progesterone and estrogen levels in serum of cycling goats measured by enzyme immunoassay

SmallRuminantResearch, 6 ( 1991 ) 95-102 95 Elsevier Science Publishers B.V., Amsterdam

Progesterone and estrogen levels in serum of cycling goats measured by enzyme immunoassay

M. Bauernfeind and W. Holtz* Institute of Animal Husbandry and Genetics, University of G~ttingen, Albrecht-Thaer-Weg 1, D-3400

Gi~ttingen Germany

(Accepted 8 December 1990)

ABSTRACT

Bauernfeind, M. and Holtz, W., 1991. Progesterone and estrogen levels in serum of cycling goats measured by immunoassay. SmallRumin. Res., 6: 95-102.

An enzyme-immunoassay (EIA) was modified to measure blood serum progesterone in female goats. Assay sensitivity was 0.1 ng/ml. Comparison of progesterone profiles in two cycling does measured by EIA and radioimmunoassay (RIA) gave excellent correlations (r=0.96 and 0.98, respectively). Progesterone profiles of six cycling does are presented. Two days prior to and on the day after estrus, progesterone levels were 0.12 and 0.10 ng/ml, respectively. Maximum levels of 7.2 to 7.3 ng/ ml were reached between day 11 and day 14 of the estrous cycle. The critical level of progesterone to distinguish between absence or presence of a functional corpus luteum was between 0.5 and 1.0 ng/ ml which indicates a suitable time for insemination. In the same six does, total serum estrogens were also determined by EIA. Lowest levels were on day 9 of the estrous cycle, at a time when progesterone levels were rising and increased continually until day 20 of the cycle, surging to a climax of 1.16 ng/ ml at the time of standing estrus.

INTRODUCTION

Ever since enzyme-immunoassays (EIA) for progesterone, which are quick, simple and cheap, have become available, they have gained in popularity for monitoring reproductive functions in cows (Arnstadt and Cleere, 1981; Sauer et al., 1981; Hoedemaker et al., 1984; Kiister and Holtz, 1985; van de Wiel and Koops, 1986; Kiister et al., 1987), sheep (Eckersall and Harvey, 1987), mares (Losert and Holtz, 1984, 1987; Losert et al., 1986; Alien and Porter, 1987 ) and, recently, goats (Braun et al., 1988 ). Due to the seasonal nature, monitoring ovarian activity in goats is of particular interest. The intention of this investigation was to employ an EIA for this purpose and, as an initial step, establish profiles of progesterone and total estrogen levels in cyclic does.

*Correspondence author

0921-4488/91/$03.50 © 1991 Elsevier Science Publishers B.V. All fights reserved.

96 S. BAUERNFEIND AND W. HOLTZ

MATERIALS AND METHODS

The experiment was conducted at the end of the breeding season, using six nuUiparous yearling German Dairy X Boer goat does ( 36-41 kg liveweight). The animals were housed together in an open barn adjacent to a sexually mature male goat. A daily allowance of 600 g of concentrates per head was pro- vided and in addition the animals had access to wheat or barley straw as well as a salt lick and water. The animals were checked twice daily for the presence of estrus using an aproned buck. Commensing 2 days after the onset of standing estrus (i.e. day 3 of the estrous cycle) blood samples were taken by jugular venipuncture at 2-day intervals for the remainder of that particular cycle and extending into day 9 of the following estrous cycle. Following centrifugation at 1050 g for 20 min at 4°C, the serum was harvested and stored in aliquots at - 20°C pending analysis for progesterone and total estrogen content.

Progesterone was determined by EIA accordifig to Arnstadt et al. ( 1982 ) with the following modifications: after thawing of samples, 100 #l of serum was added to 4 ml of petroleum-ether and shaken for 4 rain. Samples were then centrifuged for 5 rain at 1050 g and placed in alcohol bath with dry ice. After the aqueous fraction had frozen, the lipid fraction was decanted and evaporated at 45 °C under N2. This extraction was found to be necessary because measurement of untreated serum gave excessively high values and poor repeatability. This error was not eliminated by heating of the serum to 80°C for 30 rain. Other extraction procedures that were attempted (n-hexane, n- heptane and isooctane) were found to deliver less consistent results. In order to detect losses, sera containing defined quantities of progesterone were in- cluded. The standard curve (0.2 to 20 ng/ml) was derived by adding defined amounts of synthetic progesterone (Merck No. 24614, Darmstadt, FRG) to a benzol-ethanol mixture (9: 1 ). All samples were extracted and analyzed in duplicate. Assessment of intra and inter assay precision resulted in coefficients of variation of 10.2% and 11.2%, respectively. The sensitivity was 0.1 ng/ml.

Corresponding samples from two of the does were measured by radioimmunoassay (RIA) after hexane-acetic acid ethyl-ester extraction (RSL Ra- dio Assay Systems Laboratories, Carson, California, USA) as comparison.

Total estrogen content in serum was determined in all samples using the enzyme-immunoassay ESTRONOSTICON Test (Organon-Technika). This assay measures estrone, estradiol, estriol and their 16-17 conjugates. Intra and inter assay precision showed coefficients of variance of 7.0% and 11.3%, respectively, and the limit of detection was 0.4 ng/ml.

Due to slight differences in individual cycle length (20 to 22 days ), cycles were standardized to first days of the first and second estrus (day 1 = first day of standing estrus). Individual curves overlapped during mid-cycle.

Data for plasma progesterone and estrogen concentrations were analyzed

PROGESTERONE AND ESTROGEN LEVELS IN GOATS 97

by Analysis of Variance according to SAS General Linear Model Procedure. Differences between days of cycle were tested for significance by Scheff6-test (1950).

RESULTS

In Fig. 1, serum progesterone profiles of two goats, measured by EIA and RIA, are compared. They indicate close resemblance, expressed by correla- tion coefficients of r= 0.96 and r= 0.98, respectively.

Figure 2 gives progesterone profiles (EIA) of the six goats. Although maximum progesterone levels for individual does varied between 6.3 and 1 I. 1 ng/ml, the general pattern was similar for all individuals. Figure 3 has estrogen and progesterone means and standard errors. Animal 5 was omitted because her progesterone levels failed to increase after the second estrus, pre- sumably because of cessation of ovarian cyclicity at the end of the breeding

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Fig. 1. Comparison of blood serum progesterone profiles by enzyme immunoassay and radioimmunoassay of two goats during the estrous cycle.

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Fig. 2. Progesterone profiles o f six individual goats standardized to first and second estrus (day l and day 22, respectively). Overlap of curves occurred between day 13 and day 14 of the cycle.

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Fig. 3. Serum progesterone and total estrogens of five does during the estrous cycle (mean + SEM) as determined by EIA. Cycle length was standardized to 21 days, overlap of curves occurred between day 13 and day 14 of the cycle.

season. This goat was not observed in estrus until the next season. On day 3 of the cycle the progesterone concentration was 0.27 ng/ml. It increased pro- gressively until reaching maximum levels o f 7.2 to 7.3 ng/ml between day 11 and day 14 of the cycle. Thereafter it dropped rapidly to values of 0.12 and 0.10 ng/ml on days 20 and 22 (day of standing estrus), respectively. Two days later, progesterone levels increased to 0.41 ng/ml and followed a pattern


closely resembling that of the previous cycle. Progesterone concentrations on days 9 to 16 and on day 30 were significantly higher than basal levels measured on days 20 and 22 (P<0.05) . The steep decline between day 16 (6.3 ng/ml) and day 18 ( 1.8 ng/ml) was significant (P<0.05) .

Total estrogen levels in serum varied greatly. Nevertheless, a cyclic trend was discernible (Fig. 3). Animal 5 was omitted in accordance with the progesterone profile, as its estrogen level was much lower than that of the rest. Low estrogen levels were observed concomitant with rising progesterone levels. The lowest level of 0.73 ng/ml occurred on day 9. It was followed by a phase of gradual increase to values of approximately 0.9 ng/ml between day 13 and day 18. There was a sudden estrogen surge, reaching an average peak level of 1.16 ng/ml on the day of standing estrus, followed by a decrease to levels close to 0.9 ng/ml on days 28 and 30. Due to individual variability differences between means were not statistically significant (P> 0.05 ).

DISCUSSION

Enzyme-immunoassays are rapidly gaining in popularity. In comparison to radioimmunoassays they are easier to establish, require less sophisticated lab- oratory equipment and are not encumbered with the problems associated with radioactivity, and the disposal of radioactive waste. This investigation indicates that the progesterone-EIA we applied is well suited to monitoring plasma progesterone levels in goats. The resulting values compared well with those of a well-established RIA. Close agreement between plasma progesterone levels measured by EIA and RIA was also observed in cows by Hoedemaker et al. (1984).

The average progesterone level and the cyclic pattern, determined in this experiment, correspond closely with findings reported by Jones and Knifton ( 1972 ), Thorburn and Schneider ( 1972 ), Wentzel et al. ( 1979 ), Thibier et al. ( 1981 ), Chemineau et al. (1982) and Bono et al. (1983), all of whom applied radioimmunoassays. Braun et al. ( 1988 ), applying an EIA on whole blood of goats, report unusually high progesterone concentrations during the corpus luteum stage.

Progesterone levels below 0.1 ng/ml around estrus suggest that, in goats, the corpora lutea constitute the major source of progesterone. This has pre- viously been suggested by Ott et al. (1980) and Bretzlaff et al. ( 1983 ). Indi- vidual differences in progesterone level during the luteal phase (Fig. 1 ) may be associated with different numbers of functional corpora lutea as indicated by Chemineau et al. ( 1982 ). Corresponding findings have been reported for sheep by Thornburn et al. ( 1969 ), Evans and Robinson (1980) and Cahill et al. (1981).

Based on the data of this experiment, the critical level of progesterone in blood serum to distinguish between absence or presence of a functional cot-


pus luteum is 1.0 ng/ml. As in the cow (Holtz et al., 1986 ) determination of progesterone levels in blood or milk of goats may be useful for improving the efficacy of insemination programs and for distinguishing between pregnant and non-pregnant animals. Cycling animals with values lower than 1.0 ng/ml may be assumed to be in estrus or close to it. Animals with higher progesterone concentrations are in diestrus or are pregnant. In this investigation, values below 1.0 ng/ml characterized day 20 to day 24 of the cycle. Lowering the critical level to 0.5 ng/ml reduced the time span around estrus by one day from day 20 to day 23. Although assay sensitivity was 0.1 ng/ml, a further reduction of the critical level did not result in an additional narrowing down of this time span.

Although not compared with RIA it seems that the EIA for measuring total estrogens in goat blood serum also supplied useful results. There were low levels during the luteal phase and a surge due to the oncoming estrous period. The assay applied in this experiment measured total estrogens including conjugates of the physiologically relevant estrogens. Individual variation was considerable. Nevertheless, the general pattern resembled that reported by Mori and Kano (1984); Chemineau et al. (1982); Bono et al. (1983) for goats and by Hauger et al. (1977) and Pant et al. (1977) for sheep. The increase in estrogens observed as early as day 11 of the estrous cycle may indicate early follicular maturation in some individuals as is known to occur in cattle (Rajakoski, 1960) and sheep (Smeaton and Robertson, 1971; Brand and de Jong, 1973).

ACKNOWLEDGEMENT

The authors thank Ms Ernestine Seyfert for her kind assistance.

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progesterone and estrogen levels in serum of cycling goats measured by enzyme immunoassay

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