Determination Of Progesterone In Milk Research Articles

Estrategia sostenible para mejorar la eficiencia de los servicios de inseminación artificial en ganado bovino en el trópico

The aim of this study was to evaluate the efficiency of artificial insemination (AI) services in Holstein x Brahman cows under a heat induction programme. Three experiments were carried out. In Exp. 1, a retrospective analysis of the efficiency of AI insemination of 41 000 cows treated with the combination of progesterone and oestradiol benzoate or equine chorionic gonadotropin (eCG) was carried out. In Exp. 2 it was evaluated 863 cows inseminated after heat induced with progesterone and oestradiol benzoate to know the physiological events that occur before and after service in herds with a high incidence of anoestrus. Gynaecological examinations and determination of progesterone in milk were performed. The purpose of Exp. 3 was to improve the effectiveness of AI services for cows in postpartum anoestrus treated with progesterone and oestradiol benzoate combined with a technique to identify and induce the return to service of females that did not conceive. Only 30% of the cows exceeded 40% conception. The best results were obtained when using eCG (47% vs 29%). The problems associated with the low conception rate were unfertilized oestrus, embryonic mortality and anoestrus after breeding (Exp. 2). The return to service induction technique increased (p<0.001) the conception rate of the second induced service and the percentage of accumulated pregnancy in the treated group compared to the control (69.0 and 32.0%, respectively) (Exp. 3).

Magnetic nanoparticle‐based fluorescent immunoassay for determination of progesterone in milk

A sensitive and rapid magnetic nanoparticle‐based fluorescent immunoassay (MNPs‐FIA) for determination of progesterone in raw milk was developed. The PG‐3‐CMO‐BSA and PG‐3‐CMO‐BSA‐FITC conjugates were obtained. The optimal amount of immobilised antibody on MNPs (0.375 mg) and optimal concentration of PG‐3‐CMO‐BSA‐FITC conjugate (7.5 μg/mL) for MNPs‐FIA were determined. The optimal time and temperature for progesterone MNPs‐FIA were found to be – 12 min and 37 °C. The analytical parameters of immunoassay in cow milk were in the concentration range 0.25‐25 ng/mL and detection limit of 0.065 ng/mL for progesterone. The MNPs‐FIA was applied to evaluate progesterone in three types of milk (UHT, pasteurised and raw cow's milk).

Development of a Progesterone Immunosensor Based on a Colloidal Gold‐Graphite‐Teflon Composite Electrode

AbstractA novel amperometric immunosensor for the determination of progesterone, based on direct attachment of antiprogesterone on a gold nanoparticles‐modified graphite‐Teflon composite electrode is reported. Competitive assay involving progesterone labeled with alkaline phosphatase (AP), and 1‐naphtyl phosphate as the enzyme substrate, was employed. The product of the enzyme reaction, 1‐naphtol was detected amperometrically at +0.3 V vs. Ag/AgCl. Variables involved in the immunosensor preparation such as the colloidal gold loading and the amount of anti‐Prog immobilized, as well as in the immunosensing event (pH, Prog/Prog‐AP ratio and volume) were optimized. A calibration plot was obtained for progesterone with a linear range between 0 and 30 ng mL−1, and a slope value, −1.2 nA ng−1 mL, which is remarkably higher than that reported for other progesterone biosensors. A good repeatability of the amperometric measurements, RSD=3.5% (n=10), and an acceptable fabrication reproducicility, RSD=8.2% (n=3), were obtained. The developed anti‐Prog‐Aucoll‐graphite‐Teflon immunosensor was applied to the progesterone determination in milk. Recovery at the 3.5 ng mL−1 progesterone concentration level was of 101±6% (n=7).

Development of an ELISA-based kit for the on-farm determination of progesterone in milk

Effective reproductive management is a crucial concern in the dairy industry and missed oestrus and late recognition of unsuccessful insemination can lead to substantial financial loss. Progesterone levels change in a predictable manner with progress of the oestrus cycle and with the onset of pregnancy. We report here the development of a simple ELISA test for the measurement of progesterone levels in bovine milk. The method is based on the use of polyclonal antibodies raised against 11α-hydroxyprogesterone-hemisuccinate and competition for antibody binding sites between free progesterone and horseradish peroxidase-labelled progesterone. Binding is quantified by means of the colour reaction between horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine. The method has proved to be a cost effective, sensitive and easy to perform alternative to currently available methods of oestrus and pregnancy detection. The practical feasibility of the method has been proven under dairy farm conditions.

Radioimmunoassay of milk progesterone as a tool for fertility control in smallholder dairy farms

This study focused on the use of radioimmunoassay of progesterone in milk for the diagnosis of post-partum ovarian cyclicity and accurate detection of oestrus and non-pregnancy in cows in the artificial insemination (AI) programme in Bangladesh. In Investigation 1, milk samples were collected on day 0 (day of AI), day 9-13 and day 21-24 from 444 milking cows of various breeds presented for the first postpartum insemination by 413 farmers living at 182 villages/regions in Mymensingh District from 6 AI centres and sub-centres. Each cow was then examined three times after each AI until it stopped returning to oestrus. Sixty to 90 days after the last AI, the cows were examined per rectum to confirm the pregnancy. Milk progesterone data on day 21-24 contributed to a clear diagnosis with respect to non-pregnancy in 100% cows, indicating a possible use of this progesterone assay for identifying non-pregnant cows in AI programmes. In Investigation 2, milk progesterone was monitored two times in a month with a 10-day interval in 88 cows. The samples were taken between 10 days after calving and the first detected oestrus, followed by two more samples 10 days apart. The proportion of cows accurately detected in oestrus was 30%. Another 30% were stated to be in oestrus when they were not (false positive) and 40% were not detected when they were in oestrus (false negative). The mean intervals between calving and oestrus and between calving luteal activity were 40 to 362 days (median=120, n=82) and 34 to 398 (median=111, n=64) days, respectively. The body condition scores at calving and at the initiation of luteal activity influenced the interval between calving and luteal activity (p < 0.05). Cows suckled twice daily initiated luteal activity earlier than their counterparts suckled several times daily (p < 0.05). Determination of progesterone in milk on day 21-24 is a good means for detecting non-pregnant cows.

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cow's milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL(-1) and a quantification limit (LOQ) of only 2.0 pg mL(-1). These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5-15 ng mL(-1). Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL(-1) depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL(-1) for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.

Postpartum Reproductive Function: Association with Energy, Metabolic and Endocrine Status in High Yielding Dairy Cows

The effect of energetic, metabolic and endocrine status upon onset of ovarian cyclicity as well as time empty and conception at first service was assessed in 90 multiparous Holstein cows (mean 9'500 kg energy-corrected milk/305 d) in a research farm in Switzerland. Dry matter intake, milk yield and body weight were measured daily from week 2 antepartum (a.p.) through wk 20 postpartum (p.p.). Milk composition was determined 4 times/wk and milk acetone was measured weekly. Blood samples for the determination of glucose, non-esterified fatty acids (NEFA), cholesterol, creatinine, albumin, urea, s-hydroxybutyrate (BHBA), leptin, insulin, insulin-like growth factor-1 (IGF-1), growth hormone, 3,5,3'-triiodothyronine (T 3 ) and thyroxine (T 4 ) concentrations, and of activities of aspartate amino transferase (ASAT), lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH) were taken 2 wk a.p. and in wk 1-16 and 20 p.p. between 0730-0900. Cows were gynaecologically examined weekly. The onset of first ovulation was specified by skim milk progesterone determination by a radioimmunoassay (twice/wk) and by rectal palpation. Energy balance (EB) traits were calculated and expressed as accumulated negative EB from parturition to EB equilibrium (AUC), EB nadir (EBN), rate of EB recovery following EBN (EBR), time from parturition to EBN (DEBN), and time from parturition to EB equilibrium (DEBE). Results: Resumption of ovarian cyclicity postpartum was not reflected by any of the EB traits. However, a small degree of EBN and a fast EBR were related to a short period from parturition to conception. Survival analysis with timevarying covariates revealed that high plasma concentrations of T 3 and T 4 p.p. were significantly associated with a high chance of an early start of the ovarian cycle, and high concentrations of glucose and cholesterol were associated with a short time empty, respectively. Conception at first service was positively associated with EB at first service and with progesterone concentration at 10-13 d after first service. However, leptin, as most other metabolic and endocrine traits, was not associated with any of the reproductive traits. In conclusion, thyroid hormones may play an important role in the onset of the ovarian cycle p.p., and a good energy status enhances the chance of conception at first service and shortens time empty.

Postpartum reproductive function: association with energy, metabolic and endocrine status in high yielding dairy cows

This study evaluated the effect of metabolic, endocrine and energy status on onset of ovarian cycle, days open (DO), and conception at first service in 90 multiparous Holstein cows, housed at a research farm. Dry matter intake, milk yield and body weight were measured daily from Week 2 antepartum (a.p.) to Week 20 postpartum (p.p.). Milk composition was determined four times per week and milk acetone was measured weekly. Blood samples for the determination of glucose, non-esterified fatty acids, cholesterol, creatinine, albumin, urea, β-hydroxybutyrate, leptin, insulin, insulin-like growth factor-1, growth hormone, 3,5,3′-triiodothyronine (T3), and thyroxine (T4) were taken 2 weeks a.p., in Weeks 1–16, and 20 p.p. between 7:30 and 9:00h. The onset of ovarian cycle was specified by weekly gynecological examination and by skim milk progesterone determination by radioimmunoassay (twice per week). Energy balance (EB) traits were calculated and expressed as accumulated negative EB from calving to EB equilibrium, EB nadir (EBN), rate of EB recovery after EBN (EBR), and time from calving to EBN and to EB equilibrium, respectively. The onset of ovarian cycle p.p. was not related to EB. However, a low degree of EBN and a fast EBR were associated with fewer DO, and EB at first service was positively related to conception. High plasma levels of T3 and T4 p.p. were associated with an early start of ovarian cycle, and high concentrations of glucose and cholesterol with a short calving to conception interval. Conception at first service was positively related to EB at first service and progesterone concentration 10–13 days after first service. In conclusion, thyroid hormones may play an important role in resumption of ovarian cyclicity p.p., and a good energy status enhances the chance of conception at first service and shortens DO.

Relationships between body condition score (BCS), biochemical blood parameters and pregnancy rates in Prim'Holstein cows under subtropical conditions

AbstractThe relationships between body condition score (BCS), metabolic measurements and pregnancy rates were studied from 214 cows belonging to 6 herds located in Reunion Island between January and December 1998. BCS (0 to 5 scale) and biochemical blood parameters were measured before calving, 0-30, 60-90 and 120-150 days post-partum. Pregnancy status was also checked precisely after 171 AI's (n=104 cows) by EIA progesterone determination in milk 23 to 24 days after AI and pregnancy specific protein B (PSPB) measurements 30 to 45 days after AI and was further confirmed by rectal palpation by 60 days post AI. A preliminary analysis led to determine 3 groups of herds according to their pregnancy rates (40%, 26% and 14.5% respectively for group 1, 2 and 3 cows). Significant differences between the 3 groups were found for BCS, glucose and urea (p &lt; 0.0001) and a significant main effect of the post-partum stage was observed as well (p &lt; 0.0001). Mean BCS were not different in the 3 groups before calving but were higher in group 1 cows than in group 2 and 3 cows at all other stages studied. A similar trend was found for urea. Glucose concentrations were severely depressed in group 3 cows when compared to group 1 and 2 cows whatever the stage studied. These results sugggest that variables illustrating negative energy balance (BCS decrease and low glucose concentrations) or deficit in nitrogen supply (urea concentrations) on a herd basis are related to poor pregnancy rates.

First ovulation and ketone body status in the early postpartum period of dairy cows

The effect of ketone body status on occurrence of first ovulation during early lactation was assessed in 84 multiparous dairy cows under field conditions. Animals were equally distributed across 8 farms and were controlled by the same herd fertility monitoring program. Cows were visited twice antepartum and 6 times postpartum at weekly intervals between 5:30 and 8:30 AM. On these occasions, body condition scores and milk yields were measured, blood and milk samples were taken, cows were gynecologically examined, and parameters of reproduction were determined. The onset of first ovulation was specified by milk progesterone determination and rectal palpation. Cows starting postpartum ovarian cyclicity within or after 30 d were classified as early and late responders (ER and LR, respectively). Resumption of the estrous cycle within 30 d postpartum is considered optimal under practical conditions, and classification based on this threshold value resulted in groups of equal size and equal distribution of ER + LR cows within farms. Ketone bodies measured were βhydroxybutyrate in serum and acetoacetate and acetone in serum and milk. Blood serum and milk ketone body concentrations during the first 6 wk of lactation were higher in LR than in ER, whereas plasma glucose and nonesterified fatty acid and milk fat, protein and urea concentrations did not differ between groups. Maximal concentrations of ketone bodies from parturition to first ovulation were better predictors of the onset of the estrous cycle than mean or minimal concentrations over the same period. Milk acetone and serum β-hydroxybutyrate concentrations provided the most reliable information with regard to resumption of ovarian activity of all ketone bodies.

Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility

AbstractReproductive performance of 714 Holstein Friesian dairy cows was monitored between October 1995 and June 1998 using thrice weekly milk progesterone determinations. Defined endocrine parameters such as interval topost-partumcommencement of luteal activity, inter-ovulatory interval and length of luteal and inter-luteal intervals were used with a number of traditional measures of reproductive performance to investigate the current status of fertility in a sample of United Kingdom dairy herds. A comparison of the results of the 1995 to 1998 trial with those of a previous (1975 to 1982) milk progesterone database, which included 2503 lactations in British Friesian cows monitored using a similar milk sampling protocol, revealed a decline infertility between these periods.Between 1975-1982 and 1995-1998, pregnancy rate to first service declined from 55·6% to 39·7% (P&lt; 0·001), at a derived average rate approaching 1% per year. This decline was associated with an increase (P&lt; 0·001) in the proportion of animals with one or more atypical ovarian hormone patterns from 32% to 44%. There was a significant (P&lt; 0·001) increase in the incidence of delayed luteolysis during the first cyclepost partum(delayed luteolysis type I; 7·3% to 18·2%) and during subsequent cycles (delayed luteolysis type II; 6·4% to 16·8%), although the incidence of prolonged anovulation post partům (delayed ovulation type I; 10·9% to 12·9%) and prolonged inter-luteal intervals (delayed ovulation type II; 12·9% to 10·6%) did not alter significantly. These changes resulted in an increase in mean luteal phase length from 12·9 (s.e. 0·09) to 14·8 (s.e. 0·17) days and an increase in inter-ovulatory interval from 20·2 (s.e. 0·1) to 22·3 (s.e. 0·2) days. The decline infertility was also reflected in traditional measures of fertility since although interval to first service remained relatively unchanged (74·0 (s.e. 0·4) to 77·6 (s.e. 1·1) days) calving interval lengthened from 370 (s.e. 2·2) to 390 (s.e. 2·5) days. Collectively these changes may have contributed to the decline in pregnancy rates observed over the last 20 years.

Regulation of Ovarian Functions in the Post Partum Holstein Cows by Rapid Progesterone Test and Treatment of the Ovarian Dysfunctions

The effectiveness of milk progesterone determinations by rapid progesterone test (RPT) was evaluated for the control of ovarian functions and dysfunctions in postpartum cows. Total 250 healty Holstein cows, between 3-11 ages, were sampled as materials from two different official herd (herd A and B). Udder milks sampled on 24,31,38 and 45 days after calving. RPT s were carried out on total milk samples . The accuracy of RPT for determining the level of milk progesterone was controlled on 142 milk samples, using the results of quantitative laboratory EIA test. The accuracy rate was 86.61 per cent. Following the four RPT evaluation on each cow, rectal palpation, between 45-60 days postpartum, was performed individually. Progesterone profiles indicated that at the time of clinical examination 62.00 per cent of the cows examined were cycling normally and 38.00 per cent having ovarian dysfunctions. During these periods ovarian dysfunctions were classified, with 18.80 per cent delayed ovulation longer 38th days . with 2.80 per cent inactive ovaries , with 14.40 per cent persistent luteal phase and with 2.00 per cent follicular cysts. Although ovarian cycles were resumed spontaneously in delayed ovulation cases but prostaglandin F2 alpha, progestagens and progestagens +PMSG were administered in the cows with persistent luteal phase, follicular cysts and inactive ovaries respectively. Normal cyclic cows were inseminated on spontaneous estrouses between 45-60 days postpartum and the cows having ovarian dysfunctions inseminated on the first estrous following end of the treatmens. The open period of normal cylic cows in herd A (86.88±36.17 days) was shorter than herd B (94.91±40.17 days ); but the difference between two farms, looking these parameters , was not significant statistically (p>0.05). These findings suggest that, postpartum ovarian functions and dysfunctions could be effectively contolled, except the case of follicular cysts, using the RPT and rectal palpation weekly intervals.

Post-Partum Ovarian Activity in Finnhorse Mares with Special Reference to Seasonal Effects

In a previous study, times from parturition to the first ovulation were followed in 55 Finnhorse mares on the basis of milk progesterone determinations. Ninety-six per cent of mares had ovulated by day 20 post-partum. If intervals of more than 19 days are excluded from the data, the time from parturition to 1st ovulation was 117 days. However, in cases of foaling before and after the beginning of June the times were 13.0 days and 8.8 days, respectively (p less than 0.001). Long intervals (over 16 days) occurred mainly before 1st May (in 6 out of 7 cases). In a 2nd study, 25 post-partum Finnhorse mares were examined by rectal palpation and ultrasonic scanning. Five and 7 days post partum, but not 2 days post partum there was a statistically significant difference between ovulatory ovaries and non-ovulatory ovaries regarding size of whole ovary and the largest follicle. Six to 8 days before the first post-partum ovulation, the size of the preovulatory follicle was greater in mares which had foaled before the middle of May (32 mm) than in those which had foaled after the middle of May (20 mm) (p less than 0.05). Within 2 days before ovulation there was no statistical difference between the sizes (43 mm and 42 mm, respectively). The growth rate was therefore slower in cases of early foaling (1.8 mm/day) than in cases of late foaling (3.7 mm/day).

Milk progesterone determination in buffaloes post-insemination

Milk progesterone levels were studied in 17 buffaloes between post-insemination and 30 days thereafter. Six animals were confirmed pregnant. Seven of 10 animals took > 24 days to return to oestrus and the pattern of change of progesterone levels indicated a steady increase from 1 ng/ml on day 2 to 20 ng/ml on day 14 post-insemination, falling subsequently to about 5 ng/ml, 2 days prior to oestrus. In pregnant buffaloes, the milk progesterone levels showed a continuous increase up to 20 days post-insemination but did not decline thereafter. In individual buffaloes returning to oestrus, a cut-off milk progesterone level of > 10 ng/ml was considered for pregnancy diagnosis, 20, 22 and 24 days post-insemination. The test was 60, 75 and 75% accurate for detection of pregnancy on days 20, 22 and 24 respectively and 100% for non-pregnancy diagnosis on all three days. Individual animals showed a highly variable oestrous cycle length, which has been suggested as a contributory factor for false pregnancy diagnosis by milk progesterone test in this species. Milk progesterone assay may also identify silent heats, and clinical disorders.

Milk progesterone profiles in crossbred Brown Swiss × Nellore cattle following natural service

Ovarian activity and estrous behaviour were monitored through milk progesterone determinations and twice daily visual observations in 70 crossbred Brown Swiss × Nellore cows following natural service. Whole milk samples were collected on the day of estrus (Day 1), Day 11, and every 5 d thereafter until the next estrus or pregnancy confirmation. Seventy percent of the cows behaved as expected, i.e. they showed a 19-to 25-d interval between estrus and the next ovulation, or they became pregnant. Estrous cycles of regular length (18 to 24 d) were found in 54% of the cases. Prolonged luteal phases (interval from estrus to next ovulation > 28 d) were found in 15.7% of cows. Short estrous cycles (interestrous interval < 18 d) were found in 7.1% of the cases. Periods of acyclicity (basal progesterone levels for periods ≥ 15 d) were found in 5.8% of the cases, and one cow exhibited estrus while pregnant and had a high progesterone concentration. Cows with a prolonged luteal phase and those with a short estrous cycle had an interval between ovulations of 35.0 ± 6.7 d (x ± SD) and 9.6 ± 3.1 d, respectively. Signs of estrus were not detected in 33.3% of the ovulations confirmed by progesterone determinations. Low conception rates, failures in estrus detection and a high frequency of abnormal postbreeding luteal phases were found.

A comparison of alkaline phosphatase, β-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay

Assays for alkaline phosphatase, β-galactosidase, penicillinase and peroxidase were optimised for quantitation in microtitre plate wells. Their value as labels in microtitre plate enzymeimmunoassay (EIA) for progesterone was assessed following coupling with 11α-hydroxyprogesterone 11-glucuronide using an active ester procedure. Bridge-heterologous antiserum (11α-hydroxyprogesterone 11-hemisuccinate-bovine serum albumin as immunogen) was used to minimize bridge recognition. The limits of detection of the enzymes were in the order penicillinase > peroxidase > alkaline phosphatase > β-galactosidase. Under appropriate conditions it was possible to achieve > 50% displacement of label with 50 pg of progesterone for all four labels.

Milk Progesterone Concentration and Production in Superovulated Holstein Cows

Six Holstein cows in a commercial herd (three superovulated and three controls) and eight Holstein cows superovulated a total of 12 times in an experimental herd were studied. Superovulation was induced primarily by treatment with follicle stimulating hormone and prostaglandin F2α. Milk was weighed twice daily for 30 days following treatment in the commercial herd with no effect on production. Milk samples were saved on Monday, Wednesday, and Friday to determine progesterone content. Last milk at mid-cycle averaged 8.2ng/ml of progesterone for the cows in the experimental herd, and 7 days after superovulation they averaged 52.6±10.2ng/ml (mean±standard error) of progesterone. The correlation with number of embryos recovered was .86. Therefore, milk progesterone may be useful in monitoring superovulatory response. Also, injection of prostaglandin F2α into superovulated cows 9 days after a previous injection did not initiate a new estrous cycle, a fact accurately monitored by milk progesterone determinations.

Direct Enzymeimmunoassay for Determination of Progesterone in Milk from Cows

A direct enzymeimmunoassay for progesterone in whole and last milk (stripping) allows reliable detection of corpus luteum activity within one working day. The discriminatory level is 5 ng/ml.

Pregnancy diagnosis in cattle by a rapid and highly reliable method for progesterone determination in milk

A highly reliable technique for the determination of progesterone in milk has been developed, and its application to pregnancy diagnosis in cows is reported. The method is characterized by an activated charcoal treatment of the samples, extraction of the absorbed progesterone by an organic solvent, followed by RIA. The application of the method to pregnancy diagnosis, 21--23 days after artificial insemination, shows that 5 ng/ml is the most useful value to discriminate between pregnant and non-pregnant animals, with greatest precision on the 21st day, in that 93.5% of pregnant and 100% of non-pregnant animals were correctly diagnosed.

Oestrus and Pregnancy Diagnosis by Milk Progesterone Assay in the Mare

The milk progesterone profiles of four mares were followed daily for four to five weeks after foaling. Progesterone was determined by direct radioimmunoassay using iodinated progesterone as the labelled antigen. The milk progesterone concentration varied from 1 to 5 nmol/1 (0·3 to 1·6 μg/1) during the first 10 days after foaling. The first ovulation took place at about the tenth day. During the luteal phase milk progesterone levels rose dramatically, reaching a maximum level of 30 to 45 nmol/1 (9·4 to 14·2 μg/1) within five to 10 days, and then fell to low levels unless pregnancy followed, in which case milk progesterone remained high. Examples of the use of milk progesterone determinations in the diagnosis of silent heat and ovarian dysfunction are given.