Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Chemical and biological properties of the subunits of pregnant mare serum gonadotropin

The two subunits (α and β) of pregnant mare serum gonadotropin have been dissociated and partially characterized. Recombination of the biologically inactive subunits results in the restoration of both the follicle stimulating and leuteinizing activities of pregnant mare serum gonadotropin. In addition, the α subunit of pregnant mare serum gonadotropin can be combined with the β subunit of either ovine luteinizing hormone, human chorionic gonadotropin, or follicle stimulating hormone with generation of the specific activity expected of the β subunit.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits.

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Effect of long-term and short-term progestagen treatment on follicular development and pregnancy rate in cyclic ewes

The aim of this study was to evaluate the effect of the length of a progestagen treatment (12 d vs. 6 d) on follicular dynamics, estrus synchronization and pregnancy rate using medroxyprogesterone acetate (MAP) with or without an eCG dose at the end of MAP treatment. One hundred sixty Polwarth ewes were divided into four equal groups: long-term treated (LT, n=40); short-term treated (ST, n=40); long-term treated plus eCG (LTeCG, n=40); and short-term treated plus eCG (STeCG, n=40). Five ewes of each group were separated to undergo daily transrectal ultrasonography, and blood samples were taken for hormone determination. Until 96 h after sponge withdrawal the number of ewes in estrus was higher in both long-term-treated groups than in both short-term-treated groups but at the end of the observational period (144 h) no significant differences were found among groups. The pregnancy rate was higher in the ST group (87%) than in the other groups (LT, 63%; LTeCG, 67%; and STeCG, 58%; P< or =0.03). The ovulatory follicle emerged before sponge withdrawal in long-term-treated ewes (-3.8+/-0.4 d and -2.2+/-0.8 d for LT and LTeCG, respectively), whereas in short-term-treated ewes it emerges around sponge removal (0.4+/-1.1 d and 0.5+/-0.5 d for ST and STeCG, respectively; P< or =0.01). The ovulatory follicle in the LT group had a longer lifespan and attained a larger (P< or =0.05) maximum diameter than in the ST group. We conclude: a) that the lower pregnancy rate observed after long-term progestagen treatment was related to a slower follicular turnover that promoted the ovulation of persistent dominant follicles; (b) that short-term treatment resulted in a higher pregnancy rate probably due to the ovulation of newly recruited growing follicles; and (c) treatment with eCG had no advantage in association with long-term treatment and had a deleterious effect in combination with short-term treatment with MAP.     

Effects of hydroxyflutamide on rats treated with a superovulatory dose of pregnant mare serum gonadotropin

Immature female rats treated with superovulatory doses of pregnant mare serum gonadotropin (PMSG) were used to study the effects of the antiandrogen hydroxyflutamide on steroid production, particularly the biologically active androgens, in two experiments. In the first experiment, animals were given either 5 mg hydroxyflutamide or vehicle alone at 30 and 36 h following 40 IU PMSG. Compared with the vehicle group, hydroxyflutamide treatment significantly reduced the percentage of degenerate oocytes recovered from oviducts (p less than 0.05). Serum levels of testosterone and androstenedione, and their aromatized product 17 beta-estradiol, significantly decreased (p less than 0.05) in the hydroxyflutamide-treated group; however, nonaromatizable androgen, 5 alpha-dihydrotestosterone, was not affected. In the second experiment, ovaries obtained 48 h after stimulation with 4 or 40 IU PMSG were incubated with and without hydroxyflutamide (10(-5) M) and (or) testosterone (10(-7) M) to study [4-14C]pregnenolone metabolism to major steroids. In 40 IU stimulated ovaries, hydroxyflutamide significantly decreased the metabolism of pregnenolone to progesterone (p less than 0.01) and androstenedione (p less than 0.01), while the production of 17 beta-estradiol increased significantly (p less than 0.05); however, pregnenolone conversions to testosterone and 5 alpha-dihydrotestosterone were not affected. Testosterone completely reversed the hydroxyflutamide-induced alteration of pregnenolone metabolism. In contrast, there was no difference in the pregnenolone conversion patterns between untreated and hydroxyflutamide or hydroxyflutamide plus testosterone groups in 4 IU stimulated ovaries. Present results confirm our previous finding that hydroxyflutamide decreases the percentage of abnormal oocytes recovered from superovulating rats and indicates that this hydroxyflutamide effect may be partly mediated by altered ovarian steroidogenesis following inhibition of androgen binding in the ovary.     

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.     

Effect of exogenous melatonin and plane of nutrition after weaning on estrous activity, endocrine status and ovulation rate in Salz ewes lambing in the seasonal anestrus

Forty-nine Spanish Salz ewes lambing in the second fortnight of March (20 March +/- 1.5 d) were used to determine the effects of exogenous melatonin and postweaning nutrition on endocrine status, date of first estrus and ovulation rate. Experimental design was a factorial defined by 2 postweaning planes of nutrition, 1.80 (high) and 1.35 (low) times the maintenance requirements, and treatment with a single 18-mg subcutaneous implant of melatonin (M) 32 d after lambing or no treatment control (C). Mean weaning to first estrus interval was shorter in treated than in control ewes (50.8 +/- 4.2 vs 87.6 +/- 6.3 d; P < 0.01). Considering both the treated and control animals together, the ratio between mean night and daytime plasma melatonin levels was significantly correlated with the implant insertion-first estrus interval on Day 5 (0.67; P < 0.01) and Day 35 (0.63; P < 0.05) after implantation. Melatonin implants induced a significant increase of mean LH concentrations at Days 14 and 33 after implantation (P < 0.01) without any significant influence of plane of nutrition. Ovulation rate was higher for treated than control ewes in the second estrus (P < 0.05). An interaction between plane of nutrition and exogenous melatonin on ovulation rate at the second cycle after weaning was detected (P < 0.01), being close to the significance in the first, fourth and fifth cycles (P < 0.1). These results suggest that exogenous melatonin in April may be an effective way of advancing the breeding season and enhancing ovulation rate associated with a low rather than a high plane of nutrition.     

Ovulation of aged follicles does not affect embryo quality or fertility after a 14-day progestagen estrus synchronization protocol in ewes

The aim was to examine the effect of ovulation of aged follicles on embryo quality and fertility in ewes. In Experiment 1, ewes (n = 39) received a prostaglandin analogue on Day 6 of the cycle and then received either a progestagen sponge from Day 6 to 20 after estrus (Single sponge) or a progestagen sponge on Day 6 that was replaced on Day 11 and 16 and removed on Day 20 (Multiple sponges). In a subgroup of ewes, the growth of ovarian follicles was characterised using ultrasonography. Fertile rams were introduced 48 hours after sponge withdrawal; we slaughtered the ewes on Day 5 of pregnancy and recovered the embryos. The mean age of the ovulatory follicles was greater in ewes that received a single sponge compared with multiple sponges (8.7+/-0.8 days, range 4 to 14, versus 4.5+/-0.7 days, range 3 to 6; P<0.05). However, the groups did not differ (P>0.05) in ovulation rate (2.4+/-0.3 corporal lutea per ewe) or the proportion of good quality embryos recovered (71 to 82%; developed to the early morula stage or further). In Experiment 2, ewes (570 in total) received treatments similar to those in Experiment 1 but were kept until lambing. Ewes that received a single sponge came into heat earlier (P<0.05) than those that received multiple sponges, but > or = 97% of ewes in all groups (P>0.05) were bred by 48 to 72 hours after ram introduction. There was no difference (P>0.05) between groups for the proportion of ewes that lambed to first service (80 to 86%) or the number of lambs per ewe (1.94+/-0.08 lambs). We conclude that when luteolysis occurs at the beginning of progestagen synchronisation, ewes will ovulate aged follicles, but that compared to shorter duration follicles, these follicles produce oocytes that are equally competent to be fertilised and develop into good quality embryos and full-term lambs.     

Lack of effect of transrectal ultrasonography with restraint on lambing rate and prolificacy in ewes

The objective was to determine if transrectal ultrasonography for determination of pregnancy in restrained ewes increases embryonic/fetal death or loss of pregnancy. Ten flocks (N=873 ewes) bred in either the estrous or anestrous season were randomized, into control (C) or examined (E) groups within flock. Examined ewes were placed in a tilting squeeze chute and scanned by one of three operators for pregnancy by transrectal ultrasonography once between Days 25 and 100 post-breeding. Control ewes were not subjected to handling in the squeeze chute or pregnancy diagnosis. Two operators counted embryos in E ewes in six flocks. There were no differences between E and C ewes in percent ewes lambing or lambing rate on a per flock basis. Prolificacy per flock was greater in E ewes (P=0.05; 1.53 versus 1.60, C and E, respectively) than in C ewes. Eighty-five percent of exposed ewes lambed in the estrous season, while only 62% of ewes lambed in the anestrous season (P=0.05). Overall prolificacy did not differ with season (1.60 versus 1.53), and there was no season by treatment interaction for any variable tested. Losses averaged 0.02 per E ewe exposed in the estrous season and 0.20 in the anestrous season (P<0.05). Examined ewes in this study had similar pregnancy and lambing rates to C ewes in the same flocks and prolificacy was slightly greater. Based on these data, the combination of transrectal ultrasonography with restraint is safe for pregnancy diagnosis in ewes.     

Effect of type of intravaginal progestagen treatment of estrous response and reproductive performance of ewes

A comparison was made of the relative effectiveness of sponge pessaries impregnated with 40mg flourogestone acetate (FGA) or 60mg medroxyprogesterone acetate (MAP) to induce a synchronized estrus in ewes. Ewes were treated with sponge pessaries for 14 days and 500 IU pregnant mares' serum gonadotropin was injected i.m. at the time of sponge removal. The degree and pattern of mating response of ewes were similar, irrespective of the treatment used, approximately 92% of the ewes being marked by the ram by 72h after sponge removal. No significant differences in fertility or litter size were observed between the treatment groups. Ewes treated with FGA sponges had a fertility of 53% and litter size of 2.3 after mating at the synchronized estrus. The corresponding values for ewes treated with MAP sponges were 57% and 2.1. Use of MAP sponges was associated with a 17.8% sponge loss during treatment compared with 1% sponge loss in ewes treated with FGA sponges. Such losses could compromise the use of MAP sponges by reducing their overall efficacy.     

Reproductive responses of beef heifers and cows to exogenous progesterone administered in silastic coils,oestradiol benzoate and pregnant mare serum gonadotropin

The aim of this study was to investigate a Progesterone Releasing Intravaginal Device, Oestradiol benzoate and PMSG combined treatment in 81 suckling charolais cows and 56 heifers of various breeds. The former were young primiparous cows aged 2 years and treated at a means of 43 days postpartum. Most of the heifers ($\text{44}\text{56}$) were not cycling and were believed to be prepuberal.In non-cycling females, the treatment induced ovulation in 98.5% of the cows and 78 to 100% of the heifers according to breed. In those females in which ovulation was induced, pregnancy rates determined by rectal palpation were 46.9% for the cows and 66.7% for the heifers. When taking into account all treated females including those with lost Progesterone Releasing Intravaginal Devices (5.8%) and those not ovulating, the pregnancy rates determined by rectal palpation 3 to 5 months after artificial insemination were 40.7% for the cows and 52% for the heifers.     

Synchronous generation of ovarian hCG binding sites and LH-sensitive adenylate cyclase in immature rats following treatment with pregnant mare serum gonadotropin.

A concomitant increase in the activity of LH-senstive adenylate cyclase and in the number of LH/hCG binding sites was induced in ovaries of immature rats upon administration of pregnant mare serum gonadotropin (PMSG), a hormone preparation known to have predominantly follicle stimulation (FSH-like) activity. When an optimal dose of PMSG (15 i.u./rat) was administered to 25-day-old rats, specific activity of LH-dependent adenylate cyclase and the number of binding sites for LH/hCG per mg protein remained unchanged during the first 24h, but 48h after injection a 2-to 4-fold increase in both parameters was observed. By contrast, there was no change in basal adenylate cyclase activity or in the response of the enzyme to the stimulatory action of guanosine-5'-(beta gamma-imino) triphosphate (Gpp (NH)p), GTP, or NaF. Specific activity of succinate cytochrome c reductase, glucose-6-phosphatase and 5'-nucleotidase were found to be unaffected by the hormonal pretreatment, although total protein determined in these homogenates increased 3-fold in the course of this treatment. It is inferred that during follicular maturation, FSH enhances the responsiveness of ovarian adenylate cyclase to LH by stimulating the insertion of LH/hCG-receptors into the cell membrane.