S.S. King

Prolactin may play a role in stimulating the equine ovry during the spring reproductive transition

Summary Plasma prolactin and follicular development increased significantly between the 2nd and 5th weeks of March. During this time, follicular diameters were highly correlated with plasma prolactin (r=.323, dr=64, p=.008). There was no significant rise in LH during the experimental period (lst week in Jan to the 5th week of March). Plasma prolactin was increased by blocking dopamine receptors (fluphenazine decanoate, 178.6 µg/kg, im, once every 21 days) or by a single injection of ovine prolactin (200 mg/mare, iv) given during the 1st week in January. Fluphenazine accelerated follicular growth when compared to control mares but did not result in early ovulation. Exogenous ovine prolactin caused growth of ovulatory size follicles within 3 days in 3 out of 5 treated mares. One of these mares went on to ovulate 9 weeks following injection of prolactin. The remaining 2 mares continued to produce waves of follicular growth and rcgression until they ovulated, coincident with the control group. The present studies suggest that when endogenous prolactin secretion is increased during anestrus either by dopamine receptor blockade or by administering exogenous prolactin, typical transitional follicular growth commences.

Differential luteolytic function between the physiological breeding season, autumn transition and persistent winter cyclicity in the mare

There is a well-documented increase in luteolytic failure, resulting in spontaneously prolonged corpus luteum (SPCL) function, during estrous cycles of horses in autumn. The cause of this phenomenon may be due to seasonal alterations in PGF(2alpha) and/or in prolactin (PRL) secretion around luteolysis. To investigate this, progesterone (P4), 13, 14-dihydro, 15-keto PGF(2alpha) (PGFM) and PRL concentrations were compared between summer and autumn estrous cycles during natural luteolysis and luteolysis induced by benign uterine stimulation. A single estrous cycle from mares in June-July (n=12) was compared to multiple estrous cycles from these 12 mares plus 8 additional mares in September through December. Reproductive behavior was monitored by bringing a stallion in close proximity to the mare and ovarian events by ultrasonography. Blood was collected via jugular cannula every 6h from d 13 to 17 post-ovulation in untreated control mares (n=8 summer, n=9 autumn). In treated mares, blood collection occurred at 0, 15, 30, 45, 60, 90, 120, 180 and 240min followed by 6h intervals for a total of 5d following intrauterine saline infusion on d 7 (n=4 summer, n=11 autumn). Mares failing to return to estrus for 30d received intrauterine saline and the described intensive blood sampling protocol on d 30. Progesterone and PRL were determined on daily samples and PGFM on frequent plasma collections by RIA. Duration of ovarian luteal and follicular phases, P4 and PRL concentrations and PGFM secretion around luteolysis were compared between treatments and seasons by ANOVA. Mean P4 declined from June to December in all groups. Pulses of PGFM were detected on d 13-17 in controls and d 7-11 in saline-infused mares. Pulse patterns were not different between groups. The incidence of SPCL increased during autumn in the control group. PGFM pulses were absent on d 13-17 in mares with SPCL, but PGFM pulses could be induced in these mares by saline infusion at d 30. Autumn PGFM profiles were unchanged during spontaneous or saline-induced luteolysis compared with summer. Circulating PRL increased around natural or induced luteolysis. These results provide evidence that changes in luteal function during the autumn transition are not the result of alterations in the ability of the uterus to produce PGF(2alpha) nor due to changed CL sensitivity to PGF(2alpha). We conclude that seasonal changes in luteolytic function are caused by an alteration in the signal for PGF(2alpha) release.

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.

In vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method

The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 μm) compared to the control group (37.3 ± 0.7 μm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 μm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 μm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 μm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 μm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.

Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique.

The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P < 0.05) morphologically normal follicles than histology. For experiment 2, preantral follicles (N = 332) were analyzed and primordial and transitional (combined) follicles and oocytes were 36.3 ± 0.3 and 26.1 ± 0.3 μm in diameter, respectively, and primary follicles and oocytes averaged 42.9 ± 1.8 and 31.8 ± 2.1 μm. For experiment 3 (188 preantral follicles), within the same animals, the proportion of primordial versus primary follicles was higher (P < 0.03) for histological analysis (98%) compared to tissue chopper analysis (94%), and number of follicles per mg of tissue was not affected (P > 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.

Time of onset and ovarian state prior to entry into winter anestrus

Abstract The reproductive activity of 18 light horse mares was followed from September to May, 1989 and 1991. Five mares were evaluated from July to August, 1991. All mares were teased three times per week. Ovarian size, follicular growth, ovulation and corpus luteum development was followed by ultrasonography daily during estrus and once weekly during diestrus/anestrus. Progesterone (P4) was measured by RIA on plasma drawn daily or weekly. Entry into anestrus was considered complete if a mare did not exhibit cyclic estrous behavior, had no follicles > 25 mm, and maintained P4

Comparison of circulating estradiol-178 and folliculogenesis during the breeding season, autumn transition and anestrus in the mare

Abstract One of the phenomena characteristic of the vernal transition into cyclicity in the mare is the variability between follicular size and concomitant estradiol secretion. Researchers -1 have suggested that early transitional follicles of the spring may be estrogenically incompetent, gaining the ability to synthesize estrogens only immediately prior to the first ovulation of the year. It is unknown if a similar phenomenon is operating during the autumnal transition due to limited study of this period. Therefore, the associaiion between follicular development and plasma estradiol concentrations in mares during their progress into anestrus was compared to the same relationships during the breeding season. Estrous behavior, plasma estradiol-17β (E2) and progesterone (P4) concentrations, and follicular growth of 11 horse mares was evaluated from September 6 through December 31, 1989. For comparison, five summer cycling mares were monitored from July 10 through August 13, 1991. Size of the largest follicle > 25 mm in diameter during estrus was compared by regression and correlation analysis with daily plasma E2 concentrations for summer and fall cycles. Smaller E2 concentrations/mm follicular size were evident during the fall compared with summer. Correlations were higher (p

Inhibition of bacterial endometritis with mannose

Summary Prior research indicates that mannose is capable ofpreventing bacterial adherence to equine endometrial tissues in vitro. The present study was designed to test whether mannose would prevent attachment of pathogenic Escherichia coli (E. coli) to equine endometrium in vivo. Six estrogen-treated anestrous mares received intrauterine infusions of 100 mL E. coli (10 9 cells) in PBS or E. coli +50 mg mannose/mL PBS. Uterine fluid height and echogenicity were measured at 0,24 and 48 hours following infusion. Intrauterine cultures and endometrial biopsies were obtained at 24 hours post-infusion. Cultures were plated for bacterial growth and identification. Biopsies were prepared for light microscopy and evaluated for bacterial attachment to luminal epithelium. Mares receiving E. coli alone accumulated more intrauterine fluid at 24 hours than the E. coli + mannose treated group. Uterine fluid echogenicity was not different between groups, but did increase at 24 hours. Percent luminal epithelial cells with bacteria attached did not correlate with treatment. Incidence of positive culture at 24 hours was significantly reduced in the mannose-treated group. Preliminary evidence suggests that mannose may be effective in reducing bacterial infection in the equine endometrium.

Effects of arachidonic acid and oxytocin on equine endometrial PGF2α during normal cycles and pseudopregnancy

Summary The effect of excess arachidonic acid or oxytocin on equine endometrial prostaglandin F 2α (PGF 2α ) synthesis was measured in vitro under physiologic and pathophysiologic conditions. Endometrial tissues obtained by uterine biopsy at 5, 10, 12, 14, 16 and 20 days post-ovulation from cycling mares, after 0, 2, 4, 6, 8, 10, 12, 14 or 16 days of progesterone (P 4 ) in ovariectomized mares and at 30 days postovulation in mares undergoing spontaneously prolonged corpus luteum (SPCL) activity were incubated in vitro with and without added arachidonic acid or oxytocin. Endometrial PGF 2α content and synthetic capacities were determined by radioimmunoassay. PGF 2α production increased significantly at Days 12–16. Arachidonic acid did not alter this effect. Oxytocin stimulated additional PGF 2α production on Days 5, 16, and 20. SPCL tissues had minimal PGF 2α production which was increased significantly by arachidonic acid but not oxytocin. PGF 2α synthesis in ovariectomized P 4 treated mares was minimal and did not vary with length of progesterone exposure or addition of arachidonic acid. These results suggest that a) oxytocin may play a role in luteolysis in the equine, b) although arachidonic acid appears not to be limiting to PGF 2 α production under normal physiological conditions, its absence may play a role in pathophysiological conditions, c) factors in addition to progesterone and arachidonic acid are required to initiate PGF 2α synthesis in the mare.

Uterine secretions from different endometrial classifications affect the viability of early murine embryos cultured in vitro

Abstract An interspecies (murine-equine) bioassay system was employed to investigate the effect of equine endometrial secretions on embryonic survival. Uterine fluid was obtained from 15 diestrous mares by lavage with 150 ml of sterile Whittens medium. Samples were grouped according to their Kenney score for endometrial type. Samples were sterilized and protein concentration was standardized to 4 mg/ml protein with BSA. Controls consisted of either 4 mg/ml BSA in Whittens medium or 50% BSA-50% heat-treated equine serum. One hundred 2-cell mouse embryos were cultured in each sample and controls. Embryonic development was evaluated for 5 days and was expressed as the percent reaching the 4-cell, 8-cell, morula, blastocyst, expanded blastocyst, and hatched blastocyst stages. Embryonic development in Type I mare uterine fluid did not differ from controls. Development to the 4 cell, 8 cell, morula, blastocyst and expanded blastocyst stages in the Type II mare media was less (p .05) between endometrial types. Lymphocyte numbers were significantly different (p