Stimulatory and inhibitory effects of progesterone on follicular development in the hypophysectomized follicle-stimulating hormone/luteinizing hormone-treated hamster

Cyclic hamsters hypophysectomized at estrus (Day 1 of the cycle) and injected with 5 micrograms follicle-stimulating hormone (FSH) on Day 1 and 20 micrograms luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) from Days 1-4 ovulated 15.3 ova, in response to 30 IU human chorionic gonadotropin (hCG) administered at 1500 h on Day 4 (Kim and Greenwald, 1984). When 1 mg progesterone (P4) was administered daily from Days 1-4 concurrent with the above regimen, ovulation increased to 38 ova, a clearcut superovulatory response. However, daily injection of 1, 10, or 100 micrograms P4 plus FSH and LH reduced the number of antral follicles present on the afternoon of Day 4 to 3-4 per ovary, compared to 9 per ovary after FSH-LH alone, and the ovulation rate was drastically reduced with most animals being anovulatory. Substituting 1 mg 17 alpha-hydroxyprogesterone or estradiol cyclopentylpropionate for P4 on Days 1-4 did not alter the number of antral follicles on Day 4 from FSH-LH alone, whereas 1 mg androstenedione or 1 mg testosterone cyclopentylpropionate reduced the number of antral follicles to 3 or less. Hence, the stimulatory effects of 1 mg P4 are not attributable to its conversion to other P4 derivatives. After the concurrent injection of 1 mg P4 and FSH-LH, on the afternoon of Day 3, an average of only 1.8 large preantral follicles was present per ovary. By the morning of Day 4, however, the ovary contained 14 large preantral and early antral follicles in addition to 8 large antral follicles. Injection of hCG at this time resulted in the ovulation of 14.5 ova.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Secretory patterns of LH and FSH and follicular growth following administration of PGF(2)alpha during the early luteal phase in cattle

Two studies were conducted to determine the changes in gonadotropin secretion associated with growth and development of the largest follicle and the ability of the largest ovarian follicle present on Day 5 following estrus to ovulate if luteal regression is induced. In both studies, cows received either saline (i.m.) or prostaglandin F(2)alpha (PGF(2)alpha; 25 mg i.m.) on the fifth day post estrus. Frequency of LH pulses declined (P<0.01) with increasing day of cycle, while pulse amplitude and duration increased (P<0.05) in saline-treated cows. In PGF(2)alpha-treated cows, LH remained as high frequency-low amplitude pulses. Secretory patterns of FSH were similar between the two groups. In Experiment 2, the largest ovarian follicle present was marked around its periphery with sub-epithelial injections of charcoal. In saline-treated cows, the size of the charcoal marked follicles generally decreased, indicating atresia. A corpus luteum was present within the area of a previously marked follicle in three PGF(2)alpha-treated cows. The size of the marked follicles either decreased or increased in the remaining PGF(2)alpha-treated cows, with ovulation occurring at a different site. In summary, PGF(2)alpha-induced luteal regression on the fifth day of estrus subsequently alters the frequency, amplitude and duration of LH pulses, but not FSH pulses, and the largest follicle present on Day 5 either increases or decreases in size or ovulates when PGF(2)alpha is given on Day 5 following estrus.     

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.     

Pituitary and ovarian hormone secretion and ovulation in gilts injected with gonadotropins during and after oral administration of progesterone agonist (Altrenogest)

We determined changes in plasma hormone concentrations in gilts after treatment with a progesterone agonist, Altrenogest (AT), and determined the effect of exogenous gonadotropins on ovulation and plasma hormone concentrations during AT treatment. Twenty-nine cyclic gilts were fed 20 mg of AT/(day X gilt) once daily for 15 days starting on Days 10 to 14 of their estrous cycle. The 16th day after starting AT was designated Day 1. In Experiment 1, the preovulatory luteinizing hormone (LH) surge occurred 5.6 days after cessation of AT feeding. Plasma follicle-stimulating hormone (FSH) increased simultaneously with the LH surge and then increased further to a maximum 2 to 3 days later. In Experiment 2, each of 23 gilts was assigned to one of the following treatment groups: 1) no additional AT or injections, n = 4; 2) no additional AT, 1200 IU of pregnant mare's serum gonadotropin (PMSG) on Day 1, n = 4); 3) AT continued through Day 10 and PMSG on Day 1, n = 5, 4) AT continued through Day 10, PMSG on Day 1, and 500 IU of human chorionic gonadotropin (hCG) on Day 5, n = 5; or 5) AT continued through Day 10 and no injections, n = 5. Gilts were bled once daily on Days 1-3 and 9-11, bled twice daily on Days 4-8, and killed on Day 11 to recover ovaries. Termination of AT feeding or injection of PMSG increased plasma estrogen and decreased plasma FSH between Day 1 and Day 4; plasma estrogen profiles did not differ significantly among groups after injection of PMSG (Groups 2-4). Feeding AT blocked estrus, the LH surge, and ovulation after injection of PMSG (Group 3); hCG on Day 5 following PMSG on Day 1 caused ovulation (Group 4). Although AT did not block the action of PMSG and hCG at the ovary, AT did block the mechanisms by which estrogen triggers the preovulatory LH surge and estrus.     

Influence of synthetic lamprey GnRH-III on gonadotropin release and steroid hormone levels in gilts

Based on the supposition that lamprey GnRH-III (lGnRH-III) elicits FSH releasing activity in swine, synthetic lGnRH-III (peforelin, Maprelin® XP10) was used in puberal estrus synchronized gilts. The secretion of reproductive hormones FSH, LH, estradiol and progesterone was analyzed, and follicle growth and ovulation recorded. Altogether, 24 German Landrace gilts were treated after an 18-day long synchronization of the estrus cycle with Regumate® as follows: 48 h after the last Regumate® feeding they received im either 150 μg Maprelin® XP10 (lGnRH-III, group Maprelin, n = 6), 50 μg Gonavet Veyx® (GnRH-I agonist, group GnRH, n = 6), 850 IE Pregmagon® (eCG, group eCG, n = 6) or saline (group Control, n = 6). Additionally, in eight gilts the concentrations of FSH and LH were analyzed after treatment with 150 μg Maprelin® XP10 (n = 3), 50 μg Gonavet Veyx® (n = 3) or saline (n = 2) at mid-cycle (day 10 of the estrus cycle). Blood samples were collected via implanted jugular vein catheters. Ovarian features were judged endoscopically at the end of the Regumate® feeding and on days 5 and 6 after treatment. Maprelin® XP10 had no effect on FSH release in gilts; neither at the pre-ovulatory period or at mid-cycle. Furthermore, LH levels were unaffected. In contrast, GnRH-I agonist stimulates FSH release, however less compared to LH secretion. LH secretion was induced by GnRH-I both during the follicular phase and at mid-cycle. Equine CG did not stimulate the release of pituitary hormones FSH and LH due to its direct action on the ovary. Increased estradiol concentrations during days 2 to 5 after Regumate® in all treatment groups indicated pre-ovulatory follicle growth in gilts. Equine CG stimulated a higher (P < 0.01) number of ovulatory follicles compared to the other treatment groups. All together, 83 to 100 % of gilts ovulated by day 6 post treatment. In summary, results of our study on reproductive hormone secretion do not provide evidence that synthetic lGnRH-III (Maprelin® XP10) selectively releases FSH in estrus synchronized gilts.     

Modification of follicular dynamics by exogenous FSH and progesterone, and the induction of ovulation using hCG in postpartum beef cows

Follicular growth and ovulation in response to FSH, progesterone and hCG were evaluated in postpartum beef cows. In Experiment 1, on Day 21 post partum, cows received an injection of either saline (control; n = 6), FSH (200 mg; n = 6), or a PRID (n = 5) for 10 d. Both FSH and PRID prolonged maintenance of a dominant follicle (15.5 +/- 1.16 and 14.4 +/- 1.29 d, respectively, vs 8.4 +/- 1.22 d in control; P < 0.01), and increased the maximum diameter of the dominant follicle (14.0 +/- 0.91 and 16.4 +/- 1.01 mm, respectively, vs 10.9 +/- 0.95 mm in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 60% of cows, followed by normal estrous cycles (vs 0% in control; P = 0.01), whereas the dominant follicle ovulated in 33% of FSH-treated cows (P = 0.08). The PRID regimen shortened the interval to first ovulation preceding a normal cycle and continued cyclicity (44 +/- 4.1 vs 60 +/- 4.4 d in control; P = 0.02). In Experiment 2, on Day 21 post partum, cows received either saline (control), saline + PRID, or FSH + PRID (n = 16/group). Sixty hours after PRID withdrawal, cows received either saline or hCG (1,500 IU, n = 8/treatment). The FSH + PRID regimen increased the number of large (> 10 mm in diameter) follicles (3.6 +/- 0.43 vs 1.9 +/- 0.39 in control; P = 0.005). Both PRID and FSH + PRID prolonged maintenance of the largest follicle (11.0 +/- 0.82 and 11.2 +/- 0.91 d, respectively, vs 8.7 +/- 0.81 d in control; P < 0.05). The PRID-maintained dominant follicle ovulated in 50% of cows, followed by normal estrous cycles. The FSH + PRID-maintained largest follicle had become atretic at PRID withdrawal and was anovulatory. The FSH + PRID + hCG regimen increased the incidence of ovulation preceding a cycle of normal duration and continued cyclicity (100 vs 50% in PRID; P = 0.03), and reduced the interval to first ovulation preceding a cycle of normal duration and continued cyclicity (38 +/- 6.5 vs 58 +/- 6.3 d in control; P = 0.04). The area under the progesterone curve during the induced cycle was reduced after (PRID +/- FSH) + hCG than after PRID +/- FSH (P = 0.002). These results indicate that PRID alone or with FSH/hCG has the potential to modify the dominant follicle and initiate cyclicity in postpartum beef cows.     

Effect of purified llama ovulation-inducing factor (OIF) on ovarian function in cattle

Two experiments were designed to determine the effect of purified ovulation inducing factor (OIF) on ovarian function in cattle. In Experiment 1, prepubertal heifers (n = 11 per group) were treated on Day 5 (Day 0 = day of follicular wave emergence) of the follicular wave with an intramuscular dose of saline (1 mL), GnRH (100 μg), or purified OIF (1 mg/100 kg body weight). Ovulation occurred in 9/11 heifers treated with GnRH, and 1/11 heifers in each of the OIF- and saline-treated groups (P < 0.05). Compared to saline-treated controls, OIF treatment was associated with a smaller dominant follicle diameter (P < 0.01), a rise in plasma FSH concentration (P < 0.1), and earlier emergence of the next follicular wave (P < 0.05). In Experiment 2, sexually mature heifers were given either GnRH or purified OIF on Days 3, 6 or 9 of the first follicular wave (i.e., early growing, early static, or late static phase of the dominant follicle; n = 5 per group per day), or were untreated (n = 10). In heifers treated with OIF on Day 6, the dominant follicle diameter profile tended to be smaller than in controls, and was associated with a rise (P < 0.05) in plasma FSH concentrations. A similar rise in FSH was detected after OIF treatment on Day 9. Compared to untreated controls, treatment with OIF and GnRH was associated with a larger CL diameter (Days 3 and 6 groups; P < 0.05) and a greater concentration of plasma progesterone (Days 6 and 9 groups; P < 0.05). Treatment with purified OIF did not induce ovulation in heifers, but hastened new follicular wave emergence in prepubertal heifers, influenced follicular dynamics in a phase-specific manner in mature heifers, and was luteotrophic.     

Ovulation and follicular growth in gonadotropin-treated gilts followed by in vitro fertilization and development of their oocytes

We investigated whether porcine ovaries derived from FSH-pituitary (FSH-P) or hCG-treated animals can produce oocytes with better in vitro cytoplasmic maturation and in vitro embryonic development relative to those derived from saline-treated animals. The size of the follicle producing the oocyte was also studied. Each of 25 prepubertal gilts received 1 of 6 treatments by intramuscular injection: 1) saline (3 mL, once, n = 5); 2) FSH-P8-3 (8 mg, 3 times, with a 24-h interval, n = 4); 3) FSH-P16-3 (16 mg, 3 times, with a 24-h interval, n = 4); 4) FSH-P16-1-P4-2 (16 mg, once, 4 mg, twice, with a 24-h interval, n = 4); 5) FSH-P16-1 (16 mg, once, n = 4); or 6) hCG (100 IU, 3 times, with a 24-h interval, n = 4). The ovaries were removed by mid-ventral laparotomy 72 h after the first injection. The numbers of corpora hemorrhagica (CH) with each FSH-P treatment were similar (P > 0.05). However, compared with gilts treated with saline or hCG, those treated with FSH-P8-3 had a greater (P < 0.05) number of CH. Treatment with FSH-P8-3 or FSH-P16-3 induced significant growth of medium/large follicles (4 to 8 mm in diameter) compared with saline or FSH-P16-1. The same results were observed when FSH-P8-3 was compared with FSH-P16-P4-2 or hCG. After in vitro fertilization, the rates of male and female pronuclei in oocytes derived from medium/large follicles did not differ (P > 0.05) between treatments, but in oocytes derived from small follicles they were lower (P < 0.05) in saline-treated than in FSH-P16-1-P4-2-treated gilts. After 120 h in culture, the percentages of the inseminated oocytes from 1 to 3 mm or 4 to 8 mm follicles developing to > or = 2-cell did not differ (P > 0.05) between saline- and gonadotropin-treated gilts. However, a higher (P < 0.05) percentage of the inseminated oocytes from 4 to 8 mm follicles had developed to the morula stage or beyond, than those from the 1 to 3 mm follicles. In conclusion, administration of single or multiple doses of FSH-P induced ovulation, but only 8 or 16 mg FSH-P injected 3 times with 24-h intervals for 72 h induced growth of 4 to 8 mm follicles. The size of follicle from which the oocyte derived also had a significant effect on its development in vitro.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine and ovulatory responses of the gilt to exogenous gonadotropins and estradiol during sexual maturation

Three studies were conducted to investigate the endocrine and ovulatory responses of the prepubertal gilt to exogenous estradiol and gonadotropins. In Study One, prepubertal gilts of 190 days of age were injected s.c. with pregnant mare's serum gonadotropin (PMSG) or physiological saline (SAL). Following PMSG injection, circulating levels of estradiol-17 beta (E2) increased. This increase was followed by a surge of luteinizing hormone (LH), estrus, a rise in progesterone (P4) levels, and ovulation. None of the gilts given SAL had increased levels of E2, LH or P4, and none ovulated. In Study Two, prepubertal gilts of 165 days of age were treated with varying doses of PMSG. A positive correlation was observed between dose of PMSG and peak levels of E2 (r = 0.83, P less than 0.001) and between dose of PMSG and number of corpora lutea (r = 0.96, P less than 0.001). In Study Three, gilts were treated at ages of 70 to 190 days with estradiol benzoate (EB), PMSG, or corn oil plus saline (CO/SAL) followed in 72 to 96 h by human chorionic gonadotropin (hCG) or SAL. All gilts treated with EB at 100 to 175 days of age had two surges of LH at an approximately 24-h interval. Gilts responding to EB at 70 and 190 days had only one surge of LH. Gilts of 100 days of age or older responded to PMSG with a single surge or two surges of LH. Ovulation in response to treatment was observed in gilts of 100 days of age or greater but not at 70 days. The conclusions drawn from these studies are that 1) PMSG-induced ovulation is preceded by an increase in circulating levels of E2 and in some gilts by a surge of LH, and 2) prepubertal gilts are able to respond to exogenous endocrine stimulation with either a single surge or multiple surges of LH at 70 to 190 days but are unable to ovulate in response to exogenous gonadotropins until 100 days of age.     

Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares

The effects of several doses of progesterone on FSH and LH concentrations were used to study the role of the gonadotropins on deviation in growth rates of the two largest follicles during the establishment of follicle dominance. Progesterone was given to pony mares at a daily dose rate of 0 mg (controls), 30 mg (low dose), 100 mg (intermediate dose), and 300 mg (high dose). All follicles > or = 6 mm were ablated at Day 10 (Day 0 = ovulation) to initiate a new follicular wave; prostaglandin F(2alpha) was given to induce luteolysis, and progesterone was given from Days 10 to 24. The low dose did not significantly alter any of the ovarian or gonadotropin end points. The high dose reduced (P < 0.05) the ablation-induced FSH concentrations on Day 11. Maximum diameter of the largest follicle (17.2 +/- 0.6 mm) and the second-largest follicle (15.5 +/- 0.9 mm) in the high-dose group was less (P < 0.04) than the diameter of the second-largest follicle in the controls (20.0 +/- 1.0 mm) at the beginning of deviation (Day 16.7 +/- 0.4). Thus, the growth of the two largest follicles was reduced by the high dose, presumably through depression of FSH, so that the follicles did not attain a diameter characteristic of deviation in the controls. The intermediate dose did not affect FSH concentrations. However, the LH concentrations increased in the control, low, and intermediate groups, but then decreased (P < 0.05) in the intermediate group to pretreatment levels. The LH decrease in the intermediate group occurred 2 days before deviation in the controls. The maximum diameter of the largest follicle was less (P < 0.0001) in the intermediate group (27.3 +/- 1.8 mm) than in the controls (38.9 +/- 1.5 mm), but the maximum diameter of the second-largest follicle was not different between the two groups (19.0 +/- 1.1 vs. 20.3 +/- 1.0 mm). Thus, the onset of deviation, as assessed by the second-largest follicle, was not delayed by the decrease in LH. Diameter of the largest follicle by Day 18 in the intermediate group (23.1 +/- 1.6 mm) was less (P < 0.05) than in the controls (28.0 +/- 1.0 mm). These results suggest that circulating LH was not involved in the initiation of dominance (inhibition of other follicles by the largest follicle) but was required for the continued growth of the largest follicle after or concurrently with its initial expression of dominance.     

Effect of electrocautery of nonovulated day 1 follicles on subsequent morphological variation among day 11 porcine embryos

One hundred and thirteen crossbred gilts were used in three experiments to examine the relationship between the pattern or sequence of ovulation and subsequent variation in the morphology of Day 11 embryos. In the first experiment, the percentage of follicles that had ovulated was determined in individual gilts at 26, 30, 34, or 38 h after the onset of estrus (n = 20) and 39, 41, 43, 45, or 47 h post-injection of human chorionic gonadotropin (n = 25; hCG, 1000 IU). The second experiment consisted of observing the percentage of follicles ovulated in 52 additional gilts at 34 h after the onset of estrus (Day 0). In the third experiment, the morphological variation among littermate embryos was compared on Day 11 between sham-operated control gilts (n = 8) and gilts whose nonovulated follicles were destroyed by electrocautery (n = 8) on Day 1. Results of these experiments indicated that the pattern of ovulation in gilts was skewed (p less than 0.01). Ovulation, induced with hCG, appeared to occur in a majority of follicles during a short period of time, whereas the remaining ovulations occurred over a longer interval. Of the 57 gilts observed at 34 h after natural estrus, ovaries of 25 gilts contained corpora hemorrhagica (CH) and follicles; one gilt had 1 CH and 17 follicles, and 24 others had 10-17 CH with 1-4 follicles remaining. Destruction of these nonovulated follicles resulted in a more (p less than 0.01) uniform group of Day 11 embryos and with fewer (p less than 0.05) small embryos. These data demonstrated that the pattern of ovulation may affect morphological variation in embryonic development such that some of the later ovulating follicles may represent smaller embryos within a litter.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Simultaneous injection of follicle stimulating hormone (FSH) and the prostaglandin F2alpha analog cloprostenol (PGF) disrupts follicular activity in diestrous dairy cows

Simultaneous injections of PGF and FSH or saline were given to 32 Holstein cows to test their combined ability to improve estrous and ovulation synchrony beyond that of PGF alone. All the cows were randomly assigned to receive PGF on either Day 8 or Day 10 of the estrous cycle (estrus = Day 0), and all the cows in each group were further assigned to simultaneous injection of either FSH or saline. Regression of the corpus luteum (CL), return to estrus and follicular activity were monitored by plasma progesterone assay, twice-daily estrous detection and ultrasonographic examination, respectively. Plasma progesterone concentrations declined to <1.0 ng/ml at 24 hours after PGF treatment in all the cows and FSH did not affect this decline. Return to estrus was not affected by FSH treatment in cows treated on Day 8 or Day 10; however, FSH disrupted normal follicular activity and either delayed normal ovulation following estrus or induced premature ovulation or cyst formation in 4 of 8 PGF/FSH (Day 8) cows and 5 of 8 PGF/FSH (Day 10) cows. These data indicate that exogenous FSH administered simultaneously with a luteolytic does of PGF does not maintain viability of large, dominant follicles and, therefore, is not an effective method for the synchronization of estrus and ovulation.     

Ovarian follicular development following administration of progesterone or aspiration of ovarian follicles in Holstein cows

The objective of this study was to compare the effects of administration of a single injection of progesterone (P4) and follicle aspiration on Day 7 of the estrous cycle on the timing and synchrony of follicular wave emergence, time of ovulation, and concentrations of P4, estradiol and FSH in Holstein cows. Twenty cows were assigned to 4 groups (n=5 cows per group) in a 2 by 2 factorial arrangement. Cows were treated on Day 7 (Day 0 = estrus) of the estrous cycle with either sham follicular aspiration and an oil vehicle administered intramuscularly (control), aspiration of ovarian follicles (aspiration), 200 mg of P4 im, or aspiration and 200 mg of P4 im (aspiration + P4). On Day 11, PGF(2alpha)(25mg) was administered to all groups. Synchrony of ovulation was less variable in each of the treatment groups compared with the control group (P<0.05), whereas ovulation was delayed in cows in the P4 group (P<0.05). Day of follicular wave emergence was delayed in the cows of the P4 group compared with cows in the aspiration and aspiration + P4 groups (P<0.01), whereas variability in wave emergence was less among both groups of aspirated cows compared with the cows in the control group (P<0.01). More follicles 4 to 7 mm in diameter were detected in the 2 aspiration groups compared with the cows in the control and P4 group (P<0.05). No difference was detected among groups in the maximum concentration of FSH associated with follicular wave emergence. We conclude that both the administration of P4 and the aspiration of follicles on Day 7 of the estrous cycle improves the synchrony of ovulation when luteolysis is induced on Day 11 and results in similar concentrations of FSH at the time of follicular wave emergence, but the timing of wave emergence and the number of follicles post-emergence differ.     

Effects of follicular status at treatment on follicular development and ovulation in response to FSH in Spanish merino ewes

Cyclic Spanish Merino ewes were treated on Day 13 of the estrous cycle with 12 mg, i.m., FSH-P in saline (n = 9) or propylene glycol (n = 24), currently with 100 micrograms, i.m., Cloprostenol (Day 0). From Day-6 to Day 0, the ewes were observed daily by transrectal ultrasonography, after Day 0, ultrasonography was performed every 12 h for 72 h. Sizes and locations of > or = 2 mm follicles were recorded at each observation. The ovulation rate was determined by laparoscopy on Day 7 after estrus. The number of ovulations ranged from 0 to 6 in ewes treated with FSH-P in saline and from 0 to 16 in ewes receiving FSH-P in propylene glycol (P < 0.05). In the latter group, the response was bimodally distributed; about half of the females had 1 ovulation, whereas the remainder had > 4 with a mean of 7 ovulations. The ovulation rate was associated with 2 characteristics of the largest follicle present at treatment (Day 0). First, if the largest follicle on Day 0 had not changed in diameter from Day-1 to Day 0, then 7 of 9 ewes had > 3 ovulations; if the largest follicle had either increased or decreased, only 8 of 24 ewes had > 3 ovulations (P < 0.05). Second, there was a linear trend (P < 0.07) for ovulation rate to decrease as the persistence of the largest follicle at treatment increased; no ewe in which the largest follicle on Day 0 remained present for more than 36 h ovulated more than 6 follicles. As with the ovulation rate, the numbers of large follicles on Days 1.5, 2 and 2.5 varied with the interaction of change in diameter of the largest follicle on Day 0 from Day-1 to Day 0 and with vehicle. In summary, the superovulatory response was affected by the change in diameter from Day-1 to Day 0 of the largest follicle on Day 0 and the period required for that follicle to regress after treatment with FSH-P and cloprostenol.     

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.     

Evaluation of methodology for administration of porcine FSH for use in estrus induction and for increasing ovulation rate in prepubertal gilts

Ovulation rate influences production efficiency of oocytes and embryos and depends upon the amount of gonadotropin administered and the ratio of FSH:LH activity. In Experiment 1, gilts (n=135) were assigned to receive 10 or 15 Armour units (AU) of porcine FSH containing 6%, 10%, or 15% LH, whereas controls received PG600. Gilts received 1/6th the FSH dose in six sc administrations at 8-h intervals. There was no treatment effect on incidence of estrus (66%) or cysts (23.9%), or number of corpora lutea (CL, 29.6). However, treatment did affect the percentage of gilts ovulating (P<0.05) with fewer 10 AU FSH with 15% LH-treated gilts ovulating (15%) compared to controls (72%), whereas the other treatments did not differ (range, 44-65%). Experiment 2 tested whether FSH in polyvinlypyrrolidinone (PVP) could induce estrus and ovulation with reduced administration frequency. Gilts (n=105) were assigned to receive 15 AU FSH with 10% LH in one (1P) or two sc administrations (2P) whereas controls received PG600. There was no treatment effect on incidence of estrus (64%) or cysts (22%). However, the percentage of gilts ovulating was lower for 1P (56%), but did not differ (P<0.05) between 2P (83%) and controls (85%). Treatment influenced ovulation rate (P<0.05) with 2P having more CL (24) than controls (12) and 1P (19). Results indicated that 10 and 15 AU FSH induced estrus and ovulation, although high LH content proved detrimental. Further, 15 AU FSH with 10% LH in PVP allowed for reduced administration frequency without compromising ovulation.