Evaluation of two treatments in superovulation of mares

The efficiency of superovulating mares with an enriched fraction of equine follicle-stimulating hormone (feFSH) and an equine pituitary extract (EPE) with similar FSH content but differing in the LH amount was compared. Mares were randomly assigned to an feFSH (n = 5) or EPE (n = 5) treatment. The experimental period was of 2 successive estrous cycles, with the first cycle as the control. At Days 6 and 7 of the estrous cycle, the mares received 250 micrograms i.m. cloprostenol. The treatments consisted of daily injections of 25 mg feFSH or EPE beginning on Day 6 post ovulation. Mares were inseminated every other day until the last ovulation was detected. When the mares in the control and treatment cycles developed at least 1 or 2 > or = 35-mm follicle, respectively, the treatment was interrupted, and a single injection of EPE (25 mg, i.v.) was administered to induce ovulation(s). Nonsurgical embryo recovery was performed 6 or 7 d after ovulation in both control and treatment cycles. The number of ovulations per mare was not significantly different (P > 0.05) between feFSH and EPE groups, but both were higher (P < 0.05) than that of the control cycle. The number of recovered embryos per ovulation was similar (P > 0.05) for control, feFSH and EPE groups. The high amount of LH presented in EPE did not affect the superovulatory response of the mares. Superovulatory treatments increased the ovulation rate of mares but did not affect the embryo recovery rate per ovulation.     

Pulsatile release of FSH for superovulation in cattle

The studies reported here were directed towards the development of an implantable microcapsule which "pulses" release of follicle stimulating hormone, FSH, for application to superovulating cows. Final dose forms were administered using membrane-coated cylinders. The "pulse" of the FSH is achieved by membrane encapsulation of an effervescent/swelling core containing citric acid, sodium bicarbonate, glucose and FSH. Entry of water results in sufficient pressure increase (by gas generation) to rupture ("burst") the membrane. Time to rupture is dependent upon several factors, such as membrane permeability and thickness, and core composition and loading. The final dose forms were implanted by means of a trochar. This system was tested in sheep to substantiate in vivo "burst" times and then tested in cows to determine efficacy. In vivo burst times in sheep varied from 8 to 96 hr, based upon maximal FSH values in blood serum, and generally paralled the planned times resulting from in vitro tests. Multiple capsules designed to release FSH as a pulse or steady state were tested on a limited number of cows plus a control (n = 10). Four of the combinations resulted in 11, 11, 14 and 16 ovulations, indicating that further development has promise of providing a one-injection system using FSH for superovulating cattle.     

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH)

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17β, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17β, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.     

Initiation of superovulation in mares 5 or 12 days after ovulation using equine pituitary extract with or without GnRH analogue

Cyclic mares were assigned to 1 of 3 treatments (n=15 per group): Group 1 received equine pituitary extract (EPE; 25 mg, i.m.) on Day 5 after ovulation; Group 2 received EPE on Day 12 after ovulation; while Group 3 received 3.3 mg of GnRH analogue (buserelin implant) on the day of ovulation and 25 mg, i.m. EPE on Day 12. Mares in each group were given 10 mg PGF(2)alpha on the first and second day of EPE treatment. The EPE treatment was continued daily until the first spontaneous ovulation, at which time 3,300 IU of human chorionic gonadotropin (hCG) were given to induce further ovulations. Mares in estrus with a >/=35 mm follicle were inseminated every other day with pooled semen from 2 stallions. Embryo recovery was attempted 7 days after the last ovulation. Follicular changes and embryo recovery during 15 estrous cycles prior to treatment were used as control data. During treatment, the number of follicles >/=25 mm was higher (P<0.05) for Day 5 than for Day 12 or control mares, but the number for Day-5 mares was similar (P>0.05) to that of mares treated with buserelin implants (Group 3). Initiation of EPE treatment on Day 5 resulted in a greater (P<0.05) number of ovulation (2.9) than on Day 12 (1.1) or in the control mares (1.3) but not in the buserelin-treated mares (1.8). The number of embryos recovered from mares in the Day 5 (1.2), Day 12 (1.0), buserelin (0.9) and control (0.9) groups was similar (P>0.05). The conclusions were 1) EPE initiated in early diestrus increased follicular development and ovulation and 2) treatment with GnRH analogue marginally improved response to EPE treatment.     

FSH and LH concentrations in periparturient mares.

The influence of the ovaries and presence of a foal on periparturient concentrations of FSH and LH were studied in 19 Pony mares. In intact and ovariectomized mares, mean concentrations of FSH fluctuated between 1.1and 9.9 ng/ml on Days -14 to-1 before parturition (Day 0). A surge of FSH occurred in all mares in association with parturition. From Days 1 to 10, the high levels of FSH gradually decreased in the intact group to the minimal concentrations that occur during oestrus, but remained elevated in the ovariectomized mares. There were no significant pre-partum changes in LH in either type of mare. Post-partum changes in LH concentrations increased at a similar rate in ovariectomized and intact mares. The presence of a foal significantly lengthened the interval to first oestrus, depressed LH levels on Days 6--10 and decreased the FSH concentrations as averaged over the 10 days before the first ovulation after parturition.     

LH and FSH profiles at superovulation and embryo production in the cow

The variability of the superovulation response in cattle is an important problem to the commercial embryo transfer industry. Plasma LH and FSH concentrations around the time of estrus and ovulation were studied in relation to embryo production, to try and elucidate this problem. Sixteen cows were superovulated with 38 mg FSH-P and estrus synchronized with prostaglandin F(2) alpha. On the third and fourth day of superovulation increases in plasma LH but not FSH were detected. The LH and FSH profiles appeared to be normal in the size of the surge but in many cases they were were abnormal in timing. Transferable embryo production appeared to be lower in cows in which the LH and FSH surges were not coincident, and in cows where the surges were early or late with reference to estrus. FSH appeared to be primarily responsible for the number of embryos produced and LH for their quality, i.e. the number transferable.     

Efficiency of pituitary extracts (FSH) for induction of superovulation in cattle

In four experiments on 195 adult cows the number of corpora lutea, as determined by rectal palpation, and the number and quality of embryos, as determined after non-surgical recovery, were studied following 4 days of treatment with superovulatory doses of follicle stimulating hormone. Neither the total dose, the commercial origin of the hormone preparation, nor the volume of saline used as an injection medium had a significant effect on the responses. However, the number of injections per day and the pattern of doses within the injection regime were found to exert significant effects. The greatest response (15.9 ± 15.0 corpora lutea, 12.2 ± 12.3 embryos recovered and 5.3 ± 6.2 transferable embryos) was observed after the administration of the hormone twice a day at decreasing doses.     

Endometrial biopsy findings in mares with contagious equine metritis.

Endometrial biopsy samples before and after treatment were obtained from 10 mares naturally infected and one Pony mare experimentally infected with Contagious Equine Metritis in 1977. The histopathological features were a short-lived polymorphonuclear cell infiltration of the luminal epithelium and stroma followed by a very early and marked mononuclear cell infiltration of the stroma, including many plasma cells. The luminal epithelium responded with cellular proliferation which may have reflected a regenerative response. Following the initial acute response, intercellular basal vacuoles containing amorphous and granular material and degenerate leucocytes, were seen in association with the basement membrane for several weeks following infection and apparent recovery. Specimens taken from 9 mares after treatment suggested a varied response. Endometrial biopsy was useful in demonstrating an endometrial response to infection in individual mares prior to the isolation of the causal organism. Its usefullness as a screening test was limited by the ability of mares to harbour the organism in the lower genital tract whilst showing little or no evidence of acute endometritis.     

Follicular dynamics in mares treated with an equine pituitary extract

The follicular dynamics of 112 mares treated with an equine pituitary extract were studied. Follicles >10 mm in diameter at day 15 post-ovulation appeared to represent the follicles which were induced with pituitary extract to grow and ovulate. This was shown by the greater number of >10 mm follicles in mares which subsequently had higher ovulation rates and by the subsequent decrease in number of small follicles (<20 mm) which corresponded with the increase in number of large follicles (>/=20 mm). The difference in diameter (mm) between the largest and second largest follicle on day 15 post-ovulation was greater (P<0.05) for extract-treated mares which subsequently had single ovulations than for extract-treated mares which subsequently had multiple ovulations (7.7 +/-1.5 vs 2.8 +/-0.6). The observed ratio of bilateral to unilateral multiple ovulations was not different (P>0.1) from the expected ratio which was calculated on the assumption that side of ovulation occurred independently (59:19 vs 62:16, observed vs expected).     

Endocrine responses of goats after induction of superovulation with PMSG and FSH

Goats in Group A were pretreated for 9 days with a synthetic progestagen, administered via intravaginal sponge, and 1000 i.u. PMSG s.c. on Day 12 of the oestrous cycle. Goats in Group B had the same PMSG treatment, but not the progestagen pretreatment. Group C goats received a s.c. twice daily injection of a porcine FSH preparation (8 mg on Day 12, 4 mg Day 13, 2 mg Day 14 and 1 mg Day 15). Oestrus was synchronized in all animals by 50 micrograms cloprostenol, 2 days after the start of gonadotrophin treatment. The vaginal progestagen sponges were removed from Group A at the same time. Mean ovulation rate was slightly higher in FSH-treated than in the PMSG-treated animals, whereas the incidence of large follicles that failed to ovulate was significantly elevated in PMSG-treated animals in Group B. More goats in Groups A and B than in Group C exhibited premature luteal failure. Progestagen pretreatment appeared to suppress both follicular and luteal activity, as indicated by numbers of large non-ovulating follicles and by the magnitude and duration of elevated plasma oestradiol levels following PMSG stimulation, and by decreased plasma progesterone levels before and after PMSG treatment. Oestrogenic response to FSH was considerably less than that to PMSG, as indicated both by a considerably shorter duration of elevation of circulating oestradiol levels during the peri-ovulatory period, and by lower maximal oestradiol levels. Differences in the ovarian responses to PMSG and FSH may be attributed primarily to differences in the biological half-life of each preparation.     

Successful superovulation of cattle by a single administration of FSH in aluminum hydroxide gel

We investigated whether Al-gel could adsorb and release FSH effectively in vitro and in vivo, and whether a single administration of FSH in Al-gel could successfully induce superovulation (SOV) in cattle. Porcine FSH (pFSH; 30 mg) was mixed with 5 mL of Al-gel; 99.98+/-0.01% of pFSH was adsorbed by the gel and 71.6+/-1.1% of the adsorbed pFSH was subsequently released in the presence of BSA. In cattle given a single i.m. treatment of 30 mg of pFSH in 5 mL of Al-gel, the numbers of CL, total ova recovered, and transferable embryos per cow were not significantly different from conventional (twice daily for 4 d) pFSH treatment (12.3+/-1.7 versus 11.7+/-1.8, 10.0+/-2.5 versus 9.3+/-1.7, and 8.6+/-2.3 versus 8.0+/-1.8, respectively, mean+/-S.E.M.); plasma pFSH concentrations were increased for 4 d, indicating sustained release from the Al-gel. Five cows were given 30 mg pFSH in 5 mL of Al-gel i.m. on five occasions (once every 2-3 months); there was no significant difference among treatments for the number of CL (12.4+/-3.8, 13.8+/-4.8, 9.0+/-1.9, 9.8+/-3.0, 12.0+/-2.1), total ova recovered (12.0+/-3.8, 12.6+/-5.1, 6.8+/-1.9, 7.6+/-1.8, 11.4+/-2.5), and transferable embryos (11.4+/-3.9, 10.4+/-5.8, 6.6+/-2.1, 4.8+/-1.4, 10.4+/-2.6). In conclusion, a single i.m. treatment of 30 mg pFSH in 5 mL Al-gel effectively induced SOV in cattle.     

Follicular and FSH responses to parturition during the anovulatory season in mares

The ovaries of periparturient pony mares (n=9 to 16 parturitions per month for January to April) were scanned ultrasonically on the day of parturition, while those of postpartum and control mares (n=12) were examined at least twice weekly. Four mares had apparent lactational anovulation (incidence, 7%) that corrected spontaneously (1 mare) or within 14 d after the weaning of foals on August 10 (3 mares). All but 2 of the postpartum ovulations occurred after April 29; that is, parturition did not effectively stimulate ovulation in ponies foaling during the anovulatory season. Mean diameter of the largest follicle per month increased (P<0.001) progressively in the controls (means: 11.4, 14.4, 19.0 and 24.5 mm for January to April, respectively). In the parturient mares, the diameter of the largest follicle on day of parturition did not increase over months (range of means: 13.6 to 16.9 mm), indicating that a suppressive effect of pregnancy counteracted the stimulatory effect of season. Within each month of parturition, diameter of the largest follicle increased (P<0.05) between Day 0 (day of parturition) and Day 3 or 7. Blood samples for FSH assay were taken daily for 14 d from 6 mares with parturition in the middle of each month and from 6 controls on the corresponding calendar days. In periparturient mares, a significant increase in mean FSH concentrations occurred for all months of parturition between Day-2 and Day 0, followed by a significant decrease between Day 3 and Day 7. Maximum means for the periparturient FSH profile were temporally related to the beginning of follicular growth. In the controls, FSH concentrations were not affected by month or day, or by their interaction. Within each month, mean FSH concentrations were lower (P<0.05) in the periparturient mares than in the controls (averaged over all months: 3.9 +/- 0.1 versus 7.9+/-0.3 ng/ml) even though follicular growth was greater following parturition than during the corresponding calendar days in controls.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.     

The day of the estrous cycle that FSH is started and superovulation in cattle

A total of 993 commercial donor cows were superovulated with 28 mg FSH-P. The first injection of FSH-P was administered on one of days 9 through 13 of the donor's estrous cycle. The day that FSH was started did not affect total embryos collected, the number of transferable embryos or the percent transferable. These results did not support the current hypothesis that because the population of antral and preantral follicles in the ovaries on days 9 and 10 is higher, best embryo production should be achieved by starting donor cows on day 9 or 10 of the estrous cycle.     

Relationships between FSH surges and follicular waves during the estrous cycle in mares

Individual follicles >/=15 mm were monitored daily by ultrasonography in 12 mares during the estrous cycle. Follicular waves were designated as major waves (primary and secondary) and minor waves based on maximum diameter of the largest follicle of a wave (major waves, 34 to 47 mm; minor waves, 18 to 25 mm). Dominance of the largest follicle of major waves was indicated by a wide difference (mean, 18 mm) in maximum diameter relative to the second largest follicle. Dominant follicles of primary waves (n=12) emerged (attained 15 mm) at a mean of Day 12 and resulted in the ovulations associated with estrus (ovulation=Day 0). The dominant follicle of a secondary wave (n=1) emerged on Day 2 and subsequently ovulated in synchrony with the dominant follicle of the primary wave, which emerged on Day 9. The largest follicles of minor waves (n=4) emerged at a mean of Day 5, reached a mean maximum diameter 3 days later, and subsequently regressed. There was a significant increase in mean daily FSH concentrations either 6 days (primary wave) or 4 days (minor waves) before the emergence of a wave. Mean concentrations of FSH decreased significantly 2 days after emergence of the primary wave. Divergence between diameter of the dominant and largest subordinate follicle of the primary wave was indicated by a significantly greater mean diameter of the dominant follicle than of the largest subordinate follicle 3 days after wave emergence and by the cessation of growth of the largest subordinate follicle beginning 4 days after the emergence of a wave. Surges of FSH were identified in individual mares by a cycle-detection program; surges occurred every 3 to 7 days. Elevated mean FSH concentrations over the 6 days prior to emergence of the primary wave was attributable to a significantly greater frequency of individual FSH surges before wave emergence than after emergence and to an increase in magnitude of peak concentrations of FSH associated with individual surges.     

Effectiveness of an antagonist to gonadotrophin releasing hormone on the FSH and LH response to GnRH in perifused equine pituitary cells,and in seasonally acyclic mares

We wish to use a gonadotrophin-releasing hormone (GnRH) antagonist in the mare as a tool for investigating the control of the oestrous cycle. The aim of this study was to test the effectiveness of the antagonist cetrorelix by testing both in vitro, using perifused equine anterior pituitary cells, and in vivo in seasonally acyclic mares. Pituitary cells were prepared and after 3-4 days incubation, loaded onto columns and given four pulses of GnRH (at 0, 30, 60 and 90 min; dose-response study). After the second GnRH pulse, infusion of cetrorelix began (0, 100, 1000 and 2000 pmol/l) and continued until the end of the experiment. To mimic luteal phase conditions, cells were pre-incubated and perifused with progesterone (25 nmol/l) and GnRH pulses given at 0, 90, 180 and 270 min. Cetrorelix (0 or 1000 pmol/l) began after the second GnRH pulse. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were measured in 5 min fractions. Both FSH and LH response areas (above baseline) after GnRH were inhibited by 1000 pmol/l cetrorelix (P < 0.01, P < 0.01, respectively) but not by 100 pmol/l cetrorelix. Similarly, in the presence of progesterone, cetrorelix inhibited the FSH (P < 0.001) and LH (P = 0.0002) response area. Seasonally acyclic mares, pre-treated for 3 days with progesterone (150 mg i.m. per day) were given cetrorelix as (i) a loading dose of 1 microg/kg then infusion at 2.2 ng/(kg min) for 90 min, (ii) a s.c. injection at 20 microg/kg, (iii) infusion at 2.2 ng/(kg min) for 48 h, and (iv) no cetrorelix (control mares). At 90 min, 6, 24 and 48 h after cetrorelix was first administered, mares were given a bolus injection of GnRH (22.2 ng/kg i.v.) and the FSH and LH responses measured. All doses of cetrorelix inhibited the FSH response at 90 min. The response was no longer suppressed at 6 h in the 90 min infusion group, showing a rapid recovery from inhibition. At 24 h, the FSH responses in the injected and 48 h infusion group were suppressed. The LH concentrations were low and showed no significant changes. This study has defined the time course and dose of cetrorelix with respect to its effect on FSH in the horse. It is concluded that cetrorelix could be used to elucidate the role of FSH in follicular development in cyclic mares.     

Associations between FSH concentrations and major and minor follicular waves in pregnant mares

Follicular waves were detected in 19 pregnant mares (Days 11 to 40) by a significant increase followed by a significant decrease in diameters of follicles after removing large (>/=25 mm) follicles from the data sets. The waves were defined as major (largest follicle, >/=35 mm; n=18) or minor (largest follicle, <35 mm; n=17). Six mares (32%) had 2 successive major waves beginning on mean Days 15.2 and 26.8; 6 had a solitary major wave beginning on Days 11 to 20; and 6 had only minor waves occurring at irregular intervals. The mean interval between minor waves (7.8 days) was less (P<0.05) than for major waves (11.7 days). Mean divergence in diameters of the largest and second largest follicles of a wave began 4 days after the detected emergence of consecutive major waves, and was taken as the beginning of the expression of dominance by the largest follicle. The interval from emergence to divergence was several days longer (P<0.05) for solitary major waves than for consecutive waves. Dominance was not detected for the minor waves, using mean diameters of the 2 largest follicles, but was apparent on inspection of individual wave profiles in 5 of 17 (29%) minor waves. Minor waves, compared with major waves, had larger diameter of follicles on the day of wave emergence (15.0 versus 12.1 mm), and significantly greater variation in the day of attainment of maximal diameter of largest follicle and small follicles. A mean increase in FSH was temporally associated with the emergence of both major and minor waves. In mares with minor waves, concentrations of FSH were higher, on average, over Days 11 to 40, which seemed consistent with the origin of follicular waves from larger follicles in the basal populations. The lower overall FSH levels in mares with major waves seemed at least partly due to depression of FSH levels beginning at the time of divergence between the 2 largest follicles.