The influence of progesterone-induced proteins on glucose metabolism in early equine embryos.

The influence of different maternal plasma progesterone concentrations on embryonic glucose metabolism was studied. Uterine flushes were obtained after treating ovariectomized mares (n = 3) with 0 (control), 100 or 200 mg progesterone daily for 7 d. A group of progesterone-induced proteins (PIP) of Mr approximately 20,000 were identified in flushes from progesterone treatments by SDS-PAGE but were not observed in control flushes. Progesterone-induced proteins were removed from half the pooled flush in each treatment group by Sepharose blue CL-6B. In a 3 x 2 factorial (progesterone treatments, progesterone-induced proteins) experiment, 6 groups of Day 7 equine embryos (n = 6 per group) were incubated in culture media (MEM:DPBS; 1:3) containing radioactively-labeled glucose. Contributions of the Embden-Meyerhof pathway (EMP) and the pentose-phosphate pathway (PPP) to the total metabolism of glucose in early equine embryos were assessed separately. In the 200 mg progesterone treatment group, the presence of progesterone-induced proteins in the culture medium resulted in a 4-to 5-fold increase in the activities of the Embden-Meyerhof pathway and the pentose-phosphate pathway. These results lead to the following conclusions: 1)Addition of progesterone-induced uterine proteins from mares with high levels of circulating progesterone enhance the metabolic activities of the Embden-Meyerhof pathway and the pentose-phosphate pathway in Day 7.5 equine embryos in culture. 2)Uterine secretion of progesterone-induced proteins which is quantitatively and/or qualitatively adequate to modify embryonic glucose metabolism in vitro is dependent on a minimal concentration of maternal plasma progesterone.

Analysis of post-partum fertility in mares on a thoroughbred stud in southern Victoria.

This project surveys the reproductive performance of 154 foaling Thoroughbred mares on a commercial stud in southern Victoria. Of these, 96 were served on foal heat (FHS) and 58 were served at a subsequent prostaglandin-induced oestrus (PGS). The PGS group of mares performed more favourably in all aspects except the foaling-to-conception interval where there was a 9.4 day advantage to the FHS group. The first service conception rate in the FHS group was 47.9% compared with 55.2% in PGS mares. Second heat period conception rates were 46% vs 57.7% for FHS and PGS mares, respectively. Overall conception rates at the end of the breeding season were 83.3% in the FHS group and 89.7% in the PGS group. Pregnancy losses before day 45 and after day 45 were 10% and 9.3% in FHS mares and 3.9% and 3.6% in the PGS mares. Served oestrous periods per conception numbered 1.7 and 1.4 in the FHS and PGS groups, respectively.

The effect of an extended artificial photoperiod and gonadotrophin-releasing hormone infusions in inducing fertile oestrus in anoestrous mares.

The occurrence of fertile oestrus early in the breeding season is of paramount importance to the Thoroughbred industry to facilitate early conception. This paper compares 2 techniques for inducing fertile oestrus in anoestrous mares using either an extended photoperiod alone or together with gonadotrophin-releasing hormone (GnRH) infusions. Eleven mares were placed under conditions of 16 h light and 8 h darkness and 5 of these were implanted with osmotic minipumps delivering approximately 100 ng GnRH/kg/h for 28 days (treated mares). The treated mares ovulated 27.7 days earlier than and conceived 32 days earlier than the 6 mares not given GnRH. GnRH-induced ovulations were followed by a competent luteal phase. The combination of GnRH pumps implanted 2 weeks before commencement of service together with extended photoperiod from July 1 has promise in assisting the stud breeder to improve reproductive efficiency on commercial stud farms.

Changes in urinary and plasma oestrone sulphate concentrations after induction of foetal death in mares at 45 days of gestation.

Foetal death was induced in 10 Standardbred mares at day 45 of gestation by injecting 20 to 45 ml of hypertonic (24% W/V) saline into the conceptus at surgery. Ten mares underwent sham treatment and acted as controls. Blood and urine samples were collected every other day between days 30 and 45 post ovulation and at 0, 3 and 6 h relative to the infusion of saline in the treated mares, or sham treatment in control mares. Blood and urine samples were then collected daily between days 46 and 55 post ovulation. Urine oestrone sulphate (E1S) concentrations, measured by radioimmunoassay, increased between day 34 and day 36 of gestation in treated and control groups. In mares in which foetal death was induced, urine E1S concentrations declined post-operatively and were significantly (p less than .05) lower than controls by day 50. In plasma, E1S concentrations showed a major increase between days 36 and 40 in both groups. This was followed by a rapid decline after treatment in saline-injected mares, so that by day 48 plasma E1S concentrations in treated mares were significantly (P less than .05) lower than the controls. The results show that urinary and plasma E1S concentrations rise rapidly during early pregnancy, and are associated with a viable foetus after day 45 of pregnancy.

Reproductive performance of thoroughbred mares on six commercial stud farms.

The records of 1630 mare years from 6 Thoroughbred stud farms in south eastern Australia were analysed for the years 1981 to 1986. Overall pregnancy and foaling rates were 83.9% and 69.3%, respectively. When calculated per served oestrous cycle, pregnancy and foaling rates were 54.7% and 43.1%, respectively. Pregnancy and foaling rates were higher (P < 0.001) for mares 3 to 10 years of age than for older mares. There was no difference in the pregnancy rates of maiden, barren and foaling mares. The foaling rate was significantly higher (P < 0.001) in mares that became pregnant during the first served oestrous cycle (77.8%) than in mares that needed two served oestrous cycles to become pregnant (65.4%). Of all diagnosed pregnancies, 19.5% were not completed. Pregnancy loss was lower (P < 0.05) in maiden (12.4%) than in barren (19.7%) or foaling (20.9%) mares. Twins were diagnosed in 7.8% of all pregnancies. If one conceptus was lost without external interference, 84.1% of pregnancies went to term. If one conceptus was manually crushed, 55.9% of pregnancies were maintained. If prostaglandin was used to terminate twin pregnancies, 60% of mares so treated produced foals the following year.

Attempted conversion of twin to singleton pregnancy in two mares with associated changes in plasma oestrone sulphate concentrations.

The removal of one of twin embryos was attempted by infusion of 24% (w/v) saline into the gestation sac in 2 mares by laparotomy. The treatment was successful in one mare (Case 1) and the untreated embryo remained viable. However, neither foetus survived in the second mare (Case 2). Plasma oestrone sulphate (E1S) concentrations fell immediately after treatment in both mares but recovered to approximately 50% of pretreatment levels in Case 1. In Case 2 plasma E1S concentrations declined steadily and were less than 1 ng/ml within 6 days of treatment. These preliminary results suggest that the method may be useful for selective removal of one of twin embryos in mares. Furthermore, plasma E1S concentrations may be a useful indicator of embryonic viability.

Synchronization of oestus and timed insemination of mares.

Oestrus was synchronized in 116 mares by means of an i.m. injection of prostaglandin F-2 alpha (Day 0) and of fluprostenol (a PG analogue) on Day 16. Mares were then randomly divided into three groups. Group A mares (N = 30) were given 2500 i.u. hCG I.M. ON Day 20 and artificially inseminated on Day 21 without detection of oestrus. Group B mares (N = 32) were given 2500 i.u. hCG i.m. on Day 20 and inseminated on Days 21 and 23, also without oestrus detection. Group C mares (N = 54) were teased on Days 18, 19, 21, 23 and 25 and inseminated on Days 19, 21, 23 and 25 while they were in oestrus. Semen was collected by artificial vagina from 3 stallions. One-third of the mares in each group were assigned to each stallion at random. The gel-free fraction was divided equally among the mares, and used within 1 h of collection. Pregnancy rates at about 60 days of gestation were not significantly different. A high rate of synchronization of oestrus (80%) was attained within 48 h of treatment with fluprostenol.

Prostaglandin F and E levels in the conceptus, uterus and plasma during early pregnancy in the ewe.

Concentrations of prostaglandins E and F (PGE and PGF) were measured in the embryo or fetus, extra embryonic or fetal membranes (membranes), intercaruncular and caruncular endometrium and plasma collected from uterine and ovarian arterial and venous vessels from separate groups of ewes laparotomized at 5 day intervals from day 10 to day 55 of pregnancy. Our purpose was to investigate the role of prostaglandins E and F in the maternal recognition of pregnancy, implantation and early placental function. Our data suggest that the initial maintenance of the corpus luteum in the pregnant ewe does not involve a reduction in PGF production, compared to pregnant ewes; but a change in the pattern of PGF secretion. This is accompanied by an elevation in PGE production of similar magnitude to that observed in non pregnant ewes. The extra embryonic/fetal membranes appear to be the major source of elevated PGF levels in the maternal circulation prior to day 30 of pregnancy. Between days 35 and 55 of gestation the rising PGF levels in maternal serum probably come from the fetus. Over the same period PGE levels rise in the fetus and intercaruncular endometrium, but PGE secretion into the maternal circulation is not enhanced. A role for PGF and PGE in fetal, placental and uterine growth is suggested; placental and uterine endocrine function may also be targets.

Continuous infusion of gonadotrophin-releasing hormone (GnRH) advances the onset of oestrous cycles in thoroughbred mares on Australian studfarms.

Continuous infusion of gonadotrophin releasing hormone (GnRH) has been shown to induce oestrus and ovulation in experimental mares in seasonal anoestrus. The present study sought to apply this form of treatment to anoestrous mares subjected to normal management procedures on 15 commercial Thoroughbred studfarms in Australia. The GnRH was delivered via subcutaneously implanted osmotic minipumps (OMP) at dose rates of 100 ng/kg/h and 200 ng/kg/h. The ovaries of treated mares were palpated on the day of OMP insertion (Day 0) and blood samples were collected twice weekly from a proportion of mares for assay of plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone concentrations. The period from commencement of GnRH infusion to first ovulation was significantly shorter (P less than 0.001) in treated than control mares and was negatively correlated with the total ovarian volume (P less than 0.01) and the diameter of the largest follicle (P less than 0.05) at onset of treatment. GnRH infusion produced an immediate increase in plasma LH concentrations in the mares that ovulated during the infusion period and LH levels peaked at the time of ovulation. Plasma progesterone profiles indicated that the induced corpus luteum was of normal lifespan and progesterone secreting ability. The mean areas under the LH and FSH response curves in treated mares were significantly greater than in control mares (LH, P less than 0.001; FSH, P less than 0.05). However, there was no statistically significant difference in mean areas under the LH and FSH curves in the GnRH-treated groups. Continuous infusions of GnRH induced ovulation in a high proportion of seasonally anoestrous mares on commercial studfarms.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurements of glucose metabolism in single equine embryos during early development.

The contributions of 2 biochemical pathways to the total metabolism of glucose (the Embden-Meyerhof pathway [EMP] and the pentose phosphate pathway [PPP]), were assessed for equine embryos recovered on Day 4.5, 7.5 and 11.5 post ovulation. At all developmental stages studied, glucose was metabolized through both pathways. Through the EMP, the amounts of glucose metabolized per nl embryo volume per hour were 4.0, 9.9 and 3.1 pmol, whereas via the PPP, amounts were 0.9, 1.7 and 0.07 pmol for Day-4.5, -7.5 and -11.5 embryos, respectively. The ratio of EMP:PPP with age was 9.7 for Day -4.5 embryos, 7.6 for Day-7.5 embryos and 90.2 for Day -11.5 embryos. Two of the 6 unfertilized ova (UFO) exhibited metabolism through both pathways.

Prostaglandin production by ovine embryos and endometrium in vitro.

For Day-14 sheep embryos, mean (+/- s.e.m., n = 7) PGE release was 20.2 +/- 5.0, 13.0 +/- 1.9 and 6.7 +/- 1.9 ng . mg tissue-1 . h-1 after 0.5, 4.5 and 8.5 h of culture respectively, while mean PGF release rates (+/- s.e.m.) for the same periods were 98.5 +/- 24.4, 49.3 +/- 16.3 and 16.5 +/- 5.4 ng . mg tissue-1 . h-1, respectively. Total quantities of PGE and PGF released after 8.5 h of culture were 707 ng and 1833 ng per embryo, respectively. Embryos (n = 21) collected between Days 13 and 15 of gestation contained 25.4 +/- 6.9 ng PGE and 66.2 +/- 23.2 ng PGF/embryo. A total PG release 28 times the embryonic content of PG suggested active PG production by the embryos. Co-culture of embryos with endometrium resulted in PG release rates which were not significantly higher than those from embryos cultured alone. Release of PGE and PGF from embryos declined with time, that for PGF being significantly (P less than 0.05) reduced after 8.5 h incubation. Co-culture of embryos and endometrium had no effect of rate of decline of PG production over time. Day 14 endometrium from pregnant or non-pregnant ewes produced very small quantities of both PGs by comparison with embryos, and there was no consistent trend for PG production rate over time.

Some endocrine changes associated with the post-partum period of the suckling beef cow.

Seven Hereford cows with single calves were bled by jugular venepuncture, daily from parturition until 63-79 days post partum; 6 of the cows were also bled through jugular cannulae every 15 min for 8 h every 10 days. The average post-partum interval to first oestrus was 59.8 +/- 3.7 days for 5 of the cows. In cows returning to oestrus, plasma concentrations of progesterone were low until 55.5 +/- 3.0 days post partum, rose to exceed 0.5 ng/ml plasma for 4.0 +/- 0.4 days, declined for 5.0 +/- 0.5 days and then rose again to normal luteal-phase levels. First oestrus preceded the initial rise in progesterone in 1 cow and followed it is 4. Ovarian palpation revealed considerable follicular development before the initial rise in progesterone, but no clearly discernable corpus luteum until normal luteal-phase progesterone levels were detected. Plasma concentrations of oestradiol-17 beta fell after parturition and, although very variable, showed no apparent trend thereafter. Plasma concentrations of LH varied in an episodic manner, with an apparent increase in frequency and magnitude of peaks up to 10-33 days before the first elevation in plasma progesterone. Subsequently there was little change, except for a decline in peak LH concentrations after the initial elevation in plasma progesterone.

Changes in maternal hormone concentrations associated with induction of fetal death at day 45 of gestation in mares.

Pregnant Standardbred mares were allocated to 2 groups. On Day 45 of gestation, 20-45 ml saline (240 g NaCl/l) were injected into the fetal sacs of 10 mares, and the other 10 mares were given sham treatment. Post-operative plasma oestrone sulphate concentrations were lower (P less than 0.01) on Days 48-55 in saline-treated mares than in sham-treated mares. Mean plasma progesterone profiles were similar in the two groups of mares, although post-operative luteolysis occurred in 4 saline-treated mares. There was no difference in plasma CG profiles between the 2 groups, except that CG concentrations in saline-treated mares were generally lower than those of sham-treated mares. There was generally a post-operative loss of uterine and cervical tone in saline-treated mares. These results show that the maintenance of maternal plasma oestrone sulphate concentrations requires the presence of a viable embryo and confirm that luteolysis can occur despite high plasma CG concentrations after fetal death.

Infusion of gonadotrophin-releasing hormone (GnRH) induces ovulation and fertile oestrus in mares during seasonal anoestrus.

In Exp. 1, 30 Standardbred mares in deep seasonal anoestrus were divided into 3 equal groups and treated with 0, 50 (G50) or 100 (G100) ng GnRH kg-1h-1 for 28 days via osmotic minipumps. Ovulation occurred in 0/10, 3/10 and 7/10 mares respectively (P less than 0.05). Plasma GnRH profiles (Days -6, 0, 2, 6, 12, 20, 28 and 34 relative to pump insertion) were dose-dependent (P less than 0.01) and peaked on Day 12 of infusion. Mean daily plasma LH concentrations were biphasic in treated mares that ovulated, with LH peaks occurring around Day 6 and Days 16-20. By contrast, in treated mares that did not ovulate the initial LH rise was followed by a steady decline to the end of the experiment. LH pulse frequency in treated mares increased between Day 0 and Day 21 of the experiment. LH pulse frequency in G100 mares was higher (P less than 0.05) than in G50 and control mares on Day 3, and higher than the controls on Days 7 and 21 of the experiment. There were no significant differences in LH pulse amplitude between the groups on the days studied. In Exp. 2, 27 Standardbred mares in shallow seasonal anoestrus received no treatment (N = 13) or a subcutaneous infusion of GnRH (100 ng kg-1h-1) via osmotic minipump for 28 days (N = 14). Mares were served by a stallion during oestrus. Day of ovulation was earlier in treated than in control mares (18.6 +/- 2 vs 41.9 +/- 6 days; P less than 0.001). Likewise, time of conception was earlier in treated than in control mares (25.2 +/- 6 vs 49.1 +/- 9 days; P less than 0.05). One mare in the control group failed to conceive while one treated mare conceived to an undetected ovulation. The results show that constant GnRH infusion induces ovulation and fertile oestrus in mares during deep and shallow seasonal anoestrus.