Advan. Physiol. Edu. 28: 64-67, 2004;
doi:10.1152/advan.00048.2002
1043-4046/04 $5.00
ADV PHYSIOL EDUC 28:64-67, 2004
© 2004 American Physiological Society
Teaching in the Laboratory
Progesterone in milk: a simple experiment illustrating the estrous cycle and enzyme immunoassay
Pierre Rioux and
Denis Rajotte
Département de Biologie, de Chimie et des Sciences de la Santé, Université du Québec à Rimouski, Rimouski, Quebec, Canada G5L 3A1
Address for reprint requests and other correspondence: P. Rioux, Département de Biologie, de Chimie et des Sciences de la Santé, Université du Québec à Rimouski, 300, allée des Ursulines, Rimouski, QC, Canada, G5L 3A1 (E-mail: pierre_rioux{at}uqar.qc.ca)
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Abstract
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Experiments designed for students in reproductive physiology are rare. Here, we describe a simple experiment concerning a physiological aspect of the reproductive system. Milk samples are obtained from cows in estrus, in midcycle, 21 days after insemination, and in gestation. With these samples, the gestation or estrous stage is determined according to the progesterone level in milk that is measured using enzyme immunoassay. This experiment can therefore be used to demonstrate assay techniques and to illustrate the variations in progesterone concentrations during an estrous cycle and gestation. This exercise should be given after the reproduction section of the animal physiology course so that students can apply their knowledge concerning hormonal profiles during an estrous cycle.
Key words: reproduction; gestation; cow; hormonal profile
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Introduction
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EXPERIMENTS IN REPRODUCTIVE PHYSIOLOGY are rare. In some experimental physiology textbooks, experiments in reproduction are completely absent (5, 8, 27, 37). Others are limited to anatomy and histology (7, 19, 23, 35, 36, 38). When present, reproductive physiology experiments are limited to pregnancy tests in human urine (11, 26), the evaluation of estrous cycle by vaginal smear (24), and the effects of gonadectomy on uterine weight in females (24) or seminal vesicle weight in males (24).
In a recent study, we analyzed the contents of 22 experimental physiology textbooks. We compared the relative importance placed on the different systems (percentage of pages dedicated to a system vs. total number of pages in the textbook) to the total amount of space devoted to the systems in eight theory textbooks. If we take into account all types of experiments (anatomy, histology, and physiology), the reproductive system accounts for 5.0% of the physiological experiments and 6.55% of the theory texts (difference not significant). However, if we take into account only physiology experiments (excluding anatomy and histology), the relative importance placed on reproduction becomes very low (0.98%), which is highly significantly different compared with its importance in theory textbooks (6.55%; P = 0.0009).1
Context.
Traditionally, hormones have been measured by radioimmunoassay (RIA). This technique is rarely appropriate for a large group of students in a laboratory class. Radioactive materials and scintillation meters or gamma counters are frequently used in clinical and research laboratories; however, their use in laboratory classes is limited. The high costs and radioactivity involved have probably hindered the development of student exercises on the hormonal control of reproduction, an essential aspect in reproductive physiology.
Over the past few years, several biochemical measurement techniques have been developed that use antibodies instead of radioactive materials. For several years, we have used progesterone determination by enzyme immunoassay (EIA) in our animal physiology classes to demonstrate progesterone evolution during the estrous cycle. As in RIA, EIA uses a specific antibody directed against the hormone, but the labeling procedure is different. One could also use this experiment in laboratories for biochemistry courses. The technique is readily applicable to teaching laboratories.
Physiological principle of the experiment.
During the estrous cycle, one of the follicles develops in one of the ovaries during proestrus. Under the influence of gonadotropins, this follicle secretes large quantities of estrogens that cause an increase in plasma estradiol concentrations. The beginning of estrus is, hence, associated with high levels of estradiol. One will note a large increase in LH secretion and a low concentration of progesterone (1, 2). The principles of the test rest on these changes.
The length of the estrous cycle in cattle is 21 days. The average duration of estrus is 18 h, and ovulation begins 
11 h later. The size of the corpus luteum increases considerably from day 3 to day 12 of the cycle. The concentrations of progesterone in the blood and the milk follow the same pattern: the concentrations are very low from day 1 to day 3 of the cycle, increase rapidly from day 4 to day 12 (after the development of corpus luteum), remain constant until days 16–18, and fall rapidly 2–4 days before estrus. The decrease in the size of the corpus luteum in the absence of fertilization is responsible for the substantial progesterone decline (Fig. 1). The estrous cycle occurs 3 days after the progesterone drop (12). From the 4th day after the decline, the incidence of conception becomes almost null (13), and progesterone production will resume with a new cycle.
The possibility of establishing a rapid gestation diagnosis in domestic animals by means of a progesterone assay in plasma (30) and milk (18) was proposed in 1971.
Twenty-one days after insemination, the determination of the progesterone concentration allows one to predict whether the animal is pregnant (high progesterone concentration: >5.0 ng/ml) or not pregnant (
5 ng/ml) (3). The assay has to be performed between days 18 and 22 of the cycle (15). This technique correctly predicts gestation 61–88% of the time and nongestation 91–100% of the time (22).
One can accurately estimate the timing of ovulation by using the concentration of progesterone in milk. In breeding, the usefulness of this technique has been well demonstrated (20): that study showed that only 1% of ovulations had not been correctly detected, in contrast to 22% for a control group not using this test. Moreover, this technique can prevent insemination at inopportune moments, which occurs 13% of the time with traditional techniques of estrus detection. Data suggest that progesterone tests are potentially useful management aids to confirm estrus, nonpregnancy, and cyclicity (28).
Professors teaching the estrous cycle in cattle can consult the two review papers listed in REFERENCES (10, 34).
Biochemical principles of the test.
The first tests used to assay progesterone were based on RIA. The development of immunological techniques in biochemistry has led to other progesterone dosage assay techniques. EIA is similar to RIA but does not use radioisotopes. Several companies have developed kits for assaying this hormone. Many of the techniques for assaying progesterone in milk have been greatly simplified and are now routinely used (17).
The assay method we present here is based on the principle of competition. A sample of milk containing progesterone is added to a solution called a conjugate. The conjugate contains molecules of progesterone linked to an enzyme. Three types of conjugates are used: alkaline phosphatase, ß-galactosidase, and horseradish peroxidase (21).
The mixture is further inoculated with an antibody. The antibody links with both the sample and the progesterone linked to the enzyme. The quantity of antibody should be small but still plentiful enough that all antibody binding sites will be occupied. The progesterone from the sample and the conjugate therefore compete for these sites. The quantity of enzyme-labeled progesterone binding to the antibody is therefore inversely proportional to the concentration of the sample.
After incubation, the surplus of nonlinked progesterone and conjugate is removed. Only molecules linked to the antibody remain in the tube.
A chromogen solution, which is a substrate of the enzyme, is then added. It reacts with the conjugate’s enzyme (linked to the antibody) to give a coloration. The intensity of the coloration is therefore inversely proportional to the concentration of progesterone in milk.
Figure 2, A–D, shows the principle of EIAs (4) (these figures are reproduced, with slight modification, with the kind permission of Biovet).
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Fig. 2. A: the inside surface of the well is coated with a progesterone-specific antibody. The mixture of a sample of milk and the conjugate (tracer) is incubated with the antibody. B: all antibody binding sites are occupied. C: developer or chromogen (substrate for enzyme) is added to the well and reacts with the enzyme of the conjugate. D: after incubation, developer molecules have reacted with the enzyme of the conjugate to produced a colored solution.
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The reviews of Blake and Gould (6) and Gosling (14) on immunoassays constitute excellent overviews of the technique. We recommend these for students as complementary reading.
Objectives.
The pedagogical objectives of the experiment are to illustrate the progesterone changes during the normal estrous cycle, to explain bovine reproductive particularities, including the usefulness of this test in breeding as a gestation or estrus test, and to demonstrate the biochemical principle of the proposed technique.
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EXPERIMENT
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Milk samples.
We furnished students with milk samples from cows in estrus (as determined by ethological observations made by the dairyman), in the middle of the cycle (8–15 days after estrus), inseminated 20–21 days before the experiment, and pregnant. The samples (n = 17) were taken from Ayrshire dairy cows.
Milk must be taken directly from the teat, but the first five streams must be discarded to obtain a representative sample. Equal amounts of milk were collected from each quarter. Samples can be stored for 24 h in the refrigerator. If samples are to be kept for >24 h, they must be frozen at –20°C.
Progesterone determination.
The kit used (Ovucheck product no. C006) in the laboratory was adapted from the Sauer method (31). We purchased the kit from the North American distributor Biovet (Saint-Hyacinthe, QC, Canada). This kit accurately evaluates the progesterone levels in milk (9).
It requires the following simple manipulations:
- 10 µl of each standard and a milk sample (1, 5, 10, and 30 ng/ml) are pipetted into microwells
- 200 µl of conjugate (horseradish peroxidase) are added to every well
- wells are covered with a sheet of paper and incubated for 30 min at room temperature
- wells are emptied and washed three times with tap water
- 200 µl of substrate are added to all wells
- wells are covered with a sheet and incubated for 30 min at room temperature
- 100 µl of stop solution are pipetted into all wells
We used a plate reader (Kinetic Microplate Reader, Molecular Device) to read the absorbance at 405 nm.
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RESULTS AND DISCUSSION
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The progesterone concentrations are shown in Table 1. Two diagnoses of estrus, as evaluated by the dairyman, were confirmed biochemically, with values <1 ng/ml (below the detection limit of the technique). The other cow had a concentration slightly above the detection limit (5.0 ng/ml). The estrus period of this animal had therefore ended by the time the sample was taken. The values obtained for the animals that had been inseminated 21 days earlier had average values of 11.36 ng/ml and were all >5 ng/ml, indicating pregnancy. Finally, high progesterone levels were found in the cows that were in midcycle and in gestation. We evaluated the students’ comprehension with a laboratory exam. Students should be able to explain the principle of the technique and be able to interpret the different levels of progesterone concentration (estrus, midcycle, or gestation).
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Table 1. Results of a laboratory experiment estimating the reproductive status in cows by progesterone concentration as determined by enzyme immunoassay
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This experiment has many advantages. It is very simple; it is relatively inexpensive, with the price of the kit being the only cost; and it is of short duration and can therefore be used in courses having limited laboratory periods.
Milk samples are easily obtained from dairy cattle breeders and should be readily available in many countries. Moreover, several companies have marketed progesterone kits for this assay. Tests for determining progesterone in the milk of other species, goats or horses, for example, have also been developed. These species can be used if more readily available.
This experiment can be used in any program that includes physiology courses: biology, biochemistry, agriculture, and veterinary medicine. In biology and biochemistry programs, the technique is ideal for demonstrating the evolution of progesterone during an estrous cycle and to illustrate EIA.
Veterinary schools can profit from this laboratory exercise to introduce the different types of ovarian cysts, a frequent pathology in cattle: the progesterone concentration varies according to the type of cyst (16, 21).
In agricultural and veterinary sciences, one can also demonstrate the usefulness and efficiency of this test to predict estrus, to diagnose gestation, and to evaluate responses to some hormone-based medicines. Too great a gap between lactations means economic losses for breeders (25).
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Acknowledgments
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We thank Joanne Noël and Simon Lamarre for preparing the graphs and the Ferme de Fabiola (owned by Anik Gendron and Gilles Belzile) and Donat Desrosiers’ farm for samples. We thank Céline Audet and Jocelyne Pellerin for their collaboration, and Laure Devine and Khaled Chatila for help with the manuscript preparation.
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Footnotes
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1 Rioux P. Les traités de physiologie expérimentale sont-ils représentitatifs du contenu des cours théoriques? 71st Congress of Association Francophone pour le savoir, Rimouski, 19–23 May 2003. 
Received for publication September 12, 2002.
Accepted for publication January 13, 2004.
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REFERENCES
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- Anonymous. Measurement of Progesterone in Bovine Milk, Ovucheck Milk Insert. Saint-Hyacinthe, QC, Canada: Biovet, 2002.
- Anonymous. Ovucheck Bovine Fertility Diagnostics, Technical Manual. Sandy, Bedfordshire, UK: Cambridge Veterinary Sciences, 1992, p. 31.
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- Blake C and Gould BJ. Use of enzymes in immunoassay techniques. Rev Analyst 109: 533–547, 1984.[CrossRef]
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Copyright © 2004 by the American Physiological Society.
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Abstract
Introduction
