35-mm Follicle Research Articles

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8mm prehierarchal follicles were cultured with IGF1 (0, 10, 100ng/mL) in the presence or absence of FSH (0, 10ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

13 Invitro and invivo embryo production during foal heat in a mare: A case report

The objective of this case report is to describe invitro and invivo embryo production during foal heat in a mare. A 16-year-old American Quarter Horse mare was presented for breeding and nonsurgical embryo recovery days post-foaling. Upon transrectal ultrasonographic examination, the donor mare was determined to be in oestrous (foal heat) with presence of moderate uterine oedema, a relaxed cervix, absence of uterine fluid, a 35-mm dominant follicle, and multiple (n=7) subordinate follicles (10-20mm). To increase embryo production, oocytes were retrieved by ultrasound-guided ovum pickup of all subordinate follicles. Upon collection, oocytes (n=4) were placed in embryo holding medium for 18h at room temperature (22°C) and then matured in equine maturation medium (EqMat, Campos-Chillon et al. 2019 J. Equine Vet. Sci. 73, 51-55) for 30h at 38.5°C in 6% CO2, 5% O2, and 89% N2. Matured oocytes (n=3), as assessed by extrusion of the first polar body, underwent intracytoplasmic sperm injection with frozen-thawed semen and a Piezo-driven injection system. Injected oocytes were cultured in SCF1 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) for 5 days and Dulbecco's modified Eagle medium/F12 plus 5% FBS for 2 days at 38.5°C in 6% CO2, 5% O2, and 89% N2. On Day 10 post-foaling, the dominant follicle was 45mm and the donor mare was inseminated with fresh semen (500×106 progressively motile spermatozoa) and ovulation was induced with 1.8mg of deslorelin IM (SucroMate, Thorn BioScience LLC). The donor mare ovulated within normal limits by 48h post-induction of ovulation (12 days post-foaling) and embryo recovery was attempted 8 days post-ovulation (20 days post-foaling). The donor mare was then administered 250μg of Cloprostenol IM (Estrumate, Merck Animal Health) to induce oestrus and was inseminated again with cooled semen (1×109 progressively motile spermatozoa) for two consecutive cycles. Maturation, cleavage, and blastocyst rates were 57% (4/7), 67% (2/3), and 33% (1/3), respectively. The nonsurgical embryo recovery resulted in one expanded blastocyst grade 1. Both invitro- and invivo-produced blastocysts were transferred into synchronized recipients 5 days after oestrus on Day 16 and 20 post-foaling respectively. The donor mare got pregnant after the second cycle post-embryo recovery attempt. Pregnancy status of recipients and donor mare were evaluated at 14, 21, 30, and 50 days after embryo transfers or insemination with normal development for the three pregnancies. In conclusion, to our knowledge, this is the first case report describing invitro and invivo embryo production performed during foal heat increasing number of pregnancies in a post-foaling mare in a single cycle.

Differential Regulation of Gonadotropins in Response to Continuous Infusion of Native Gonadotropin-Releasing Hormone in the Winter Anovulatory Mare and Effects of Treatment With Estradiol-17β

We tested the hypotheses that in winter anovulatory mares (1) both chronic daily injections of estradiol-17β (E2) and subcutaneous E2 implants could enhance pituitary secretion of gonadotropins in response to continuous subcutaneous infusion of native gonadotropin-releasing hormone (GnRH); and (2) the secretory pattern of follicle-stimulating hormone (FSH) in response to continuous subcutaneous infusion of native GnRH is similar to that of luteinizing hormone (LH) but differs between mares that develop or fail to develop an estrogen-active, preovulatory follicle. In Experiment 1, 20 winter anovulatory mares (n = 5 per group) in early February received twice-daily injections of corn oil (control) or 5 mg of E2 with or without continuous subcutaneous treatment with native GnRH (100 μg/hr) or received GnRH only for up to 14 days. In Experiment 2, 24 winter anovulatory mares (n = 6 per group) were treated with a full-length (high dose) or quarter-length (low dose) E2 implant (Compudose) in combination with continuous GnRH infusion (100 μg/hr) for up to 28 days or served as sham controls. Mares developing 35-mm follicles were induced to ovulate with human chorionic gonadotropin. Mares not developing a 35-mm follicle within 14 days received a replacement 14-day GnRH pump. In Experiment 1, E2 enhanced the response to GnRH beginning on Day 3, with mean LH greater (P < .001) in GnRH + E2 than in GnRH only and control mares. In Experiment 2, plasma E2 and estrone sulfate were increased in association with the development of a large (35 mm) follicle but did not increase in response to either E2 implant despite marked increases in uterine edema following their insertion. A sustained increase (P < .0001) in plasma LH was observed in all GnRH-treated mares, but this effect was not modified by implant treatment. By Day 28, six of six GnRH, five of six GnRH + low E2, two of six GnRH + high E2, and zero of six control mares developed 35-mm follicles and were induced to ovulate. A marked increase (P < .0001) in plasma FSH was observed within 24 hours in all GnRH-treated mares, returning to baseline by Day 4. In summary, twice-daily injection of 5 mg E2 enhanced pituitary secretion of LH in response to continuous administration of GnRH, but commercial E2 cattle implants failed to duplicate these effects. Continuous infusion of GnRH produced a differential but consistent pattern of FSH secretion (short-term increase) compared with LH (sustained increase). Differences in ovarian responses to GnRH treatment could not be explained by variation in gonadotropin secretion.

Effects of Combined Estradiol-Sulpiride Treatment and Follicle Ablation on Vernal Transition in Mares: Evaluation of Plasma and Follicular Fluid Hormones and Luteinizing Hormone Receptor Gene Expression

This experiment assessed the hormonal production, secretory aspects, and changes in luteinizing hormone (LH) receptor gene expression of early induced ovulatory-sized follicles relative to the first ovulatory-sized follicles occurring naturally in the spring. Anovulatory mares were treated on January 21 with (1) 50 mg of estradiol cypionate (ECP, n = 8) alone or (2) with ECP followed by two 3-g sulpiride injections (n = 8), 5 and 12 days later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned regularly until detection of a 32–35 mm follicle; follicular fluid was recovered via aspiration for analysis of hormonal concentrations. Blood was collected regularly to characterize plasma prolactin, LH, follicle stimulating hormone, progesterone, and estradiol concentrations. Mean date to first 35-mm follicle was earlier (P < .05) in sulpiride-treated mares: five of eight (63%) responded within 28 days of the first sulpiride injection. Ablation did not affect ovarian response. Plasma prolactin was stimulated (P < .0001) in ECP-sulpiride–treated mares for 16 days but did not dictate ovarian response. Estradiol stimulated plasma LH (P < .05), which was higher (P < .05) in treated mares that responded. There was no effect of treatment or ablation on follicular fluid concentrations of estradiol, progesterone, leptin, or insulin-like growth factor 1 or on LH receptor gene expression. These latter similarities indicate that ECP-sulpiride early induced follicles have apparently reached a degree of maturity equivalent to naturally occurring ovulatory-sized follicles later in the spring.

Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches.

125 CHANGES IN INTRAFOLLICULAR CONCENTRATIONS OF FREE INSULIN-LIKE GROWTH FACTOR-1, ACTIVIN-A, INHIBIN-A, VASCULAR ENDOTHELIAL GROWTH FACTOR, ESTRADIOL, AND PRL BEFORE OVULATION IN MARES

Intrafollicular factors and hormones during the process of follicle deviation have been studied extensively in mares. However, knowledge is still lacking concerning the dominance and preovulatory (postdeviation) phases of the oestrous cycle. Furthermore, information about intrafollicular growth factors and hormones during postdeviation growth is critical for understanding the ovulatory and post-ovulatory physiological mechanisms that have important in vivo and in vitro applications, such as oocyte maturation, ovulation, and embryo development and survival. Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Hormone and growth factor concentrations were determined by ELISA or RIA. Mares (n = 30) were randomly assigned to 5 experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Data were analysed by one-way ANOVA, paired-test, and Pearson correlation. Data belonging to 2 or more proximal diameter groups that were not different were combined and regrouped for each factor separately. When the data were regrouped (all results shown below), follicular fluid-free insulin-like growth factor-1 was higher (P &lt; 0.003) in 35-mm follicles than in the 25- to 30-mm and 40-mm impending ovulation follicle groups. The 25- to 30-mm follicle group had the lowest insulin-like growth factor-1 concentration. However, concentrations of insulin-like growth factor binding protein-2 in follicular fluid did not differ (P &gt; 0.05) among groups. Additionally, follicular fluid activin-A tended (P &lt; 0.06) to be higher in impending ovulation follicles when compared to the 25- to 40-mm follicle group. Concentrations of intrafollicular oestradiol were higher (P &lt; 0.0001) in 40-mm impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin-A and vascular endothelial growth factor were lower (P &lt; 0.02–0.002) in the 40-mm impending ovulation follicle group when compared to the 25- to 35-mm follicle group. Systemic and intrafollicular PRL were lower (P &lt; 0.05) in the impending ovulation group when compared to the 25- to 40-mm follicle group. PRL concentrations were higher (P &lt; 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular free insulin-like growth factor-1, inhibin-A, vascular endothelial growth factor, and PRL during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches. Overall, the elucidation of the role of follicular fluid factors and hormones and their interactions need to be studied further to allow a better understanding of the ovulatory process in mares. This knowledge will potentially assist in obtaining better outcomes during the use of assisted reproductive technologies and will also help to understand mechanisms of ovulatory dysfunctions in mares and perhaps in other species.

Pharmacologic application of native GnRH in the winter anovulatory mare, II: Accelerating the timing of pregnancy

Onset of the winter anovulatory period in mares is associated with a marked diminution in adenohypophyseal synthesis and release of LH. Native GnRH, unlike its synthetic agonists, stimulates the synthesis and secretion of LH in mares without pituitary refractoriness. Herein we tested the hypotheses that (1) the average Julian day of pregnancy can be accelerated by up to 2 months in winter anovulatory mares treated continuously with native GnRH beginning on February 1 and (2) mares will sustain luteal function and pregnancy after treatment withdrawal. Forty-two winter anovulatory mares were stratified by age, body condition score, and size of the largest follicle across two locations in a randomized design and assigned to one of three groups (n = 14 per group): (1) Control: untreated, (2) GnRH-14: GnRH delivered subcutaneously in saline at a rate of 100 μg/h for 8 weeks (February 1–March 29) using four consecutive 14-day pumps (Alzet 2ML2), or (3) GnRH-28: GnRH delivered as in (2), but using two 28-day pumps (Alzet 2ML4). On development of a 35-mm follicle and expression of estrus, mares were bred the following day and treated with hCG. Pregnancies were confirmed using transrectal ultrasonography on Days 14, 24, 33, and 45, with blood samples collected to assess luteal function. Mares treated with GnRH (GnRH-14 and GnRH-28) did not differ reproductively in their responses and data were pooled for statistical comparisons. Mares treated with GnRH exhibited marked increases (P ≤ 0.04) in the frequency of development of a 35-mm follicle, submission rate for live cover and/or artificial insemination, ovulation, and pregnancy compared with control mares on treatment Day 56 (March 29). Interval to the first 35-mm follicle was 51.8 ± 4.9 and 19.3 ± 3.5 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively. Interval to pregnancy was 65.3 ± 6.7 and 28.6 ± 4.8 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively, excluding one GnRH-14 mare that failed to become pregnant over four cycles. By the end of the treatment period (March 29), only 21% of control mares were pregnant compared with 79% of GnRH-treated mares. Furthermore, mean serum concentrations of progesterone were similar to (GnRH-28; P = 0.26) or greater than (GnRH-14; P = 0.01) that of control mares from Day 0 to 46 postbreeding. Data illustrate that continuous administration of native GnRH is a highly efficient option for managing seasonal anovulation in mares and could be effectively used in the breeding industry if a user-friendly delivery option were available.

12 FERTILITY OF FROZEN EQUINE SPERM IN SYSTEMS FOR CRYOPRESERVATION

Optimizing cryopreservation of equine sperm will facilitate genetic banking and propagation of important horse strains through assisted reproduction. This study aimed to evaluate the motility pattern using computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen frozen in 0.5 mL straws at different freezing rates; also, a fertility trial was performed according to the freezing protocol. Three ejaculates from four stallions of various breeds (Mangalarga Marchador, Westfallen, Hanovarian and Arabian) and ages (5 to 20 years) were collected and processed for cryopreservation. The stallions were housed at the CERBEQ, Reproduction Centre of the Department of Animal Reproduction and Veterinary Radiology, UNESP. The ejaculates were filtered and submitted to analysis by CASA (HTM IVOS 12, Hamilton Thorne Research, USA). In addition, the plasma membrane integrity was determined by fluorescent probes. After evaluation, the ejaculates were diluted at 1:1 (extender:semen) with skim milk extender Botu-Semen™ and centrifuged at 600 × g for 10 min. The supernatant was removed and the pellet resuspended to a final concentration of 100 × 106 sperm mL–1 with milk-egg yolk freezing extender (Botu-Crio™). Semen was packaged in 0.5-mL straws (IMV, LAigle, France) and was placed in nitrogen for 20 min and then from room temperature to 5°C and then frozen in two different cooling systems: an isothermic box (42 cm × 28 cm × 12.5 cm) was placed upon racks suspended 6 cm above liquid nitrogen or other 20 min then immersed into nitrogen and automated system Mini Digitcool™ (IMV Technologies, France), cooling at a –40°C min–1 rate. All straws were stored in liquid nitrogen until thawing and analysis. The straws were thawed in a water bath at 46°C for 20 s and the samples were evaluated for progressive motility, angular progressive velocity, progressive velocity, track speed, percentage of rapid sperm and percentage of sperm with plasma membrane integrity. For the fertility trial, 65 clinically healthy mares had their oestrous cycle monitored by ultrasound and inseminated postovulation with sperm into the uterus. Ovulation was induced with 1 mL of deslorelin acetate (GnRH) injected IM when a 35-mm follicle was detected. Thirty-six hours later, mares were monitored every 6 h until ovulation was detected. When it was detected, mares were inseminated with 800 × 106 total sperm. Pregnancy was confirmed via ultrasound examination 15 days after ovulation. Pregnancy rate was 52.2% using the isothermic box and 60% using the automated machine. Statistical analysis from the frozen–thawed semen evaluated parameters was performed using the statistics software Proc. MIXED of SAS 9.1 and for the fertility trial, logistic regression using the Proc GENMOD from SAS 9.1. The conventional method using the isothermic box was similar to the automated machine with a fast freezing rate. Additionally, AI with 800 × 106 sperm frozen in the isothermic box or automated system resulted in similarly acceptable conception rates.

Embryo Transfer Efficiency of Quarter Horse Athletic Mares

This study was designed to compare embryo recovery rates and pregnancy rates of athletic and breeding Quarter Horse mares in a tropical warm climate. Thirty-nine barrel racing mares in training and 135 breeding mares as control donors were included. During the training period, the ambient temperature ranged from 31°C to 36°C and the average humidity from 70% to 90%. After the detection of a 35-mm follicle by ultrasound, ovulation was induced with 1 mg of deslorelin acetate (i.m), and insemination was performed 24 hours later with cooled and fresh semen from different fertile stallions. Embryos were collected on day 8 postovulation. The body temperature (rectal) was evaluated from eight athletic donor mares randomly selected from the same studied group. A total of 138 and 657 embryo collections were carried out on training and breeding mares, respectively, with a total of 105 (76%) and 466 (71%) embryos collected ( P > .05). Similarly, no differences ( P > .05) were observed for the pregnancy rates on day 15 (82/105, 78% vs. 370/466, 79%), and day 40 (73/105, 69% vs. 328/466, 70%) between the training and breeding donor mares. Just after training, the body temperature increased to an average of 39.4°C and the respiratory rate from 14.5 to 35.3 breaths per minute. The results of the present study showed that embryo production from appropriately trained donor mares in good condition were similar to non-athletic broodmares.

175 REPRODUCTIVE PERFORMANCE OF MANGALARGA MARCHADOR MARES AFTER INDUCTION OF OVULATION WITH hCG OR DESLORELIN

The use of biotechnology, such as AI, embryo transfer, and hormonal protocols, have accelerated the genetic improvement in the equine specie, and inducers of ovulation, such as hCG, are important tools in implementing them. However, the repeated use of ovulation inducers can promote the development of antibodies and undermine their purpose. Thus, a synthetic GnRH, deslorelin, has been used. In this study, we compared the reproductive performance of 44 Mangalarga Marchador donor mares, aged between 5 and 21 years and with sound reproductive history after treatment with hCG or deslorelin. During estrus, mares were examined by transrectal ultrasonography every other day until a follicle of 30 mm in diameter was detected and then daily until detection of a &gt;35-mm follicle and moderate uterine edema. At this time, mares were treated with either 2 mL of saline, i.m. as controls (CON = 16); 2500IU of hCG, i.v. (Chorulon, Intervet Inc., Millsboro, DE, USA; hCG = 14); or 2 mg mL-1 deslorelin in BRT vehicle (BET Pharm, Lexington, KY, USA), i.m. (DES = 14). After treatments, mares were examined at 12-h intervals until ovulation and were inseminated with fresh semen 12 h after ovulation. On Day 8, embryos were recovered and evaluated. All mares were ovulated, and follicle diameters were not different (P &gt; 0.05) at the time of treatment (38 ± 0.029 v. 39 ± 0.029 v. 38 ± 0.029 mm). Embryo recovery was similar among groups (CON, 37.5%; hCG, 42.8%; DES, 50.0%, respectively). The interval from treatment to ovulation did not differ (P &gt; 0.05) between hCG (1.43 +0.56 days) and DES mares (1.14 + 0.36 days), and both were less (P &gt; 0.05) than for controls (CON, 2.94 + 0.77 days). The number of mares that ovulated per ovulation interval were as follows: &lt;24 h = CON: 0%, hCG: 7.14%, and DES: 14.29%, respectively; from 24 to 48 h = 6.25%, 64.29%, and 78.57%; from 49 to 72 h = 37.5%, 28.57%, and 7.14%; &gt;72 h = 56.25%, 0%, and 0%. Time to ovulation was negatively correlated (P &lt; 0.05) with follicle diameter in CON (r = -0.50, P &lt; 0.05) but not for hCG (r = 0.05) or DES (r = 0.022) and was positively correlated with embryo recovery for DES (r = 0.46, P &lt; 0.10), but not hCG (r = 0.23) or CON (r = 0.09). Based on the results, we concluded that hCG and DES effectively synchronize the time of ovulation. Follicle size did not affect the time of ovulation in hCG- and DES-treated mares. We thank the Farm Water supply for the animals and drugs.

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19 degrees 45'45'S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post-ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35-mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35- to 40-mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively--one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low-dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.

Effect of intrauterine treatment with prostaglandin e 2 prior to insemination of mares in the uterine horn or body

Two trials were conducted to investigate the effects of intrauterine infusion of PGE 2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 million total spermatozoa from either a stallion with good semen quality (Trial 1) or poor semen quality (Trial 2). In each trial, mares were assigned to 1 of 4 treatment groups as follows: Group PGE— infusion of 0.25 mg PGE 2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; Group PGE-BI — infusion of 0.25 mg PGE 2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body; Group SAL-HI — infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; or Group SAL-BI — infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body. After breeding, mares were examined daily by transrectal ultrasonography to confirm ovulation, and were re-examined 14 to 16 d after ovulation for pregnancy status. Data were analyzed by Chi-square. Overall pregnancy rates were 59% for stallion 1 and 29% for stallion 2. Group pregnancy rates did not differ for mares bred by either stallion (P > 0.10). Pregnancy rates were not altered by horn insemination for either stallion (P > 0.10). Intrauterine infusion of PGE 2 improved pregnancy rate in mares bred by the stallion with good quality semen (P < 0.05), but did not alter pregnancy rate in mares bred by the stallion with poor quality semen (P > 0.10). Further research is warranted to determine if intrauterine infusion of PGE 2 will enhance spermatozoal colonization of the oviduct and pregnancy rates in mares, and if PGE-treatment will improve pregnancy rates achieved by subfertile stallions

Efficacy of short-term administration of altrenogest to postpone ovulation in mares

Estrogen from a growing follicle stimulates the preovulatory surge of luteinizing hormone (LH) while progesterone (P) is known to suppress LH. The possibility exists that administration of P, in the presence of an ovulatory follicle, would sufficiently suppress LH and, therefore, delay ovulation. The objective of this research was to elucidate the potential for oral administration of altrenogest (17-Allyl-17β-hydroxyestra-4,9,11-trien-3-one) to postpone ovulation of a preovulatory follicle (35 mm) for approximately two days. Fourteen light-horse mares, ranging in age from two to 19 years, were randomly assigned to one of three treatments (A-.044 mg/kg BW altrenogest for two days; B-.088 mg/kg BW altrenogest for two days; and C- no altrenogest). Mares began treatment when a 35-mm or greater follicle was observed via real-time transrectal ultrasonography. Both number of days until ovulation and follicular maintenance differed between treated and control mares. Number of days until ovulation was increased (P<.05) for mares in treatment A when compared with the control mares. Follicular diameter maintenance, a measurement of follicular diameter throughout treatment, also increased (P<.05) for mares in treatment A when compared with the control mares. Mean LH concentration was not different between mares treated with altrenogest at either treatment dose when compared with the control mares. Pregnancy rates and embryonic vesicle size change were also measured to determine potential effects of altrenogest administration. No differences (P>.05) were found in either characteristic. Short-term administration of altrenogest increased the number of days to ovulation. Further study is warranted to prove conclusively that altrenogest increases follicular maintenance, alters the preovulatory LH surge, and has no detrimental effects upon reproductive efficiency.

Effects of gonadotropin-releasing hormone infused in a pulsatile or continuous fashion on serum gonadotropin concentrations and ovulation in the mare.

Studies were conducted to compare continuous vs pulsatile i.v. infusion of GnRH on serum gonadotropin concentrations and ovulation in seasonally anestrous mares and in cycling mares. Anestrous mares (Exp. 1) received no treatment (control; n = 3), 2, or 20 micrograms of GnRH/h continuous infusion (CI) (n = 4 and n = 6, respectively), or 20 micrograms of GnRH/h pulsatile infusion (PI) (n = 5). After initiation of GnRH infusion, serum LH levels increased earlier, and to a greater extent, in the PI group than in other groups (P less than .05). In contrast, serum FSH concentrations did not differ among groups. The number of days to development of the first 35-mm follicle was not different among GnRH treatment groups; however, mares receiving PI ovulated on d 9.4 of treatment, 2.8 d earlier than those receiving 20 micrograms of GnRH/h CI (P less than .05). Mares given 2 micrograms of GnRH/h CI failed to ovulate spontaneously after 16 d of treatment, but each one ovulated within 2 to 4 d after injection of 2,000 IU of hCG on d 16. Control mares did not ovulate or show any significant follicular development throughout the experiment. Cycling mares (Exp. 2) received no treatment (control; n = 6), 20 micrograms of GnRH/h CI, or 20 micrograms of GnRH/h PI (n = 4) beginning on d 16 of an estrous cycle (d 0 = day of ovulation). Serum LH concentrations in all groups increased after initiation of treatment; however, on the day of ovulation LH concentrations were lower in the CI group than in the PI or control groups (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of chemotactic factors in preovulatory follicular fluid from mares

Summary Ovulation has been likened to an inflammatory process. Inflammatory cells accumulate in the ovulating follicle, presumably because of chemotactic factors. Chemotactic activity was measured in fluid aspirated from follicles of estrous mares 0, 12, 24, and 36 hours after ultrasonographic detection of a 35-mm follicle and iv treatment with 2,500 IU of human chorionic gonadotropin. Chemotaxis was assessed by measuring directional migration of equine neutrophils under agarose. Follicular fluid acted as a chemoattractant for neutrophils, but there was no significant difference in chemotactic activity among different time intervals after administration of human chorionic gonadotropin. On the basis of results of various treatments, chemotactic properties of serum and follicular fluid were similar. Chemotactic activity was significantly reduced by heating (56 C for 30 minutes) and by trypsinization and was virtually removed by charcoal treatment. Dialyzing the follicular fluid (3,500 and 8,000 molecular weight cut-off) significantly reduced the chemotactic activity of follicular fluid and serum. The importance of chemotactic factors in the process of ovulation in the mare is yet to be established.

Effect of aspiration of follicular fluid on subsequent luteal function in the mare

Ultrasonography was performed daily on mares during estrus to assess follicular growth. All mares received 2,500 IU human chorionic gonadotropin (hCG) intravenously when the preovulatory follicle reached 35 mm in diameter. Follicular fluid was aspirated when a 35-mm follicle was detected (0 h) or 12, 24, or 36 h later. Blood samples were collected daily from the mares until they returned to estrus. When data were pooled for early 0 + 12 h) or late (24 + 36 h) aspirations, progesterone concentrations were lower on Days 2 (P < 0.01), 3 (P < 0.01), 4 (P < 0.05), and 5 (P = 0.1) in mares in which follicular fluid had been aspirated early. However, when progesterone concentrations were compared relative to administration of hCG, time of aspiration did not affect subsequent luteal function.

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20°C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20°C or 3) insemination with semen stored for 24 h at 5°C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 × 10 6 progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20°C or from semen stored for 24 h at 5°C were the same ( 11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 ± 2.9, 19.6 ± 2.6 and 20.5 ± 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 ± 2, semen stored for 24 h at 20°C was 57 ± 11 and semen stored for 24 h at 5°C was 80 ± 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5°C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20°C than that stored at 5°C.

Comparison of pregnancy rates achieved with frozen semen using two packaging methods

The pregnancy rate achieved with frozen semen using two packaging methods was compared in two separate experiments. The sperm-rich fraction of the semen was collected and immediately extended in an egg-yolk based extender to a final concentration of 100 × 10 6 sperm/ml. Half of each ejaculate was packaged in 4.0-ml plastic straws and the other half in 10-to 12-ml flattened aluminum tubes. After detection of a 35-mm follicle, mares were inseminated daily until ovulation. Pregnancy was detected, and embryonic vesicle size was measured by ultrasonic echography Days 14 to 26 after ovulation. The one-cycle pregnancy rate achieved using both packaging methods was 50% overall ( 23 46 ), with 46% ( 12 26 ) and 55% ( 11 20 ) rates achieved, respectively, using straws and aluminum tubes. These rates were not significantly different.

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20°C

The effects of extender and storage at 20°C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 × 10 6 progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37°C with insemination in <1 h; b) E-Z Mixin extender at 37°C with insemination in <1 h; and c) E-Z Mixin extender at 37°C with cooling to 20°C and insemination after storage for 12 h at 20°C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37°C with insemination in <1 h or b) E-Z Mixin extender at 37°C with cooling to 20°C and insemination after storage for 24 h at 20°C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20°C for 24 h without a marked decline in fertility.