Short communication: Validation of a novel milk progesterone-based tool to monitor luteolysis in dairy cows using cost-effective, on-farm measured data.

The progesterone (P4) monitoring algorithm using synergistic control (PMASC) uses luteal dynamics to identify fertility events in dairy cows. This algorithm employs a combination of mathematical functions describing the increasing and decreasing P4 concentrations during the development and regression of the corpus luteum and a statistical control chart that allows identification of luteolysis. The mathematical model combines sigmoidal functions from which the cycle characteristics can be calculated. Both the moment at which luteolysis is detected and confirmed by PMASC, as well as the model features themselves, can be used to inform the farmer on the fertility status of the cows.

Assessment of luteal function in the vervet monkey as a means to develop a model for obesity-related reproductive phenotype.

The objective of the current study was to characterize luteal function in vervet monkeys. Urine from 12 adult female vervets housed at an academic research center was collected for 10 weeks from single-caged monkeys in order to assess evidence of luteal activity (ELA) as determined by urinary excretion of pregnanediol glucuronide (Pdg) and estrone conjugates (E1c). Dual energy X-ray absorptiometry (DXA) was performed on the monkeys to assess body composition, bone density, and fat mass. Menstrual cyclicity was determined using records of vaginal bleeding. ELA was observed in 9 monkeys and was characterized by a late follicular rise in E1c followed by a progressive increase in Pdg excretion. Mean menstrual cycle length was 26.7 ± 3.8 days and the average day of luteal transition was 14 ± 1.8. Three monkeys without ELA had a clearly defined E1c rise (mean 12-fold from nadir) followed by an E1c drop that was not accompanied by Pdg rise and coincided with vaginal bleeding. Among the 9 ELA monkeys, excretion of E1c tended to negatively associate with fat mass, although this finding did not reach statistical significance (r = -0.61, p = 0.08). Similar to women, vervet monkeys experience an increase in E1c late in the follicular phase of the menstrual cycle which is followed by a subsequent luteal Pdg peak. Assessment of urinary reproductive hormones allows for identification of cardinal menstrual cycle events; thus, the similarity of vervet cycles to human menstrual cycles makes them a useful model for obesity-related human reproductive impairment.

Assessment of superovulatory responses in terms of palpable corpora lutea and embryo recovery using plasma progesterone in yaks (Poephagus grunniens L.).

Plasma progesterone profiles were used to assess superovulatory responses in cyclic yaks (n=10) in terms of the number of ovulations and the number of embryos recovered. The animals were synchronized into oestrus following Ovsynch treatment. All the animals received a total of 200 mg Folltropin divided into morning and evening and spread over 4 days, beginning on day 10 of the oestrus cycle (day of expected oestrus=day 0). Plasma samples for progesterone estimation were collected daily starting from the day of expected synchronized oestrus to the day of flushing. All the animals were palpated per rectum on the day of flushing in order to record the number of corpora lutea. Of an estimated 27 ovulations from the nine yaks, only 16 embryos were recovered. Plasma progesterone profiles from individual yaks suggested that a poor superovulatory response in terms of embryo recovery in some animals was caused by the lysis of corpora lutea before flushing which was carried out 7 days after superovulatory oestrus. It was suggested that flushing 5 days post superovulatory oestrus could improve the superovulatory response in this species.

Assessment of luteal function by ultrasonographic appearance and measurement of corpora lutea in goats.

In order to characterize the evolution pattern of the corpora lutea (CL) and to compare luteal function with their ultrasonographic appearance, 37 estrous cycles of Serrana goats (n=22) were studied during breeding season. A daily transrectal ultrasound scanning was performed through two successive estrous cycles. Both solid and fluid-filled CL were observed and measured in both ovaries of each goat. Additionally, each CL was classified as CL(ICHE) (CL with irregular contours and heterogeneous echotexture) or CL(RCGE) (CL with regular contours and granular echotexture). Ovarian cyclic activity and luteal function were evaluated by biweekly plasma progesterone (P4) determination. The CL (n=60) were first visualized on day 2.9+/-1.0 after the day of ovulation (day 0), showing 7.1+/-1.8mm of diameter and reach their maximum size (12.5+/-1.6mm) on day 10.7+/-3.2 (P<0.001). Two days before the following ovulation (day -2), the CL regressed to 8.4+/-1.3mm (P<0.001). The central cavity was found in 78.3% of CL, and had a persistence of over 50% until the last days of estrous cycle. The ratio CL length/cavity length was low during the first-third and high during the remaining two-thirds of estrous cycle. On day 2, the percentage of CL(ICHE) was 33.3%, and began to decrease to 16.7% on day 6, reaching the minimum of 3.3% on day 10 (P<0.001). This proportion increased on day -3 to 48.3% and reached 90% on day -1 (P<0.001). The correlation between CL size and plasma P4 levels was r=0.63 (n=87; P<0.001). A negative correlation between the daily proportion of CL(ICHE) and plasma P4 levels was found (r=-0.95; n=18; P<0.001). These results suggest that the ultrasonographic appearance of CL is a reliable parameter for the assessment of luteal function in goats. Both the characterization of echotexture and size of central cavity could be valuable tools to differentiate between phases of normal estrous cycles.

Validation of a sensitive enzymeimmunoassay for 13,14-dihydro-15-keto-PGF2alpha in buffalo plasma and its application for reproductive health status monitoring.

A simple, sensitive and direct enzymeimmunoassay (EIA) procedure on microtitre plates using the second antibody coating technique was standardized and validated for the determination of 13,14-dihydro-15-keto PGF2alpha (PGFM) in unextracted buffalo plasma. The assay was carried out directly in 20 microl of buffalo plasma. PGFM standards prepared in charcoal stripped hormone-free plasma were used. The sensitivity of the assay was 0.4 pg/well, which corresponded to 20 pg/ml plasma. Plasma volumes for the assay ranging from 10 to 50 microl did not influence the PGFM standard curve; however, a slight drop in the OD450 was observed with higher plasma volumes. Biological validation of the assay was carried out in buffalo plasma samples obtained during physiological states of cyclicity, peri-estrus, post-insemination, reproductive tract infection and persistent corpus luteum conditions. A pulsatile pattern of plasma PGFM release was observed prior to estrus when PGFM was determined in blood samples collected at hourly intervals of time. The PGFM pulsatility was not observed when blood sampling frequency of either 4 or 12 h was considered. The PGFM levels stayed high in peripheral circulation of buffaloes with reproductive tract infections and remained low throughout the sampling period in buffaloes having persistent corpus luteum. After an initial increase post-insemination, the plasma PGFM levels showed minor fluctuations. The assay was found to be sufficiently reliable and specific for estimation of PGFM levels in buffaloes. The standardization and validation of PGFM assay in buffalo opens the prospects of using PGFM levels as an indicator for reproductive health status monitoring in this species.

Effect of exogenous prostaglandin F2 alpha in clinically normal postparturient dairy cows with a palpable corpus luteum.

In 228 clinically normal cows with a palpable corpus luteum 20 to 40 days after parturition and a mean 305-day mature equivalent milk production of 8,970 kg, prostaglandin F2 alpha was administered in a randomized, controlled clinical trial to determine whether such treatment enhanced their subsequent reproductive performance. Although the treatment reduced median time to first breeding by 4.5 days (P = 0.0025) from 57.0 days, median time to conception was not significantly different between the treatment and nontreated control group (87.0 vs 88.5 days) and conception rate by 110 days after parturition was not significantly different (64.7 vs 69.6%). Use of prostaglandin was associated with a significant (P = 0.0459) decrease in conception rate at first breeding from 42.0 to 29.3%. This study suggested that prostaglandin treatment of cows with a normal reproductive tract and a palpable corpus luteum at a median of 25 days after parturition does not enhance their reproductive performance and thus is not cost-effective.

The assessment of normal early pregnancy by transvaginal color Doppler ultrasonography.

Transvaginal color Doppler was performed in 198 volunteer pregnant women whose menstrual age ranged from the fifth to the twelfth week. In all patients an attempt was made to obtain signals from both uterine arteries, peritrophoblastic/retroplacental vessels, umbilical arteries, fetal aorta, intracranial vessels, and corpus luteum flow. With the combination of color and pulsed Doppler transvaginal sonography, detection of vascular structures was greatly facilitated and the amount of time for examination significantly reduced. Flow velocity waveforms were measured and results were analyzed by calculation of the Resistance Index. During the early stage of pregnancy, we were able to locate both uterine arteries in all cases and continuous diastolic shift signal was found. Flow in the peritrophoblastic/retroplacental area was observed with an overall success rate of 94%. Blood flow in the umbilical artery and fetal aorta was visualized by color Doppler starting from the seventh week. Intracranial blood flow could be visualized starting from the tenth week in some cases. Diastolic flow in these vessels was detectable starting from the twelfth week. Corpus luteum flow was found in 148 cases (75%) and the Resistance Index decreased as pregnancy progressed.

Clinical importance of endometrial histology and progesterone level assessment in luteal-phase defect.

In order to clarify the relationship between endometrial histology and progesterone (P4), plasma P4 and estradiol levels in the luteal phase were measured in 126 cases of unexplained infertility. Endometrial biopsies were performed in the midluteal period of menstrual cycles. Forty-three of the 126 cases showed retarded endometrium. Of these 43 cases, 23 exhibited three different types of abnormal P4 secretion. Type A showed low P4 levels throughout the luteal period. Type B showed low P4 levels only in the early luteal period. Type C showed normal P4 levels in the early luteal period followed by a prompt decline. These findings indicated that P4 determination during the early, mid- and late luteal phases is necessary to assess P4 secretion. However, 20 of the 43 cases had normal P4 levels through the entire luteal phase, demonstrating an insufficient response of the endometrium to P4. Consequently, histological examination of the endometrium is required to investigate the luteal phase defect.

Assessment of luteal function after surgical tubal sterilization.

To evaluate ovarian luteal function after tubal occlusion, a group of women who underwent Pomeroy sterilization were studied. A prospective group I (n = 16) were followed for one year and scheduled for blood sampling every other day during their luteal phase before surgical procedure and at 3 and 12 months thereafter. Group II (n = 15) included women who were studied during their luteal phase at 1 or 5 years post-surgery. Mid-luteal progesterone and estradiol serum levels were calculated by estimating the average of at least 3 values of serum samples obtained in days 20-25 of a menstrual cycle. The data suggest that no major changes occur in ovarian function after surgical tubal occlusion, as assessed by the mid-luteal hormone serum levels, and underscore the safety of this procedure.

The integrated luteal progesterone: an assessment of luteal function.

An integrated luteal progesterone (ILPL) was calculated on the basis of a luteal progesterone (P) level with the assumption that the daily plasma P level in the luteal phase closely approximates a sine curve. The midluteal P-amplitude (K) was also obtained mathematically. Daily luteal P levels from five normal ovulatory cycles were assessed for the biologic variation of ILPL and K, then compiled to construct a normogram of the ILP during the luteal phase. The coefficient of variation of K and total ILPL in each cycle ranged from 9.7% to 24.3% and 3.5% to 13.2%, respectively. Fifty-two infertility patients were evaluated for their luteal function by the luteal P and estradiol (E2) level, K, ILPL, endometrial biopsy (EBX)-lag-day, as well as the lengths of follicular phase, luteal phase (L#), and cycle. Thirty-nine patients had EBX-lag day less than or equal to 2 days and were designated as infertile-normal (INF-NL) luteal phase, while the remaining 13 patients who had EBX-lag day greater than 2 days were considered as luteal phase defect (LPD). Significant (P less than 0.05) differences were observed between INF-NL and LPD in: luteal length (13.2 +/- 0.31 versus 11.0 +/- 0.58 days, respectively), and total ILPL (170 +/- 8.3 versus 113 +/- 8.5 ng/ml-day, respectively). No differences were seen in luteal P, E2 and K levels, nor in follicular and cycle length. Significant (P less than 0.05) correlations were observed between total ILPL and luteal P, E2, L#, and K; while a negative correlation was noted between follicular and luteal length.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of penicillinase linked ELISA of pregnanediol glucuronide for detection of ovulation and assessment of corpus luteal function.

A penicillinase linked enzyme immunoassay was developed for the estimation of pregnanediol-3 alpha-glucuronide (PdG) in urine. The immunoassay satisfied all the validity criteria and was used in detecting ovulation and in the assessment of corpus luteal function (CLF) during spontaneous or induced cycles. Reference values were established by estimating PdG levels in daily early morning urine samples during 31 menstrual cycles obtained from 17 regularly menstruating women. A PdG value of 1.7 micrograms/mg creatinine (micrograms/mgC) (90th Centile of follicular phase) in any MLP (mid-luteal phase) sample was considered as indicating ovulation. A value of 4.6 micrograms/mgC (20th centile of MLP) was considered to be evidence of sufficient CLF. When this approach was applied to 20 infertile cases, detection of the occurrence of ovulation/anovulation was made correctly in 19 out of 20 cases (95%). Accuracy was poor (55.6%) when the aim of the diagnosis was corpus luteal deficiency. Higher accuracy (88.9%) for corpus luteal deficiency/corpus luteal adequacy was obtained when the sum of PdG concentrations in three MLP samples were taken into consideration. A total of 13.8 micrograms/mgC (thrice the 20th centile for MLP) indicated probable corpus luteal deficiency, and values above this limit were considered to indicate corpus luteal adequacy.

Assessment of corpus luteum function by direct radioimmunoassay for progesterone in blood spotted on filter paper.

In this specific, direct RIA for progesterone in capillary blood dried on filter paper, progesterone is eluted, with phosphate buffer containing bovine serum albumin, from 5.9 microL of blood dried on 5.0-mm (diameter) discs of filter paper. The eluate is assayed, with 125I-labeled progesterone-11 alpha-glucuronyl-tyramine as tracer, with separation by a double-antibody solid-phase technique. The sensitivity of the assay is 4.7 pg per tube, corresponding to 2.5 nmol per liter of blood. Within- and between-batch CVs averaged 7.0 and 9.2%, respectively, over the working range of the assay (4.5-64 nmol/L). Concentrations of progesterone in blood spots (y) correlated well with those in serum (x) as measured by an established direct RIA (Clin Chem 28:1314, 1982): y = 0.430x - 2.44 (r = 0.972, n = 104). Progesterone is stable in the blood spots for at least 15 weeks at 25 degrees C. The convenience of multiple sampling of blood by finger prick and the simplicity of the assay make this approach useful in investigating serial progesterone concentrations in outpatients.

Pituitary alpha subunit mRNA amounts during the sheep estrous cycle. Assessment by cDNA hybridizations.

Pituitary glycoprotein hormones exhibit a dimeric structure consisting of a common alpha subunit and similar beta subunits. In this study, alpha subunit mRNA amounts have been examined in sheep pituitaries during defined times of the normal estrous cycle. These times were designed to include events prior to, and including the beginning of, the preovulatory luteinizing hormone surge. Criteria such as serum and pituitary luteinizing hormone, serum progesterone, and ovarian morphology were used to classify the groups as: 1) Day 12 of the cycle; 2) 24 h before behavioral estrus (E-24); and 3) 5 h after estrus (E + 5). RNA was extracted from the pituitaries and amounts of alpha subunit mRNA quantitated using cell-free translations and cDNA hybridizations. Both Northern transfers and RNA dot blots were used. The amount of alpha subunit mRNA in the Day 12 group was the lowest of the three groups and was similar to that seen in the pituitary from an anestrous ewe. The amount observed in the E-24 animals was only slightly increased over the Day 12 (approximately 2-fold); however, a greater increase was observed when the E + 5 group was examined (approximately 4-8-fold). These results suggest that the amount of alpha subunit mRNA increases during the time of the preovulatory luteinizing hormone surge in the normal estrous cycle of the sheep and thus probably plays a role in the important physiological event.

Radioimmunoassay of pregnanediol concentrations in early morning urine specimens for assessment of luteal function in women.

A radioimmunoassay has been developed that employs a readily available radioligand 3H-20alpha-hydroxy-4-pregnen-3-one for the determination of pregnanediol glucuronide in urine. The unextracted steroid is assayed directly after dilution of urine with the use of an antiserum produced to a thyroglobulin conjugate of pregnanediol glucuronide. The assay has been validated, and the ability to assess luteal function by measurement of the concentration of pregnanediol glucuronide in early morning urine specimens has been demonstrated.

Radioimmunoassay of progesterone in saliva: application to the assessment of ovarian function.

We report a specific radioimmunoassay that has the required sensitivity (7 pg per assay tube) for determining progesterone concentrations in 400 microL of mixed saliva collected from normal women. The assay is precise: intra and inter-assay variation (CV) never exceeded 11.0 and 8.0%, respectively. The assay was used to determine progesterone in saliva samples collected daily for not less than 28 days by normal women and by patients having abnormal ovarian function. Four normal women provided matched saliva and plasma samples for accurate dating of the menstrual cycle by plasma progesterone, estradiol, lutropin, and follitropin. Nine further subjects collected saliva samples only, and from these data a provisional "normal range" was established. Progesterone concentrations in saliva during the follicular phase of the cycle were low (less than 100 pmol/L) but rose beginning on day 12 to reach peak values of 230-550 pmol/L on day 21. Thereafter, progesterone concentrations in saliva declined to values generally less than 170 pmol/L at the commencement of menses. Saliva samples from three patients attending an infertility clinic were also studied to assess ovarian function.

PIP: A radioimmunoassay technique is reported which has the sensitivity required to determine progesterone concentrations in 400 muL of mixed saliva collected from normal women. The assay is precise; variations between tests were minimal. The assay was used to determine progesterone concentration in saliva samples collected daily for not less than 28 days by normal women and by patients with abnormal ovarian function. 4 of the normal women provided matched saliva and plasma samples for accurate dating of the menstrual cycle through plasma progesteorne, estradiol, lutropin, and follitropin. 9 others provided only saliva samples. Progesterone nccnentrations in saliva were found to be low (100pmol/L) during the follicular phase; they rose beginning on day 12 to a high of 230-550 pmol/L on day 21; afterward, they declined to values near 170 pmol/L at the start of menstruation. Saliva samples from 3 patients attending an infertility clinic were studied to assess ovarian function.

Dominance and reproduction in Baboons (Papio cynocephalus).

This monograph reports on a 14 month study of yellow baboons (Papio cynocephalus) in the Masai-Amboseli Game Reserve, Kenya. The study was an attempt to determine the relationship between agonistic dominance and reproductive success in male baboons and centered around testing a priority-of-access model of mating behavior. Explicit criteria for determining dominance in baboons are presented and the consistency of dominance relationships through time is analyzed for all classes of individuals. Related data on reproductive cycle length, perineal and behavioral indications of the optimal day for mating, changes in female behavior during estrus, and effects of the presence of estrous females on group organization are also included. This work constitutes the first comprehensive field study of baboon mating systems and social organization and emphasizes the use of systematic behavior sampling techniques in the field and quantitative models in the study of primate social behavior.