The influence of nutrition on the insulin-like growth factor system and the concentrations of growth hormone, glucose, insulin, gonadotropins and progesterone in ovarian follicular fluid and plasma from adult female horses (Equus caballus).

BACKGROUND: Feed intake affects the GH-IGF system and may be a key factor in determining the ovarian follicular growth rate. In fat mares, the plasma IGF-1 concentration is high with low GH and a quick follicular growth rate, in contrast to values observed in thin mares. Nothing is known regarding the long-term effects of differential feed intake on the IGF system. The objective of this experiment was to quantify IGFs, IGFBPs, GH, glucose, insulin, gonadotropin and progesterone (P4) in blood and in preovulatory follicular fluid (FF) in relation to feeding levels in mares. METHODS: Three years prior to the experiment, Welsh Pony mares were assigned to a restricted diet group (R, n = 10) or a well-fed group (WF, n = 9). All mares were in good health and exhibited differences in body weight and subcutaneous fat thickness. Follicular development was scanned daily and plasma was also collected daily. Preovulatory FF was collected by ultrasound-guided follicular aspiration. Hormone levels were assayed in FF and plasma with a validated RIA. RESULTS: According to scans, the total number of follicles in group R was 53% lower than group WF. Insulin and IGF-1 concentrations were higher in WF than in R mares. GH and IGF-2 concentrations were lower in plasma from WF mares than from R mares, but the difference was not significant in FF. The IGFBP-2/IGFBP-3 ratio in FF was not affected by feeding but was dramatically increased in R mare plasma. No difference in gonadotropin concentration was found with the exception of FSH, which was higher in the plasma of R mares. On the day of puncture, P4 concentrations were not affected by feeding but were higher in preovulatory FF than in plasma. CONCLUSIONS: The bioavailability of IGF-1 or IGF-2, represented by the IGFBP2/IGFBP3 ratio, is modified by feed intake in plasma but not in FF. These differences partially explain the variability in follicular growth observed between well-fed mares and mares on restricted diets.

Luteoprotective role of equine chorionic gonadotropin (eCG) during pregnancy in the mare.

The effects of repeated cloprostenol administration were compared in mares impregnated by horses and mares impregnated by donkeys in order to assess the role of eCG on the development of pregnancy-associated resistance to the luteolytic and abortifacient effects of PGF2α. Eleven mares impregnated by donkey (mule pregnancy) and 9 mares impregnated by horse (horse pregnancy) were used. Six mares with mule pregnancy and four with horse pregnancy were injected with cloprostenol (0.25 mg) when they were between day 65 and day 75 of pregnancy, and the treatment was repeated 48, 72 and 96 h latter. The rest of the mares remained as controls. Concentrations of eCG were 10 times higher (p < 0.001) in mares impregnated by horses than in mares impregnated by donkeys, and they were not affected by cloprostenol treatment. Luteolysis was completed 30 h after the first cloprostenol injection in mule pregnancies, while mares with horse pregnancies required 96 h and three cloprostenol injections to complete luteolysis. Regression analysis revealed significant associations between eCG concentrations at time 0 and the time required for completion of luteolysis (p < 0.001), foetal death (p < 0.01) and foetal expulsion (p < 0.05). It is concluded that high eCG concentrations in mares impregnated by horses protect the corpora lutea of pregnancy against the luteolytic effects of PGF2α. Low eCG concentrations in mares carrying mule foetuses afford them less protection against the luteolytic effect of PGF2α, and this may be a cause of the increased foetal mortality that occurs between days 60 and 90 of pregnancy in these mares.

Follicle stimulating hormone secretion and dominant follicle growth during treatment of Bos indicus heifers with intra-vaginal progesterone releasing devices, oestradiol benzoate, equine chorionic gonadotrophin and prostaglandin F(2α).

The aim of this study was to investigate the effects on follicle stimulating hormone (FSH) secretion and dominant follicle (DF) growth, of treatment of Bos indicus heifers with different combinations of intra-vaginal progesterone releasing devices (IPRD), oestradiol benzoate (ODB), PGF2α and eCG. Two-year-old Brahman (BN; n=30) and Brahman-cross (BNX; n=34) heifers were randomly allocated to three IPRD-treatments: (i) standard-dose IPRD [CM 1.56g; 1.56g progesterone (P4); n=17]; (ii) half-dose IPRD (CM 0.78g; 0.78g P4; n=15); (iii) half-dose IPRD+300IU eCG at IPRD removal (CM 0.78g+G; n=14); and, (iv) non-IPRD control (2×PGF2α; n=18) 500µg cloprostenol on Days -16 and -2. IPRD-treated heifers received 250µg PGF2α at IPRD insertion (Day -10) and IPRD removal (Day -2) and 1mg ODB on Day -10 and Day -1. Follicular dynamics were monitored daily by trans-rectal ultrasonography from Day -10 to Day 1. Blood samples for determination of P4 were collected daily and samples for FSH determination were collected at 12h intervals from Day -9 to Day -2. A significant surge in concentrations of FSH was observed in the 2×PGF2α treatment 12h prior and 48h after follicular wave emergence, but not in the IPRD-treated heifers. Estimated mean concentrations of total plasma P4 during the 8 days of IPRD insertion was greater (P<0.001) in the CM 1.56g P4 treated heifers compared to the CM 0.78g P4 treated heifers (18.38ng/ml compared with 11.09ng/ml, respectively). A treatment by genotype interaction (P=0.036) was observed in the mean plasma P4 concentration in heifers with no CL during IPRD insertion, whereby BN heifers in the CM 1.56g treatment had greater plasma P4 than the BNX heifers on Days-9, -7, -6, -5, and -4. However, there was no genotype effect in the CM 0.78g±G or the 2×PGF2α treatment. Treatment had no effect on the DF growth from either day of wave emergence (P=0.378) or day of IPRD removal (P=0.780) to ovulation. This study demonstrates that FSH secretion in B. indicus heifers treated with a combination of IPRD's and ODB to synchronise ovulation was suppressed during the period of IPRD insertion but no significant effect on growth of the DF was observed.

Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells.

The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive ß-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities.

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares.

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = -0.834, P < 0.05; day 35: r = -0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.

Effect of exogenous progesterone and eCG treatment on ovarian follicular dynamics in vicunas (Vicugna vicugna).

The aim of the present study was two-fold. First, to evaluate the effect of exogenous progesterone on ovarian follicular dynamics in order to assess its ability to synchronize ovarian activity in the vicuna. Secondly, to evaluate the ovarian response to the treatment with eCG through the observation of the structures developed in the ovaries. Follicular dynamics was monitored daily by transrectal ultrasonography in 12 adult, non-pregnant vicunas. Plasma progesterone and estradiol-17beta concentrations were measured in blood samples collected daily. In experiment 1, intravaginal devices containing 0.33g of progesterone were inserted into the vagina and kept in place for 5 days (treatment group, n = 8). After progesterone withdrawal, five animals were further monitored in order to evaluate the efficacy of the CIDR to synchronize the emergence of a dominant follicle. In experiment 2, four females received 750IU of eCG IM. Two were previously monitored ultrasonographically to confirm the absence of a dominant follicle at the beginning of the superstimulatory treatment (group A). The other two animals had a CIDR inserted into the vagina for 5 days and the superstimulatory treatment was applied 24h after device withdrawal (group B). Females from both groups were surgically explored 96 h after eCG injection; the ovaries were exposed and the number of newly formed structures produced by each ovary was counted. Peak progesterone concentrations (25.9 +/- 5.29 nmol l(-1), mean +/- S.E.M.) were attained on day 1 after device insertion, remained high until the day of device withdrawal (9.7 +/- 1.98 nmol l(-1)) and decreased to 5.5 +/- 1.13 nmol l(-1) the day after. There was no follicle development to the state of dominance after device insertion. Moreover, mean follicle diameter steadily decreased after insertion of the device until the minimum mean value (1.85 +/- 0.17 mm) was recorded on day 5 (P = 0.006). Similarly, plasma concentrations of estradiol-17beta remained below 35 pmol l(-1) during the period of progesterone treatment in all animals and the mean estradiol-17beta declined with the lowest value (22.1 +/- 2.19 pmol l(-1)) being recorded on day 4 after device insertion. After superstimulation of follicular development with eCG, the total number of follicles that developed was 33 in group A and 58 in group B and the mean number of newly developed ovarian structures per female was 22.75 +/- 4.26. In conclusion, progesterone released by the CIDR exerts a negative effect on ovarian follicular development and function suggesting intravaginal devices could be used to synchronize the beginning of follicular waves during a superstimulatory treatment. There was also a tendency for greater ovarian follicular development when the animals were previously treated with progesterone.

Successful non-surgical transfer of horse embryos to mule recipients.

Mules, hybrids resulting from the mating of a horse mare (Equus caballus, 2n = 64) to a Jack donkey (E. asinus, 2n = 62), are generally infertile. Five horse embryos were transferred non-surgically to two cyclic and one acyclic recipient mules. In the mares and cycling mules, oestrus and ovulation were induced with, respectively, d-cloprostenol and human chorionic gonadotrophin (hCG). The acyclic mule, on the other hand, received oestradiol benzoate when the embryo donor was showing oestrus and progesterone after the donor had ovulated and until pregnancy diagnosis. Non-surgical embryo collections were attempted on day 7 after ovulation and recovered embryos were transferred transcervically into the mules' uteri. Mules that became pregnant were blood sampled serially for equine chorion gonadotrophin (eCG), progestagen and total conjugated oestrogen concentrations until around 6 months of gestation. The three embryos transferred to the acyclic mule did not produce any pregnancies whereas both embryos transferred to the cycling mules resulted in the birth of live foals. The peak concentration and duration of secretion of eCG differed markedly between the two pregnant mules, although both animals appeared to develop secondary corpora lutea beyond day 40 of gestation, as in normal intraspecies horse pregnancy. Moreover, the rise in serum oestrogen concentrations from around day 90 was also similar to that seen in normal pregnant mares. Parturition occurred spontaneously on day 348 of gestation in both mules and the resulting colt foals developed normally to weaning. Thus, cycling mules can carry a horse conceptus after non-surgical embryo transfer and give birth to a normal mature foal.

Persistence of pregnant mare serum gonadotrophin in plasma of Japanese quail (Coturnix coturnix japonica).

Pregnant mare serum gonadotrophin (PMSG) is currently being used to develop a hormone regime that will stimulate reproductive development in Japanese quail (Coturnix coturnix japonica). In this study, the persistence of PMSG in quail plasma was examined after a single injection of the hormone (500 IU). Plasma concentrations of PMSG increased to a peak 12 h after injection and declined to approximately 50% of peak concentrations 24 h after injection. Pregnant mare serum gonadotrophin concentrations declined gradually thereafter and had not returned to basal levels by 96 h after injection. Cloacal diameter, and ovarian and oviducal mass, had increased significantly by 96 h after injection. The persistence of PMSG in quail plasma has implications for the use of the hormone in future regimes stimulating reproductive activity in birds.

Effect of the gonadotrophins treatment on morphological alterations in ovary and peripheral plasma concentrations of steroid hormones in gilts.

The present study was designed to examine the influence of gonadotrophins treatment on the ovarian morphology changes and plasma concentrations of steroid hormones in peripheral blood. The experiment was performed on sexually pubertal gilts (Large White x Landrace) of similar age (7-8 months) and body mass (100-110 kg) with two controlled subsequent estrous cycles. The animals were randomly divided into four groups: two control consisting of pigs with the luteal phase (n = 9, the 10th day of the estrous cycle) and the follicular phase (n = 6, the 20th day of the estrous cycle) and two experimental ones consisting of animals with both mentioned periods (n = 7 and n = 9) treated with gonadotrophins (PMSG and hCG). The gilts in the luteal phase were injected (s.c.) with gonadotrophins at a daily dose of PMSG 400 and hCG 200 IU from the 16th to the 27th day (the 6th day of the next estrous cycle). The gilts in the follicular phase, were injected with the same dose of gonadotrophins but from the 8th to the 19th day of the estrous cycle. Plasma concentrations of P4, A4, T, E1, E2 and metabolite of PGF2 alpha-PGFM were determined by radioimmunoassay (RIA) method. Injections of PMSG and hCG in both experimental groups produced several times enlarged: weight, size and volume of ovaries and alterations in a number of structural elements as compared with those found in the control animals. The morphological elements presented in ovaries: corpora haemorrhagica, corpora lutea, regular and atretic follicles and first of all cysts by distinctly differentiation thickness of the walls are characteristic for cystic ovarian degeneration. Plasma concentrations all determined hormones after gonadotrophins treatment in experimental groups were increased except E1 (insignificant decrease) in luteal phase as compared with those found in the control groups. Statistically significant increase (p < 0.001) in plasma concentrations of P4, A4, and T in both experimental groups and E2 (p < 0.001) in luteal phase were noted. In peripheral plasma concentrations increase of E1 and E2 in follicular phase of the estrous cycle were insignificant.

The influence of maternal size on placental, fetal and postnatal growth in the horse. II. Endocrinology of pregnancy.

Within-breed artificial insemination and between-breed embryo transfer were carried out in small pony (P) and large Thoroughbred (Tb) mares to create 4 types of horse pregnancy in which the fetus experienced spatial and nutritional deprivation (Tb-in-P; n=8), luxury (P-in-Tb; n=7) or normality (Tb-in-Tb; n=7 and P-in-P; n=7) in utero. Measurement of equine chorionic gonadotrophin (eCG), total conjugated oestrogens and progestagen concentrations in serial peripheral serum samples recovered from all the mares throughout gestation showed that the amount of eCG produced during the first half of gestation was dependent upon the breed of the mare rather than the breed of the fetus being carried. In contrast, the mean total amounts of oestrogens produced, as measured by area under the curve, were significantly greater (P=0.003) in the two types of pregnancy in which a Thoroughbred fetus was being carried (Tb-in-Tb and Tb-in-P) than those in which a pony fetus was gestated (P-in-P and P-in-Tb); the evidence suggests that the Tb fetus may have larger gonads than the P fetus and thereby secrete more C-19 precursor steroids for aromatisation to oestrogens by the placenta. In the final weeks of pregnancy mean plasma progestagen concentrations rose much earlier, and to significantly higher levels (P<0.001), in the Tb-in-P than in the P-in-Tb pregnancies, thereby reflecting the increased fetal stress in the former causing premature maturation of the fetal adrenal gland. This, in turn, resulted in increased secretion of pregnenolone by the adrenal cortex for conversion to progestagens by the placenta.

Increase of plasma eCG binding rate after administration of repeated high dose of eCG to cows.

Equine chorionic gonadotrophin (eCG) is still used to promote follicular growth in cattle and, more recently with an increased frequency of administration, in ovum pick-up protocols. The aim of this experiment was to verify the possible effect of high frequency of administration on the immune response to eCG. The profiles of eCG binding rate, in the blood of two groups (A, B) of 4 primiparous cross breed beef cows (3-3.5 years old) submitted weekly for 5 to 10 weeks to repeated high doses (1000-2000 IU) of equine chorionic gonadotrophin, are presented in this paper. A sensitive radiometric method was used to detect antibodies in plasma. The profiles clearly indicated a marked increase of eCG binding rate after 3 to 5 injections of the exogenous hormone to the females. The statistical analysis of the results established that treatments induced a significant increase (P < 0.01) in binding rates after 6 and 3 injections in group A and B respectively. These binding rates remained elevated for at least 1 week following the last injection and decreased afterwards. The values of plasma binding rates following repeated eCG administration differed significantly between groups (0.90+/-1.04 and 1.04+/-0.11 for groups A and B before treatment versus 11.77+/-0.92, 6.70+/-0.85 for groups A and B after treatment, P < 0.01) and from one cow to another (P < 0.01) with some cows presenting no significant immune response while others were more reactive against the hormone (at least 3 injections).

Luteal deficiency and embryo mortality in the mare.

Four separate components combine to produce the progesterone and biologically active 5 alpha-reduced pregnanes needed to maintain pregnancy in the mare. The primary corpus luteum (CL) is prolonged beyond its cyclical lifespan by the down-regulation of endometrial oxytocin receptors to prevent activation of the luteolytic pathway and its waning progesterone production is supplemented from day 40 of gestation by the formation of a series of accessory CL which develop in the maternal ovaries as a result of the gonadotrophic actions of pituitary FSH and the equine chorionic gonadotrophin (eCG). From around day 100 the allantochorion secretes progesterone and progestagens directly to the endometrium and underlying myometrium and, in the last month of gestation, the enlarging foetal adrenal gland secretes appreciable quantities of pregnenelone which is also utilized by the placenta to synthesize progestagens. Between 10 and 15% of mares undergo foetal death and abortion at some time in gestation and the majority of these losses occur during the first 40 days of gestation when the primary CL is the sole source of progesterone. Yet, all the available evidence suggests that untoward luteolysis is not common in this period and the losses that do occur have other underlying causes. Beyond day 40 the secondary CL receive powerful luteotrophic support from eCG and from day 80-100 until term the supply organ (placenta) and target tissues (endometrium and myometrium) are in direct contact with each other over their entire surface. In the face of this interlocking and failsafe system for progestagen production throughout pregnancy, and despite a paucity of evidence that a deficiency of progesterone production is a cause of pregnancy loss in the mare, it is surprising, and worrying, that annually many thousands of pregnant mares throughout the world are given exogenous progestagen therapy during part or all of their gestation as a form of preventative insurance against the possibility of pregnancy failure. Basic investigative research is required urgently to validate or debunk the practice.

Neurosteroid withdrawal model of perimenstrual catamenial epilepsy.

PURPOSE: Perimenstrual catamenial epilepsy, the increase in seizure frequency that some women with epilepsy experience near the time of menstruation, may in part be related to withdrawal of the progesterone metabolite allopregnanolone, an endogenous anticonvulsant neurosteroid that is a potent positive allosteric gamma-aminobutyric acidA (GABA(A)) receptor modulator. The objective of this study was to develop an animal model of perimenstrual catamenial epilepsy for use in evaluating drug-treatment strategies. METHODS: A state of prolonged high serum progesterone (pseudopregnancy) was induced in 26-day-old female rats by sequential injection of pregnant mares' serum gonadotropin and human chorionic gonadotropin. Neurosteroid withdrawal was induced by treatment with finasteride (100 mg/kg, i.p.), a 5alpha-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone. Plasma progesterone and allopregnanolone levels were measured by gas chromatography/electron capture negative chemical ionization mass spectrometry. Seizure susceptibility was evaluated with the convulsant pentylenetetrazol (PTZ). RESULTS: Plasma allopregnanolone levels were markedly increased during pseudopregnancy (peak level, 55.1 vs. control diestrous level, 9.3 ng/mL) and were reduced by 86% 24 h after finasteride treatment (6.4 ng/mL). Progesterone levels were unaffected by finasteride. After finasteride-induced withdrawal, rats showed increased susceptibility to PTZ seizures. There was a significant increase in the number of animals exhibiting clonic seizures when challenged with subcutaneous PTZ (60 mg/kg) compared with control pseudopregnant animals not undergoing withdrawal and nonpseudopregnant diestrous females. The CD50 (50% convulsant dose) was 46 mg/kg, compared with 73 mg/kg in nonwithdrawn pseudopregnant animals and 60 mg/kg in diestrous controls. The threshold doses for induction of various seizure signs, measured by constant intravenous infusion of PTZ, were reduced by 30-35% in neurosteroid-withdrawing animals compared with control diestrous females. No change in threshold was observed in pseudopregnant rats treated from days 7 to 11 with finasteride, demonstrating that high levels of progesterone alone do not alter seizure reactivity. CONCLUSIONS: Neurosteroid withdrawal in pseudopregnant rats results in enhanced seizure susceptibility, providing an animal model of perimenstrual catamenial epilepsy that can be used for the evaluation of new therapeutic approaches.

Study of early pregnancy factor (EPF) in equine (Equus caballus).

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. CONCLUSIONS: The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.

Extraction of equine chorionic gonadotrophin from pregnant mare plasma by direct adsorption on chromatographic media.

Equine chorionic gonadotrophin (eCG) is a hormone of practical value in veterinary medicine and animal production. Here we report a novel preparation procedure based on its direct adsorption onto anionic-exchange resins in a batch-wise mode. The active plasma is previously conditioned to reduce pH and ionic strength to required levels. After the adsorption stage, a 90% recovery of the initial eCG is achieved, with a concentration factor of about 50 and an enrichment factor around 500, with high preservation of biological activity. Further purification is carried out by cation-exchange column chromatography. The recovery for the whole process is higher than 70%, and the final potency of the preparation is close to 4000 IU/mg. The process is well suited for its application to the industrial scale.

Comparison of the merits of measuring equine chorionic gonadotrophin (eCG) and blood and faecal concentrations of oestrone sulphate for determining the pregnancy status of miniature horses.

The relative merits of measuring blood concentrations of equine chorionic gonadotrophin (eCG, previously known as pregnant mare serum gonadotrophin (PMSG)), or oestrone sulphate (OS), or faecal OS concentrations for determining pregnancy status in miniature horses were investigated. Pregnant mares between 40 and 140 days after mating had serum eCG concentrations > 1 I.U. mL-1, with the highest concentrations occurring between days 50 and 120. However, eCG measurements were susceptible to returning a 'false positive' diagnosis of pregnancy. Plasma OS concentrations ranged from 0.1 to 3.6 ng mL-1 in non-pregnant mares, whereas pregnant mares beyond 100 days post-mating all had plasma OS concentrations > 30 ng mL-1. Faecal OS concentrations ranged from 4 to 89 ng g-1 in non-pregnant mares. For faecal samples collected from pregnant mares 150 days or more after mating, 97% of samples had OS concentrations > 85 ng g-1, the value 3 standard deviations above the mean non-pregnant value. None had values below 67 ng g-1, the value 2 standard deviations above the mean non-pregnant value. These results show that measurement of eCG is suitable for determining pregnancy status in miniature mares between 40 and 100 days post-mating. However, mares returning a 'pregnant' diagnosis should undergo a blood OS test 100 or more days after mating to eliminate the possibility of a 'false positive' diagnosis. Measuring blood OS is recommended as the method of choice for determining pregnancy status in miniature mares 100 or more days after mating. Faecal OS measurements provide a non-invasive alternative to blood OS testing from 150 days post-mating. However, the discrimination between 'pregnant' and 'non-pregnant' levels of OS is not as great in faeces as it is in blood.

Studies on aromatase inhibitors. IV. Synthesis and biological evaluation of N,N-disubstituted-5-aminopyrimidine derivatives.

In order to study the potency of the 5-aminopyrimidine skeleton as an aromatase inhibitor, we synthesized various N,N-disubstituted-5-aminopyrimidine derivatives and evaluated their aromatase-inhibitory activity (in vitro) and their inhibitory activity on pregnant mare serum gonadotropin (PMSG)-induced estrogen synthesis (in vivo). Compounds with the fluoro-substituted benzyl group showed potent aromatase inhibition. Among them, 5-[(4-cyanophenyl)(3,5-difluorobenzyl)amino]pyrimidine (5w, YM553) was a highly potent compound with an IC50 value of 0.038 nM for aromatase from human placenta. Its inhibitory effect was approximately four times greater than that of YM511. In addition, YM553 was a weak inhibitor of other enzymes involved in steroid hormone synthesis. These results indicate that YM553, as well as YM511 (a 4-amino-4H-1,2,4-triazole derivative), is a promising agent for the treatment of estrogen-dependent diseases.

Effects of follicular aspiration and flushing, and the genotype of the fetus on circulating progesterone levels during pregnancy in the mare.

When aspirating ovarian follicles in pregnant mares to obtain oocytes for in vitro fertilisation (IVF), the effect of the manipulation on circulating concentrations of progesterone may be an important consideration in terms of the maintenance of pregnancy. The object of this study was to compare the effects of 3 different forms of transvaginal ultrasound-guided follicle aspiration (Treatment 1, no aspiration, n = 4; Treatment 2, aspirate only follicles > or =20 mm in diameter, n = 7; Treatment 3, aspirate all visible follicles, n = 7) on peripheral plasma progesterone concentrations between Days 21 and 150 of gestation in 9 mares carrying intraspecies horse and 9 mares carrying interspecies mule conceptuses. The 3 follicle aspiration treatments were applied at the peak of each follicular wave as determined by follicular mapping by means of transrectal ultrasonography on alternate days. The plasma progesterone profile in mares undergoing Treatment 1 was in close agreement with those reported previously in pregnant mares. A decline in plasma progesterone levels occurred after Day 53 of gestation in Treatments 2 and 3 mares, indicating that the follicular aspiration procedures did interfere with the formation of secondary corpora lutea. However, the levels in individual mares never dropped low enough to endanger the pregnancy. Mares carrying mule pregnancies exhibited higher mean plasma progesterone concentrations between Days 39 and 45 of gestation than mares carrying horse pregnancies, equivalent levels between Days 46 and 66 despite the lower circulating concentrations of chorionic gonadotrophin (mule CG) in their blood during this period and lower progesterone levels between Days 67 and 150 of gestation. The results indicate that the primary corpus luteum in the pregnant mare may be more sensitive to mule CG than horse CG. Furthermore, the earlier disappearance of CG from the circulation in mares carrying mule fetuses is reflected by an earlier decline in plasma progesterone concentrations in this type of equine pregnancy.

Anti-PMSG antibody levels in sheep subjected annually to oestrus synchronisation.

Estimation of the long-term consequences on reproduction performance of the oestrus synchronisation treatments that are annually applied to ewes was carried out on nine officially controlled dairy flocks in the Roquefort region of France. A hormonal treatment combining the insertion of a vaginal fluoro-gestone acetate (FGA) sponge for 14 days and the injection of about 500 IU of pregnant mare serum gonadotropin (PMSG) at withdrawal was applied to the ewes in seven of the nine flocks. The ewes in the two other flocks were used as controls. Blood samples were taken from each female just before the treatment (to test for the presence of residual antibodies) and 20 days after the PMSG injection. Anti-PMSG antibody binding rates were calculated for each blood sample. The residual binding rate increased with age and induce negative effects on the following years reproduction performances, i.e., they increased the probability that the ewes would not become pregnant.

In vitro and in vivo evidence on the site of neutralization of equine chorionic gonadotrophin (eCG) by an eCG antiserum.

This study was designed to determine whether the major site of eCG neutralization by an antiserum to the hormone is at the peripheral or ovarian level. Hamsters hypophysectomized at oestrus were injected s.c. with 25 iu eCG. Three days later, preovulatory follicles were dissected and cultured for 5 h and the medium was changed every hour. At the end of the first hour of incubation, oestradiol and androstenedione accumulation was high, with a sharp drop over the next 4 h, whereas progesterone concentrations did not change over the entire period. Addition of eCG antiserum to the incubated follicles did not affect steroidogenesis. Addition of 1.0 iu eCG in the second hour or every hour sustained oestradiol production at supraphysiological amounts. However, addition of eCG plus eCG antiserum every hour eliminated the stimulatory effects of eCG on oestradiol production. In another experiment, hamsters injected with eCG were treated 3 days later by i.p. injection of eCG antiserum and groups of animals were killed over the next 8 h. Serum samples before and after injecting eCG antiserum were incubated overnight with a goat anti-rabbit immunoglobulin to separate free, unbound eCG from bound eCG. At time zero (before injecting the antiserum) free eCG was increased, but within 1 h after eCG antiserum there was an eightfold decrease of the hormone, and these concentrations were maintained over the next 7 h. The fall in unbound eCG in vivo coincided with the decay in serum oestradiol and androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)