast, our values were in the range of 3.5-7.5 ng/ml peaks as obtained by Rhind et
PLASMAPROGESTERONEOF CYCLINGETHIOPIANMENZSHEEP MEASUREDBYELISA
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al. (1985), who like Yenikoye (1983) used RIA for progesterone determination. Irrespective of differences in assay techniques, it was evident that progesterone levels were under 1.0 ng/ml in all studies from 2 days before to 4 days after oestrus. This level can therefore be adopted as the minimum (discriminatory) value to indicate anoestrus or the follicular and early luteal phase of the oestrous cycle. This level may, therefore, be used in studies of prenatal reproductive wastage, detecting silent oestrus and to assist in the better reproductive management of Ethiopian Menz sheep. ACKNOWLEDGEMENTS
Thanks are due to Mr. Tekeste Tibebu and Mr. Girma Abebe in sample collection and processing, and Mr. Solomon Zewde for assistance in data analysis. REFERENCES Agyemang, K., Negussie, A., Voorthuizen, A. and Anderson, F.M., 1985. A rapid survey of reproduction in the traditional sector of Debre Berhan, Ethiopian Highlands. In: R.T. Wilson and D. Bouzart (Editors), Proc. Conf. Small Ruminants in African Agriculture. ILCA, Addis Ababa, Ethiopia, 30 Sept.-4 Oct., pp. 175-185. Bassett, J.M., Oxborrow, T.J., Smith, I.D. and Thorburn, G.D., 1969. The concentration of progesterone in the peripheral plasma of the pregnant ewe. J. Endocrinol., 45: 449. Boland, M.P., Foulkes, J.A., MacDonnell, H.F. and Sauer, M.J., 1985. Plasma progesterone concentrations in superovulated heifers determined by enzymeimmunoassay and radioimmunoassay. Br. Vet. J., 144: 409-415. El-Wishy, A.B., EI-Sawaf, S.A. and E1-Mikkawi, F., 1971. Some aspects of reproduction in fattailed sheep in sub-tropics. I. Reproductive behaviour of local Ausimi and imported Awassi ewes. Vet. Med. J. United Arab Rep., 19: 131-155. FAO, 1985. Production Yearbook, Vol. 39. Food and Agricultural Organization, Rome, Italy. Farid, A. and Makarechian, M., 1987. Onset of oestrus and length of oestrous cycle during breeding season in five breeds of fat-tailed sheep. World Rev. Anim. Prod., 23(2): 39-45. Foulkes, J.A., Cookson, A.D. and Saner, M.J., 1982. Artificial insemination of cattle based on daily enzyme immunoassay of progesterone in whole milk. Vet. Rec., 111: 302-303. Gaillard, Y., 1979. Caracteristiques de la reproduction de la brebis Oudah (Reproductive characteristics of the Uda ewe). Rev. Elev. Med. Vet. Pays Trop., 32: 285-290. Hafez, E.S.E., 1952. Studies in the breeding season and reproduction of the ewe. J. Agric. Sci. (Camb.), 42: 189-265. Hambolu, J.O. and Ojo, S.A., 1985. Ovarian activity of Sokoto Red goats using abattoir specimens. Theriogenology, 23 (2): 273-282. Hambolu, J.O., Ojo, S.A., Jamdar, M.N. and Molokwu, E.C.I., 1985. Ovarian activity of Yankasa sheep using abattoir specimens. Theriogenology, 23 (2): 263-272. Harvey, W.R., 1976. User's guide for mixed model least-squares and maximum likelihood computer program (LSML 76). Ohio State University, Columbus, OH. ILCA, 1979. Small Ruminant Production in the Humid Tropics. ILCA Systems Study No. 3. International Livestock Centre for Africa, Addis Ababa, Ethiopia, 122 pp. Jollans, J.L., 1960. A study of West African dwarf sheep in the closed forest zone of Ashanti. West Afr. J. Biol. Appl. Chem., 3(4): 74-78. Joubert, D.M., 1962. Sex behaviour of purebred and crossbred Merino and Blackface Persian ewes. J. Reprod. Fertil., 3: 41-49.
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