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 tech- nique 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 be- tween 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 ru- minant reproductive performance in tropical Africa (Jollans, 1960; Wilson, 1975; ILCA, 1979; Mulokwu and Umunna, 1980; Agyemang et al., 1985; Ham- bolu 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 re- productive physiology of ewes (Bassett et al., 1969; Thorburn et al., 1969; Rob-

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58 E. MUKASA-MUGERWA ET 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 mini- mum (discriminatory) progesterone values that could be adopted in investi- gations on anoestrus, pregnancy diagnosis and prenatal reproductive wastage.

MATERIALS AND 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 Teke- lye, 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 excel- lent 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 hepar- inised vacutainers (Becton Dickinson). Sampling was continued until ewes had exhibited two subsequent oestrous periods. Plasma was recovered by cen- trifugation 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 im-

munosorbent 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 sam- ples and a known quantity of enzyme alkaline phosphatase (AP)-labelled pro- gesterone 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 (p- nitrophenyl phosphate: prepared by dissolving 3 X 40 mg tablets of the sub- strate 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 (sup- plied 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 val- ues 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 coef- ficients 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.

R E S U L T S

Mean length of oestrous cycles was 17.2+1.0 days (n=36) . 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

6 0 E . M U K A S A - M U G E R W A E T AL.

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A

~6

~5 o ~4 o~

b9

I0 12 14 16 0 2 4 6 8 I0 12 14 16 0 2 4

Doy of the oestrous cyc{e

Fig. 1. Mean daily plasma progesterone concentration during the oestrous cycle of Ethiopian High- land 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 trop- ical 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. Pat- terns 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 Yeni- koye ( 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

PLASMA PROGESTERONE OF CYCLING ETHIOPIAN MENZ SHEEP MEASURED BY ELISA 61

al. (1985), who like Yenikoye (1983) used RIA for progesterone determina- tion. Irrespective of differences in assay techniques, it was evident that pro- gesterone 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 (dis- criminatory) 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 repro- ductive 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.

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