Comparison of equine pituitary extract and follicle stimulating hormone for superovulating mares

Sixty light-horse, nonlactating mares were used to compare the efficacy of equine pituitary extract versus follicle stimulating hormone (FSH-P) for inducing multiple ovulations. On Day 12 of diestrus, mares were assigned to receive 1) no treatment, controls; 2) subcutaneous injections of 750 Fevold rat units of equine pituitary extract once daily; or 3) intramuscular injection of 150 mg of FSH-P twice daily. Ultrasound was used twice daily to visualize follicular changes and ovulation. For mares in Groups 2 and 3, treatment was initiated when two or more follicles > 20 mm were detected, and it continued until all large follicles (> 30 mm) had ovulated or regressed. Five milligrams of prostaglandin F(2)alpha (PGF(2)) were administered to mares in Groups 2 and 3 on the first day of treatment. Human chorionic gonadotropin (3,300 IU) was given to all groups of mares during estrus when a 35-mm follicle was detected. Ovulation rate was greater (P < 0.05) for mares treated with pituitary extract (2.2) compared to FSH-P treatment (1.6) or no treatment (1.0). Thirteen of 18 mares treated with the extract had more than one ovulation versus only four of nine FSH-treated mares. Mares in the pituitary extract group were given injections for an average of 6.4 d compared to 6.8 d (13.7 injections) for FSH-treated mares. Intervals to estrus and ovulation from initial injection of extract were 2.9, 7.6; and 2.6, 9.2 d for FSH-treated mares. The mean number of medium-sized follicles (25 to 30 mm) was greater (P < 0.05) in extract-treated mares compared to the FSH-treated mares. Both extract and FSH increased (P < 0.05) the number of follicles > 30 mm and the size of the second largest follicle 1 and 2 d prior to ovulation when compared to controls. Overall, mares with multiple ovulations had more (P < 0.05) follicles 25 to 30 mm and > 30 mm on Day -6 through -1 (Day 0 = day of ovulation) than single ovulating mares. Those mares that had multiple ovulations had less (P < 0.05) size difference between the largest and second largest follicle when compared to single ovulating mares. In summary, FSH-P at the one dose studied was less effective than equine pituitary extract in inducing follicular activity and multiple ovulation in the mare.     

Induction of multiple ovulations during the ovulatory season in mares

The efficacy of an equine pituitary extract for induction of multiple ovulations during the ovulatory season was studied in 112 horse mares in four experiments. Combined for all experiments, 70% of the mares (78 112 ) had multiple ovulations for an average of 3.0 ovulations per mare. The interval between first and last ovulation was decreased (P<0.01) when human chorionic gonadotropin (hCG) was included in the treatment regimen (0.0 +/- 0.0 versus 1.6 +/- 0.4 days). Ovulation rate was lower (P<0.01) when extract treatment was initiated at day 19 (1.3 +/- 0.2) than when initiated at day 15 post-ovulation (2.9 +/- 0.5). Initiation of extract treatment at day 11 was as effective as initiation at day 15, with or without an injection of prostaglandin F(2alpha) on the first day of extract treatment. Increasing the dose of extract from 750 to 1500 or 2250 units per day did not significantly increase the ovulatory response.     

Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare

The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mares were treated similarly but with saline instead of rhFSH. The response was monitored by daily scanning of the mares' ovaries and assay of systemic oestradiol-17beta and progesterone concentrations. When the dominant follicle(s) exceeded 35 mm, ovulation was induced with human chorionic gonadotrophin; embryos were recovered on day 7 after ovulation. After an untreated oestrous cycle to 'wash-out' the rhFSH, the groups were crossed-over and treated twice-daily with 900 i.u. rhFSH, or saline. At the onset of treatment, the largest follicle was <25 mm in all mares, and mares destined for rhFSH treatment had at least as many 10-25 mm follicles as controls. However, neither dose of rhFSH altered the number of days before the dominant follicle(s) reached 35 mm, the number of follicles of any size class (10-25, 25-35, >3 mm) at ovulation induction, the pre- or post-ovulatory oestradiol-17beta or progesterone concentrations, the number of ovulations or the embryo yield. It is concluded that rhFSH, at the doses used, is insufficient to stimulate multiple follicle development in mares.     

Fertilization rates in superovulated and spontaneously ovulating mares

Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Group 1 received 40 mg of equine pituitary extract (EPE; i.m.) daily beginning on Day 5 after ovulation; mares assigned to Group 2 served as untreated controls. All mares were given 10 mg PGF(2alpha) on Day 5 and Day 6, and 3,300 IU of human chorionic gonadotropin (hCG) were administered intravenously once mares developed 2 follicles >/=35 mm in diameter (Group 1) or 1 follicle >/=35 mm in diameter (Group 2). Mares in estrus were inseminated daily with 1 x 10(9) progressively motile spermatozoa once a >/=35 mm follicle was obtained. Two days after the last ovulation the ovaries and oviducts were removed. Ovaries were examined for ovulatory tracts to confirm ovulation, while the oviducts were trimmed and flushed with Dulbeccos PBS + 10% FCS to recover fertilized oocytes. All fertilized oocytes (embryos) recovered were cultured in vitro for 5 d using TCM-199 conditioned with equine oviductal cells. Ninety-two percent of the CL's from EPE mares resulted from ovulations compared with 94% for mares in the control group (P>0.05). The percentages of ovulations resulting in embryos were 57.1 and 62.5% for EPE-treated and control mares, respectively (P>0.05). Eighty-eight (Group 1) and 91% (Group 2) of the freshly ovulated oocytes recovered were fertilized (P>0.05). After 5 d of culture, 46.4 and 40.0% of the embryos from EPE-treated and control mares developed to the morula or early blastocyst stage (P>0.05). In summary, the CL's formed in superovulated mares were from ovulations not luteinizations. Although embryo recovery was less than expected, fertilization rates and embryo development were similar (P>0.05) between superovulated and control mares.     

The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model

The efficacy of a recently engineered single chain recombinant equine follicle stimulating hormone (reFSH) was investigated in estrous cycling mares whose gonadotropins and follicular activity had been suppressed by concurrent treatment with progesterone and estradiol (P&E). Time of estrus was synchronized in 15 estrous cycling mares during the breeding season with prostaglandins F_2α (PGF_2α). The day after ovulation, mares were treated once daily with P&E for 14 days. Mares received a second injection of PGF_2α on day 6 of the synchronized estrous cycle to induce luteolysis. On day 8 post-ovulation mares were randomly assigned to three groups: small dose reFSH-treatment group (0.5 mg reFSH IV, twice daily); large dose reFSH-treatment group (0.85 mg reFSH IV twice daily); control group (saline IV, twice daily). reFSH treatment occurred concurrently with the last week of P&E treatment. After a follicle or cohort of follicles reached 35 mm in diameter, mares were injected with 0.75 mg of recombinant equine luteinizing hormone (reLH) to induce ovulation. Post-treatment ovulation was assessed. Daily blood samples were collected for analysis of FSH, LH, estradiol, progesterone, and inhibin by radioimmunoassay (RIA). On the first day of reFSH/saline treatment, blood samples were collected periodically from 1 h prior to treatment to 6 h post-injection via an indwelling jugular catheter to determine acute changes in FSH concentrations. Monitoring of follicular activity, estrus, and ovulation was performed daily by utilizing a stallion and transrectal ultrasonography.A difference (p ≤ 0.05) between the largest diameter follicle in the reFSH-treatment groups compared to controls occurred on day 14 post-ovulation, the day treatments ended, and the difference continued until day 21 post-ovulation. reFSH-treatment groups had larger (p ≤ 0.05) numbers of 20–29 mm follicles (days 13–18), 30–34 mm follicles (days 15–20) and ≥35 mm follicles (days 16–21) than controls. Mares treated with reFSH, at either dose, took less time (average: 2.95 ± 0.42 days) to develop 2–3 times more pre-ovulatory follicles than control mares (7.8 ± 0.51 days) (p ≤ 0.05). The number of ovulations between treated mares and controls were similar due to a greater incidence of ovulation failure in reFSH-treated mares. During reFSH treatment, concentrations of plasma FSH, inhibin and estradiol were greater (p ≤ 0.05) compared to control concentrations. Plasma LH concentrations in reFSH-treated mares were suppressed and did not exhibit the ovulatory surge of controls (p ≤ 0.05). Plasma progesterone concentrations were not different across groups.These findings demonstrate the specific effects of reFSH to increase number of total follicles including pre-ovulatory follicles in mares with endogenous pituitary gonadotropins and follicular growth suppressed by a regimen of P&E.     

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25mg EPE and treated with 2500 IU hCG, (2) superovulation with 25mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles >10mm were aspirated 24h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group, 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%; p<0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15+/-4 days for control to 27+/-15 days for EPE (p<0.05) and to 23+/-13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations.     

Spontaneous multiple ovulation in the mare and its effect on the incidence of twin embryo collections

Records from 183 nonlactating mares that experienced spontaneous multiple ovulation were examined to determine if: 1) double ovulations are as likely to be unilateral as bilateral; 2) the interval between two ovulations is shorter when the ovulations are unilateral than when they are bilateral; 3) the mean diameter of the two follicles on the day prior to ovulation is less when the ovulations are synchronous and unilateral; 4) for both unilateral and bilateral ovulation, twin embryos are more likely to be detected when double ovulations are asynchronous; and 5) for both synchronous and asynchronous ovulations, twin embryos are more likely to be detected when the ovulations are bilateral. Mares were teased daily with a stallion and follicular development was assessed daily during estrus by ultrasonography. Mares were inseminated daily during estrus and embryo recovery attempts were performed 6 to 7 d post ovulation. Double ovulations occurred as frequently from the same, as from opposite ovaries. The interval between the double ovulations was not shorter (P > 0.05) in unilateral versus bilateral ovulations. In addition, size of the largest and second largest preovulatory follicles was not altered (P > 0.05) by type of ovulation (bilateral vs unilateral) or synchrony of ovulation. Synchrony of ovulations had no affect (P > 0.05) on the incidence of twin embryos recovered. However, more (P < 0.05) twin embryos were recovered from bilateral ovulators compared to unilateral ovulators.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Intrauterine embryo reduction in the mare

The site of a hypothesized embryo reduction mechanism was studied. The number of normal-sized embryos and undersized embryos (>2 standard deviations below the mean embryo diameter in control mares) was determined at day 7 and 11 post-ovulation in single-ovulating control mares and in multiple-ovulating pituitary extract-treated mares in which all ovulations occurred on a single day. In two additional groups (control and treated), the embryonal enlargement was monitored by rectal palpation until day 49. An average of 0.7 normal-sized embryos per control mare at day 7 and at day 11 and 0.6 vesicles per control mare at day 49 was found. More normal-sized embryos (P<0.05) were recovered per extract-treated mare at day 7 (2.9 embryos/mare) than at day 11 (0.7), and fewer undersized embryos per extract-treated mare (P<0.05) were recovered at day 7 (0.1) than at day 11 (1.1). The total number of embryos (normal-sized plus undersized) per treated mare was greater (P<0.05) at day 7 and at day 11 than the number of palpated vesicles per treated mare at day 49. The number of mares with more than one normal-sized embryo in the day 7 treated group (10/14) was greater (P<0.05) than in the day 11 treated group (1/14) and was greater than the number of mares with more than one palpated vesicle in the day 49 treated group (2/14). Intrauterine reduction was therefore manifested between days 7 and 11 in multiple-ovulating mares, as demonstrated by the number of multiple normal-sized blastocysts recovered at day 7 and by the reduced number of normal-sized and the increased number of undersized blastocysts recovered at day 11.     

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.     

A biological embryo-reduction mechanism for the elimination of excess embryos in mares

Only one embryo was found in each of 15 pregnant mares in which multiple, synchronous (0 or 1 day apart) ovulations were induced with a pituitary extract. In the brood-farm mares, fewer (P<.05) twin pregnancies were associated with synchronous, double ovulations (0 39 ), than with asynchronous, double ovulations (9 57 ). Higher (P<.05) pregnancy rates (number of mares pregnant, regardless of number of embryos) were obtained in pituitary extract-treated mares with multiple, synchronous ovulations (15 18 ) than in treated mares with single ovulations (6 14 ). Similarily, pregnancy rates were higher (P<.01) in brood-farm mares with double, synchronous ovulations (39 47 ) than in brood-farm mares with single ovulations (1320 2426 ). Our interpretation is that ova produced by synchronous, double ovulations are viable and fertilizable (indicated by higher pregnancy rates), but that one of the resulting embryos is eliminated (indicated by the absence of twins).     

Ovarian follicles, ovulations and progesterone concentrations in aged versus young mares

The objectives of this study were: 1) to document age-related ovulation failure in mares and 2) to contrast the number of ovarian follicles, occurrence of ovulations, and postovulatory concentrations of progesterone in aged versus young mares. In Experiment 1, 4 of 10 aged (25- to 33-years-old) mares were anovulatory between July 1 and September 1, 1989. In Experiment 2, two of 25 aged (20- to 30-years-old) and none of 21 young (3- to 12-years-old) mares were anovulatory between February 1 and June 30, 1990. The average (+/- SEM) day of the first ovulation was later (P<0.05) for aged versus young mares (May 9 +/- 7.1 vs April 25 +/- 7.4 days, respectively). There tended (P<0.10) to be fewer 11- to 20-mm ovarian follicles in aged versus young mares (2.8 +/- 0.2 vs 5.3 +/- 0.1, respectively), but there was no difference (P>0.10) in the total number of ovarian follicles in aged versus young mares (21.0 +/- 0.3 vs 26.1 +/- 0.2, respectively) during the pooled periovulatory period of the first and second (single) ovulations. The number of ovulatory cycles during the study period was less (P=0.01) for aged versus young mares (2.2 +/- 0.3 vs 3.2 +/- 0.3). Plasma progesterone concentrations on Days 10 and 15 of the first ovulatory cycle were higher (P<0.05) in aged versus young mares.     

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles > or = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.     

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.     

Effects of unilateral ovariectomy on follicular development and ovulation in cattle

In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers.     

Ultrasonic anatomy of equine ovaries

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for studying follicular and luteal status in mares, and the ultrasonic properties of equine ovaries were characterized. Follicular diameters were estimated in vivo and after removing and slicing six ovaries. Correlation coefficients between the two kinds of determinations were 0.91 for number of follicles >/=2 mm in diameter and 0.95 for diameter of largest follicle. The ovaries of five mares were examined daily until all mares had been examined from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of largest follicle and second largest follicle and for number of follicles 2-5 mm, 16-20 mm, and >20 mm. Differences seemed to be caused by the presence of many 2- to 5-mm follicles during early diestrus, initiation of growth of large follicles at mid-cycle, selective accelerated growth of an ovulatory follicle beginning five days before ovulation, and regression of large nonovulatory follicles a few days before ovulation. In one of the five mares, the corpus luteum was identified throughout the interovulatory interval, and the corresponding corpus albicans was identified for three days after the second ovulation. In the other four mares, the corpus luteum was last identified an average of 16 days after ovulation or five days before the next ovulation. In a blind study, the location of the corpus luteum (left or right ovary) as determined by ultrasonography agreed with a previous determination of side of ovulation by palpation in 88% of 40 mares on days 0-14. In the remaining 12% and in all of 12 estrous mares, the location was recorded as uncertain. The ultrasound instrument was judged effective for monitoring and evaluating follicles and corpora lutea.     

Ultrasonic evaluation of the preovulatory follicle in the mare

Ultrasonically visible characteristics of preovulatory follicles in mares which single ovulated were studied daily for 79 preovulatory periods in 40 mares. The preovulatory follicle became the largest follicle in the ovary from which ovulation later occurred six or more days before ovulation in 65 of 79 (82%) preovulatory periods; the mean was day -7 (range, day -14 to day -4). The increase in mean diameter of the preovulatory follicle was linear (R(2)=99.5%) over day -7 (29.4 +/- 0.8 mm) to day -1 (45.2 +/- 0.5 mm; growth rate, 2.7 mm/day). Follicles which double-ovulated were smaller (P<0.05) on day -1 (36 +/- 1.6 mm; n=12 follicles). Preovulatory follicles exhibited a pronounced change in shape from a spherical to a conical or pear-shaped structure in 84% of the preovulatory periods. Remaining follicles retained a spherical shape. Scores representing thickness of the follicular wall increased (P<0.05) as the interval to ovulation decreased. There was no significant difference among days in mean gray-scale value of the follicular wall or in echogenicity of the follicular fluid. Although diameter and shape of the follicle and thickness of the follicular wall changed during the preovulatory period, no reliable ultrasonically visible predictor of impending ovulation was found.     

Age and pasture effects on vernal transition in mares

The objectives of the present study were to determine if follicular activity was less in old than in young mares during the spring transition and if green pasture would hasten onset of the ovulatory season. Experiments were conducted over 2 sequential years using young mares (3 to 7 yr) and old mares (> or =14 yr). In Experiment 1, growth of the largest and second-largest follicles were compared for young mares (5 to 7 yr) and old mares (> or =14 yr) for 21 d prior to the first ovulation of the year. More follicular activity was noted in young than in old mares. Main effect of age was significant for diameter of the largest follicle, and interaction of day-by-age was significant for diameter of the second-largest follicle. Prior to the beginning of the breeding season, the mares were randomly divided into dry-lot and pasture groups. The interval from May 2 to ovulation was shorter (P < 0.005) for mares put on pasture on May 2 than for mares kept in dry lot (means +/- SEM, 14.5 +/- 2.7 and 21.3 +/- 3.2 d, respectively). In Experiment 2, follicular activity was compared among 3 age groups (3 to 7, 17 to 19, and > or =20 yr). The total number of follicles > or =10 mm was higher (P < 0.05) for young mares and lower (P < 0.05) for old mares than for mares of an intermediate age. Main effect of age and interaction of day-by-age were significant for diameter of largest and second-largest follicles, being smaller for mares > or =20 yr than for younger mares. The interval from development of a follicle > or =30 mm to ovulation was shorter (P < 0.05) for mares placed on pasture when a > or =30 mm follicle developed than the interval for mares kept in dry lot (5.7 +/- 0.7 and 8.2 +/- 0.9 d, respectively). In summary, less follicular activity occurred in old than in young mares during the transitional period, and mares pastured on green grass ovulated sooner in the spring than mares housed on dry lot and fed hay.     

Effects of a new injectable short-term release deslorelin in foal-heat mares

Mares treated with subcutaneous deslorelin implants on the first postpartum estrus early in the breeding season had significant reductions in the number of large follicles at early pregnancy examinations and delayed return to estrus (in mares that failed to become pregnant); these adverse effects were attributed to a prolonged release of the drug from the implant. In 2003, an injectable short-term release (<24 h) deslorelin product became available. The objective of this study was to determine if this product would hasten ovulation in early foaling first postpartum estrus mares without reducing the number of large follicles at early pregnancy examination (14-15 days postovulation). Beginning 5-6 days postpartum, first postpartum estrus (foal-heat) mares were teased daily and examined thrice weekly (Tuesday, Thursday and Saturday) by transrectal ultrasonography. Mares in estrus with a follicle > or = 34 mm diameter on Tuesdays or Thursdays were alternately assigned to: Treatment 1, n = 17; 1.5 mg injectable short-term release deslorelin, or Treatment 2, n = 16; Control (no treatment). The schedule allowed accurate determination of the number of mares ovulating within 2 days of treatment (i.e., ovulations detected on Thursday or Saturday). Mares were mated on the day of treatment and at 2-day intervals until either ovulation was confirmed or until behavioral estrus ceased. Transrectal ultrasonography was done 14-15 days after ovulation to assess ovarian follicles and pregnancy status. Fewer covers were required and more mares ovulated within 2 days of treatment in deslorelin-treated versus Control mares (P < 0.01). Pregnancy rates were normal (69%) in deslorelin-treated mares. The number of large follicles 14-15 days after ovulation did not differ between deslorelin-treated and Control mares (P > 0.10), suggesting follicular suppression did not occur with this formulation of deslorelin.     

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily; (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of >35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation, or two doses of a GnRH-a agonist upon detection of a follicle >35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles >35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF2, and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF2alpha, but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles > or = 35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose, improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates.