Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro fertilization of gonadotropin-stimulated leopard cat (Felis bengalensis) follicular oocytes

Based on techniques developed for the domestic cat, in vitro fertilization (IVF) studies were conducted in the taxonomically related leopard cat (Felis bengalensis). Adult females received pregnant mares' serum gonadotropin (PMSG) followed 80 or 84 h later by human chorionic gonadotropin (hCG) on two to four occasions over a 40-day to 27-month interval. Oocytes were collected laparoscopically from ovarian follicles 25-27 h after hCG and co-cultured with processed, homologous spermatozoa (37 degrees C, 5% CO2 in air, humidified atmosphere) for 30-36 h. There was no apparent ovarian refractoriness to repeated treatments with exogenous gonadotropins. Overall, the mean number of mature follicles present and the total number of oocytes and proportion of immature oocytes collected did not differ (P greater than 0.05) between the 80 h (4.9 +/- 0.9; 4.7 +/- 1.2; 14.9%, respectively) and 84 h (5.6 +/- 1.4; 5.4 +/- 1.7; 22.2%, respectively) gonadotropin interval groups. However, the proportion of mature leopard cat oocytes fertilized in vitro, as determined by embryonic cleavage, was increased (P less than 0.005) by extending the interval between PMSG and hCG from 80 (17.5%) to 84 (52.4%) h. These data 1) demonstrate that, compared to the domestic cat, the ovaries of the leopard cat are less responsive to a given PMSG/hCG treatment; 2) indicate that leopard cat follicular oocytes can be recovered readily by laparoscopy and are capable of becoming fertilized in vitro; and 3) suggest that IVF may be a viable approach for producing embryos and perhaps enhancing captive propagation of rare Felidae.     

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.     

Endocrine, luteal and follicular responses after the use of the short-term protocol to synchronize ovulation in goats

The effect of the so-called Short-Term Protocol (5-day progesterone treatment+PGF(2)alpha) on ovarian activity and LH surge was studied in goats. The goats received 250IU eCG at the time of device withdrawal (eCG group; n=7), or 200microg of EB (estradiol benzoate) 24h after device withdrawal (EB group; n=8), or received neither eCG nor EB (control group; n=8). The Short-Term Protocol induced greater (4.1+/-1.1ng/ml) progesterone serum concentrations at 24h after start of the treatment, that declined to 0.2+/-0.1ng/ml at 12h after device withdrawal. In all of the groups, the maximum concentration of estradiol-17beta was reached at about 36h after device withdrawal. Maximum concentration was greater in the EB group (76.9+/-24.6pmol/l) than in the control group (41.8+/-9.0pmol/l; P<0.01), with the eCG group showing intermediate concentration (70.3+/-32.5pmol/l; P=NS). The LH peak occurred earlier in the eCG group (38.4+/-2.0h after device withdrawal) and in the EB group (41.0+/-4.1h), than in the control group (46.3+/-5.1h; P<0.05). Ovulation occurred earlier in the eCG group (5/7) and in the EB group (8/8) (58.8+/-2.7h and 63.0+/-5.6h, respectively), than in the control group (7/8) (70.2+/-8.3h; P<0.05). In summary, the Short-Term Protocol induced similar concentrations of progesterone among treated goats. In addition, eCG or EB resulted in a similar increase in estradiol-17beta and a similar LH surge, which induced ovulation in most females (86.7%) in a consistent interval (about 60h) after the end of progesterone exposure.     

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris)

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.     

Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-17 beta, progesterone, luteinizing hormone, and follicle-stimulating hormone associated with follicular development in cows induced to superovulate with equine chorionic gonadotropin.

Changes in concentrations of bioactive and immunoreactive (ir-) inhibin, estradiol-17 beta, progesterone, LH, and FSH in peripheral blood were determined in cows induced to superovulate with eCG. The pattern of follicular growth was also characterized by daily ultrasonographic examination. Hormonal profiles and follicular development during the intact estrous cycle of the same animals before eCG treatment served as controls. Equine CG increased the number of follicles of various sizes (small, greater than or equal to 4 less than 7, medium, greater than or equal to 7 less than 10; large, greater than or equal to 10 mm in diameter) by 4 days after administration. The second growth of large follicles occurred within 1 day after superovulation. Inhibin bioactivity in jugular vein blood was detectable 48 h after eCG injection (44 h before LH peak), whereas it was not detected before administration of eCG or during control cycles. Circulating levels of bioactive inhibin further increased during the two waves of growth of large follicles. The highest activity of inhibin was noted at the time of the preovulatory LH peak (0 h). Thereafter, bioactivity of inhibin in peripheral plasma dropped from 0 to 24 h after the LH peak, and the activity increased again at 72 h compared to the value at -44 h. Plasma levels of ir-inhibin showed a pattern similar to changes in bioactive inhibin in the eCG-treated cows. Plasma concentrations of estradiol-17 beta also increased concomitantly with two waves of growth of large follicles. There was no correlation between plasma levels of progesterone and inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental competence of domestic cat follicular oocytes after fertilization in vitro

Empirical evaluation of variables affecting oocyte collection, in vitro fertilization, and embryo transfer resulted in establishing a successful procedure for the artificial production of offspring in the domestic cat. Female cats were treated with pregnant mare's serum gonadotropin (PMSG, 150 IU) followed 72 or 80 h later with 100 or 200 IU human chorionic gonadotropin (hCG). After laparoscopic collection, follicular oocytes were inseminated in vitro with ejaculated, processed spermatozoa, cultured (37 degrees C, 5% CO2), and then examined for evidence of fertilization. Two- to 4-cell stage embryos were transferred to the oviducts of oocyte donors. Oocyte donor cats and naturally mated controls also were subjected to sequential laparoscopic examinations and blood sampling to assess corpora lutea (CL) function. At 24-30 h of culture, fewer (p less than 0.001) degenerate oocytes were observed in cats receiving 100 IU hCG (8.2%) compared to those receiving 200 IU (20.6%), regardless of the PMSG-hCG interval. Overall fertilization (48.1%) and cleavage (45.2%, at 30 h post-insemination) rates were greatest following an 80-h PMSG-hCG interval combined with the 100 IU hCG dose. Five of the 6 cats receiving 6 to 18 embryos became pregnant and produced from 1 to 4 kittens/litter. Gonadotropin-treated females subjected to follicular aspiration produced morphologically normal CL and circulating progesterone patterns that were qualitatively similar (p greater than 0.05) to control cats. These data indicate that domestic cat follicular oocytes are capable of fertilization in vitro, but success is dependent on both the timing and dose of the hCG stimulus. Follicles subjected to aspiration appear capable of forming normal, functional CL and the birth of live young after embryo transfer unequivocally demonstrates, for the first time, the developmental competence of in vitro-fertilized carnivore oocytes.     

PGE2 induces its own secretion in vitro by bovine 270-day placenta but not by 200-day placenta.

Two separate experiments were conducted to determine whether prostaglandin (PG) E2 stimulates the secretion of progesterone by 270- or 200-day Brahman placentas in vitro. Secretion of progesterone, PGF2alpha, pregnancy specific protein B, or estradiol-17beta by 270-day Brahman placentas was not affected (p > or = 0.05) by PGE2, during the 4-h incubation period at the doses tested. Indomethacin or meclofenamic acid decreased (p < or = 0.05) 270-day Brahman placental secretion of PGE and PGF2alpha by 98 and 60%, respectively. However, PGE2 induced (p < or = 0.05) its own secretion, but not the secretion of PGF2alpha (p > or = 0.05), by 270-day Brahman placentas, even in the presence of indomethacin or meclofenamic acid at a dose of 100 ng/mL. Also, secretion of 8-Epi-PGE2 by Day 270 Brahman placentas was increased (p < or = 0.05) by PGE2. Secretion of progesterone, estradiol-17beta, or pregnancy specific protein B by 200-day Brahman placentas was not affected by PGE2, 8-Epi-PGE2, PGF2alpha, estradiol-17beta, or trichosanthin during the 4- or 8-h incubation period (p > or = 0.05). Secretion of estradiol-17beta at 8 h was lower (p < or = 0.05) in all treatment groups and did not differ (p > or = 0.05) among the 8-h incubation treatment groups. Secretion of PGE by 200-day Brahman placentas was reduced (p < 0.05) by indomethacin 72 and 82% and by meclofenamic acid 72 and 96%, respectively, at 4 and 8 h when compared to controls. Secretion of PGF2alpha was reduced (p < or = 0.05) 71 and 86% by indomethacin or 89 and 89% by meclofenamic acid at 4 and 8 h, respectively, and did not differ (p > or = 0.05) between 4 and 8 h of incubation. PGE2 did not (p > or = 0.05) induce secretion of PGE above what was added in any treatment group. PGE in culture media was increased (p < or = 0.05) by 8-Epi-PGE2, pregnancy specific protein B, and the 100 ng/mL PGF2alpha dose (p < or = 0.05), but not by PGE2, progesterone, estradiol-17beta, 8-Epi-PGF2alpha, or trichosanthin. Secretion of PGF2alpha by 200-day Brahman placentas was not affected (p > or = 0.05) by 8-Epi-PGE2, progesterone, or estradiol-17beta, but PGF2alpha secretion was increased (p < or = 0.05) by trichosanthin or PGE2, even in the presence of indomethacin or meclofenamic acid. It is concluded that PGE does not affect secretion of progesterone by 200- or 270-day bovine placentas, but, pregnancy specific protein B may regulate placental secretion of PGE. Also, indomethacin and meclofenamic may affect enzymes converting PGH to PGE rather than acting only on cyclooxygenase because indomethacin and meclofenamic acid lowered PGE secretion by 270-day Brahman placentas more than they lowered PGF2alpha. In addition, it is concluded that PGE2 can induce bovine placental secretion of PGE, but this is dependent upon the stage of gestation.     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of PGF2alpha, indomethacin, tamoxifen, or estradiol-17beta on incidence of abortion, progesterone, and pregnancy-specific protein B (PSPB) secretion in 88- to 90-day pregnant sheep

One objective of this experiment was to evaluate our hypotheses that estradiol-17beta regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2alpha (PGF2alpha), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-beta; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P < or = 0.05) by PGF2alpha, indomethacin, PGF2alpha + tamoxifen, PGF2alpha + indomethacin, and PGF2alpha + estradiol-17beta but not (P > or = 0.05) by tamoxifen or estradiol-17beta alone. No ewe treated with PGF2alpha alone aborted (P > or = 0.05). Forty percent of ewes treated with PGF2alpha + estradiol-17beta aborted (P < or = 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) among control, indomethacin-, tamoxifen-, and PGF2alpha + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P < or = 0.05) by 24 h, followed by a quadratic increase (P < or = 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2alpha-, PGF2alpha- + tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + estradiol-17beta-, and tamoxifen-treated ewes was reduced (P < or = 0.05) by 18 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P < or = 0.05) at 72 h in PGF2alpha-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + tamoxifen-, and PGF2alpha + estradiol-17beta-treated ewes. Weights of placentomes did not differ among treatment groups (P > or = 0.05). Profiles of PSPB in inferior vena cava blood differed (P < or = 0.05) among control, estradiol-17beta-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, and PGF2alpha + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P < or = 0.05) in indomethacin-, estradiol-17beta-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated ewes were greater (P < or = 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P > or = 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88-90 days of gestation in sheep and that there is no luteal source of PSPB.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

Evidence for estrogen-dependent blastocyst formation in the pig

The physiological significance of estradiol-17beta for the early embryonic development in the pig was investigated in vitro by four different experimental designs. A total of 1635 morphologically intact morulae were cultured in vitro in Krebs-Ringer bicarbonate solution supplemented with 10% heat-inactivated lamb serum, and the blastocyst formation rate (BFR) was recorded after 24 or 48 h. The addition of estradiol-17 beta (0.1 nM, 1 nM, 100 nM), progesterone (100 nM, 500 nM) or cortisol (100 nM) to the culture medium did not affect BFR (95 to 100%, Experiment 1). Similarly, adding charcoal-stripped lamb serum to the medium instead of normal lamb serum in the absence or presence of 1 nM estradiol-17 beta had no effect (93 to 95% BFR, Experiment 2). The antiestrogen Nafoxidine, however, at a concentration of 15 micrograms/ml, significantly (p less than 0.01) reduced BFR to 13.3 +/- 5.8% compared to controls (93.3 +/- 4.2%, Experiment 3). Supplementation with estradiol-17 beta (1 nM) in the presence of 15 micrograms/ml Nafoxidine significantly (p less than 0.01) improved BFR to 57.2 +/- 8.9%. Higher concentrations of estradiol-17 beta (100 nM, 100 microM) did not further enhance BFR. The stimulatory effects of estradiol-17 beta were specific since the BFR remained low in the presence of 100 nM progesterone (10.0 +/- 4.5%) or 100 nM cortisol (3.3 +/- 3.3%). Addition of 5% estradiol-17 beta-antiserum to the culture medium (Experiment 4) significantly (p less than 0.01) reduced BRF to 51.9 +/- 6.7% compared to controls (93.1 +/- 2.2%).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.