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Comparison of serial prostaglandin F2α protocols for control of the estrous cycle in mares
Journal of Equine Veterinary Science 52 (2017) 110e113
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Reproductive Physiology Oral Presentations 161 Addition of carnitine and pyruvate to commercial stallion semen extenders A. Safley, G. Webb* Missouri State University, Springfield, MO, USA
stored in equine express containers was higher than that of spermatozoa stored in either BOTU or VMD-Z. Progressive motility of spermatozoa stored in SKMG was higher than that of those stored in VMD-Z. When stored at 10 C, motility of spermatozoa diluted in VMD-Z was significantly lower (P < 0.05) than that of spermatozoa stored in INRA (P < 0.05). Key Words: stallion, carnitine, pyruvate
Recently the inclusion of carnitine and pyruvate in semen extenders has been shown to improve post storage motility of stallion spermatozoa. Three experiments were conducted to determine if inclusion of these 2 compounds in commercially available diluents would be beneficial. In each experiment, 3 stallions were collected 3 times for a total of 9 ejaculates per experiment. In experiments 1 and 2, following collection, aliquots were diluted in test extenders and evaluated for motility after 2, 24, 48 and 72 h of storage in Equine Express containers (Exodus Breeders Supply, York, PA). When storage time exceeded 24 h coolant packs were changed daily. In the third experiment (EXP 3) aliquots were also stored in refrigerated cooler 10 C for the same amounts of time. Separate aliquots were stored for evaluation at each time. Diluted samples were evaluated for motility, velocity and pattern of movement using computer assisted sperm analysis (CASA). Data were analyzed using the GLM procedure of ANOVA across all stallion/ejaculate/times for extender effects. Ejaculate was nested within stallion and time was nested within ejaculate. Pairwise comparisons were made with Tukey’s test. For experiment 1 (EXP1) aliquots were diluted with INRA 96 (control), skim milk glucose (SKMG), SKMG + carnitine (C), SKMG + pyruvate (P) or SKMG + both (CP). Carnitine was added at 0.00806 g/mL and pyruvate was added at 0.0011004 g/mL. Post storage total and progressive motility and velocity were higher (P < 0.05) for spermatozoa diluted in INRA as compared with any other diluent. In experiment 2 (EXP 2) aliquots were diluted in INRA 96 (control) and BOTU semen (BOTU, Exodus Breeders Supply, York, PA) or VMD-Z (Reproduction Resources, Walworth, WI) extenders. Each of the latter extenders were also tested with C, P and CP added. In EXP2, there was no advantage to adding, C, P, or both to either VMD-Z or BOTU. In experiment 3 (EXP3) INRA, SKMG, BOTU and VMD-Z were tested with and without CP added. Within base extender (BOTU, INRA, SKMG, VMD-Z) inclusion of CP did not improve post-storage motility of stallion spermatozoa stored in either Equine Express II containers or at 10 C (P < 0.05). Between extender groups, total and progressive motility and velocity of spermatozoa diluted in INRA and
0737-0806/$ e see front matter
Graduate Student Competition 162 Comparison of serial prostaglandin F2a protocols for control of the estrous cycle in mares K. Potter*, G. Webb, W. Boyer Missouri State University, Springfield, MO, USA Prostaglandin analogs are commonly administered to manipulate the estrous cycle of mares. Previous reports have indicated that serial administration of prostaglandin F2a (PGF2a) beginning on the day of ovulation will prevent CL development and in turn decrease the interval from ovulation to ovulation. The standard dose of dinoprost tromethamine (Lutalyse) is 10 mg per mare, administered IM. It has been reported that a lower dose is not only effective but results in less side effects. Therefore, objectives of the current study were to compare 3 PGF2a treatment protocols utilizing a lower dose and to determine the most effective treatment in terms of number of injections, intensity of protocol, time allocated, and labor. Eleven Quarter Horse mares were used for the study, in a crossover design with each mare subjected to all treatments. A dose of 1.1 mL was used to insure that mares received at least 5 mg of dinoprost tromethamine per injection. Treatment protocols were as follows: (1) PGF2a administered once on d 6 post-ovulation; (2) PGF2a administered twice daily on d 0, 1, and 2, then once on d 3 and 4 post-ovulation; (3) PGF2a administered twice daily on d 2, then once on d 3 and 4 post-ovulation. Teasing scores were assigned daily. Rectal palpation and ultrasound were performed to measure follicular growth and detect ovulation. Blood samples were collected on d 3 and 6 postovulation to analyze changes in plasma progesterone (P4) levels. Minitab 17 software was used for data analysis. Plasma P4 values were not statistically different between treatments on d 3 and 6 post ovulation. However, P4 numerically increased within treatment from 3 d to 6 d for treatment 1, and decreased between those days for the other treatments. Mean intervals from ovulation to ovulation for treatments 1, 2, and 3 were 14.8 d ± 3.1, 12.9 d ± 6.3 d and 14.3 d ± 1.4 respectively. Mean intervals from first day of
Abstracts / Journal of Equine Veterinary Science 52 (2017) 110e113
treatment to ovulation for treatments 1, 2, and 3 were 9.8 d ± 2.0, 12.9 d ± 6.3, and 12.6 d ± 1.7. Although not statistically different, these results agree with a previous study that utilized a dose of 10 mg dinoprost tromethamine. One mare on treatment 2 displayed estrus and maintained follicle > 30 mm for 13 d and had an intraovulatory interval of 29 d. Also, one mare on treatment 3 failed to return to estrus or develop a follicle >30 mm. Further research is needed to determine if manipulation of serial dosages and timing of treatment will result in results that are more consistent. Key Words: mare, prostaglandin, estrus synchronization
163 Evaluation of phytoestrogens in equine feeds and broodmare serum A.M. Adkin*, L.K. Warren, C. Silva-Sanchez, C.J. Mortensen University of Florida, Gainesville, FL, USA Phytoestrogens are a family of polyphenolic plant compounds that bind estrogen receptors and negatively impact female reproduction. Research in several species has demonstrated that dietary phytoestrogens can interrupt female cyclicity and may induce a sub-fertile state; however limited data exists on the amount of phytoestrogens in equine feeds and ultimately, the circulating concentrations within the mare. The objectives of this study were to: 1) quantify specific phytoestrogens in commonly fed concentrate ingredients, hay and legumes in the equine diet and 2) quantify serum phytoestrogen concentrations in broodmares. Feed samples analyzed in this study included alfalfa hay, perennial peanut hay, bahiagrass pasture, Coastal bermudagrass hay, soybean meal, soy hulls, and grain concentrate. Monthly venous blood samples were collected from 8 (mean ± SE, 530 ± 19 kg) mature non-pregnant stock breed mares for 3 mo (JanuaryeMarch). Basal diets of the mares included grain concentrate and ad libitum bahiagrass pasture and Coastal bermudagrass hay. All feeds and serum samples were analyzed for phytoestrogen composition using targeted high performance liquid chromatography-multiple reaction monitoring mass spectrometry to determine genistein, daidzein, genistin, daidzin, glycitin, formononetin, biochanin A, and coumestrol concentrations. Data were analyzed using one-way ANOVA or a mixed model ANOVA with repeated measures. All phytoestrogens evaluated were detected in soy products, but only coumestrol, genistein, and genistin were detected in legume and grass samples. Due to low concentrations in feeds, phytoestrogens were combined to compare total phytoestrogen concentration between feeds. Soybean meal was highest (P < 0.0001) in total phytoestrogens followed by soy hulls, grain concentrate, alfalfa hay, bahiagrass pasture, perennial peanut hay and Coastal bermudagrass hay. Total phytoestrogen concentration was higher in soybean meal (P < 0.0001) compared with soy hulls, and higher in alfalfa (P < 0.0001) compared with perennial peanut hay and Coastal bermudagrass hay, which contained negligible amounts. Concentrations of total phytoestrogens in bahiagrass pasture peaked in February with only trace amounts detected in January and March. Most phytoestrogens evaluated were not within detectable limits in mare serum. Genistein was the predominant phytoestrogen detected in serum, and was highest (P < 0.05) in March. Results confirm that equine feeds contain phytoestrogens which are
111
reflected in serum concentrations; however, further studies are needed to link dietary phytoestrogens to the reproductive health of broodmares. Key Words: genistein, coumestrol, horse
164 Effects of combined estradiol cypionatesulpiride treatment and follicle ablation on vernal transition in mares: Evaluation of plasma and follicular fluid hormones E.L. Oberhaus*1, D.L. Thompson, Jr. 1, C.R. Pinto 2, B.A. Foster 1, N. Arana-Valencia 1 1 Louisiana State University Agricultural Center, Baton Rouge, LA, USA; 2 Louisiana State University School of Veterinary Medicine, Baton Rouge, LA, USA This experiment was designed to assess the hormonal production and secretory aspects of early-induced ovulatory-sized follicles to those naturally occurring in the spring. Seasonally anovulatory mares were treated on Jan 21 with (1) 50 mg estradiol cypionate (ECP, n ¼ 8) alone or (2) with ECP on Jan 21 followed by 2 sulpiride injections (3 g, s.q., in vegetable shortening, n ¼ 8) 5 and 12 d later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned via ultrasonography regularly until detection of a 32e35 mm follicle; follicular fluid was then recovered via aspiration and analyzed for prolactin, estradiol and progesterone. Blood was collected every 4 d to characterize prolactin, LH, FSH and estradiol. Also, on the day of aspiration and for 6 more days, blood was collected to characterize LH and progesterone. Mean date to first 35-mm follicle was advanced (P < 0.05) in sulpiride-treated mares: 5 of the 8 (63%) responded within 28 d of the first sulpiride treatment. Complete follicle ablation had no effect (P > 0.05) on the ovarian response. Plasma prolactin was stimulated (P < 0.0001) in ECP-sulpiride treated mares for 16 d. There was no difference (P > 0.05) in plasma prolactin in mares that responded versus mares that did not. Estradiol cypionate stimulated plasma LH in all mares (P < 0.05) and ablation had no effect (P > 0.05) on LH response to ECP. Plasma LH was higher (P < 0.05) in treated mares that responded compared with mares that did not respond on d 8 and 12 post sulpiride treatment. In plasma samples collected daily after aspiration, both LH and progesterone were higher (P < 0.01) in mares that were treated with ECP-sulpiride and were not ablated compared with ECP-only treated mares (both ablated and not) and to ECP-sulpiride treated mares that were ablated. Plasma estradiol was similar (P > 0.05) in all mares on day of aspiration. There was no effect (P > 0.05) of treatment or ablation on follicular fluid prolactin, estradiol or progesterone. In conclusion, combined ECP-sulpiride treatment stimulated circulating prolactin and hastened the date to first preovulatory follicle, but degree of prolactin response was not a predictor of ovarian response. Sulpiride-treated mares that were not ablated had elevated plasma progesterone and LH after aspiration. Ablation may inhibit maturity of the first preovulatory follicle based on decreased progesterone and LH production, but does not inhibit growth and development of the first preovulatory follicle. Key Words: seasonal transition, prolactin, sulpiride
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Reproductive Physiology Oral Presentations 161 Addition of carnitine and pyruvate to commercial stallion semen extenders A. Safley, G. Webb* Missouri State University, Springfield, MO, USA
stored in equine express containers was higher than that of spermatozoa stored in either BOTU or VMD-Z. Progressive motility of spermatozoa stored in SKMG was higher than that of those stored in VMD-Z. When stored at 10 C, motility of spermatozoa diluted in VMD-Z was significantly lower (P < 0.05) than that of spermatozoa stored in INRA (P < 0.05). Key Words: stallion, carnitine, pyruvate
Recently the inclusion of carnitine and pyruvate in semen extenders has been shown to improve post storage motility of stallion spermatozoa. Three experiments were conducted to determine if inclusion of these 2 compounds in commercially available diluents would be beneficial. In each experiment, 3 stallions were collected 3 times for a total of 9 ejaculates per experiment. In experiments 1 and 2, following collection, aliquots were diluted in test extenders and evaluated for motility after 2, 24, 48 and 72 h of storage in Equine Express containers (Exodus Breeders Supply, York, PA). When storage time exceeded 24 h coolant packs were changed daily. In the third experiment (EXP 3) aliquots were also stored in refrigerated cooler 10 C for the same amounts of time. Separate aliquots were stored for evaluation at each time. Diluted samples were evaluated for motility, velocity and pattern of movement using computer assisted sperm analysis (CASA). Data were analyzed using the GLM procedure of ANOVA across all stallion/ejaculate/times for extender effects. Ejaculate was nested within stallion and time was nested within ejaculate. Pairwise comparisons were made with Tukey’s test. For experiment 1 (EXP1) aliquots were diluted with INRA 96 (control), skim milk glucose (SKMG), SKMG + carnitine (C), SKMG + pyruvate (P) or SKMG + both (CP). Carnitine was added at 0.00806 g/mL and pyruvate was added at 0.0011004 g/mL. Post storage total and progressive motility and velocity were higher (P < 0.05) for spermatozoa diluted in INRA as compared with any other diluent. In experiment 2 (EXP 2) aliquots were diluted in INRA 96 (control) and BOTU semen (BOTU, Exodus Breeders Supply, York, PA) or VMD-Z (Reproduction Resources, Walworth, WI) extenders. Each of the latter extenders were also tested with C, P and CP added. In EXP2, there was no advantage to adding, C, P, or both to either VMD-Z or BOTU. In experiment 3 (EXP3) INRA, SKMG, BOTU and VMD-Z were tested with and without CP added. Within base extender (BOTU, INRA, SKMG, VMD-Z) inclusion of CP did not improve post-storage motility of stallion spermatozoa stored in either Equine Express II containers or at 10 C (P < 0.05). Between extender groups, total and progressive motility and velocity of spermatozoa diluted in INRA and
0737-0806/$ e see front matter
Graduate Student Competition 162 Comparison of serial prostaglandin F2a protocols for control of the estrous cycle in mares K. Potter*, G. Webb, W. Boyer Missouri State University, Springfield, MO, USA Prostaglandin analogs are commonly administered to manipulate the estrous cycle of mares. Previous reports have indicated that serial administration of prostaglandin F2a (PGF2a) beginning on the day of ovulation will prevent CL development and in turn decrease the interval from ovulation to ovulation. The standard dose of dinoprost tromethamine (Lutalyse) is 10 mg per mare, administered IM. It has been reported that a lower dose is not only effective but results in less side effects. Therefore, objectives of the current study were to compare 3 PGF2a treatment protocols utilizing a lower dose and to determine the most effective treatment in terms of number of injections, intensity of protocol, time allocated, and labor. Eleven Quarter Horse mares were used for the study, in a crossover design with each mare subjected to all treatments. A dose of 1.1 mL was used to insure that mares received at least 5 mg of dinoprost tromethamine per injection. Treatment protocols were as follows: (1) PGF2a administered once on d 6 post-ovulation; (2) PGF2a administered twice daily on d 0, 1, and 2, then once on d 3 and 4 post-ovulation; (3) PGF2a administered twice daily on d 2, then once on d 3 and 4 post-ovulation. Teasing scores were assigned daily. Rectal palpation and ultrasound were performed to measure follicular growth and detect ovulation. Blood samples were collected on d 3 and 6 postovulation to analyze changes in plasma progesterone (P4) levels. Minitab 17 software was used for data analysis. Plasma P4 values were not statistically different between treatments on d 3 and 6 post ovulation. However, P4 numerically increased within treatment from 3 d to 6 d for treatment 1, and decreased between those days for the other treatments. Mean intervals from ovulation to ovulation for treatments 1, 2, and 3 were 14.8 d ± 3.1, 12.9 d ± 6.3 d and 14.3 d ± 1.4 respectively. Mean intervals from first day of
Abstracts / Journal of Equine Veterinary Science 52 (2017) 110e113
treatment to ovulation for treatments 1, 2, and 3 were 9.8 d ± 2.0, 12.9 d ± 6.3, and 12.6 d ± 1.7. Although not statistically different, these results agree with a previous study that utilized a dose of 10 mg dinoprost tromethamine. One mare on treatment 2 displayed estrus and maintained follicle > 30 mm for 13 d and had an intraovulatory interval of 29 d. Also, one mare on treatment 3 failed to return to estrus or develop a follicle >30 mm. Further research is needed to determine if manipulation of serial dosages and timing of treatment will result in results that are more consistent. Key Words: mare, prostaglandin, estrus synchronization
163 Evaluation of phytoestrogens in equine feeds and broodmare serum A.M. Adkin*, L.K. Warren, C. Silva-Sanchez, C.J. Mortensen University of Florida, Gainesville, FL, USA Phytoestrogens are a family of polyphenolic plant compounds that bind estrogen receptors and negatively impact female reproduction. Research in several species has demonstrated that dietary phytoestrogens can interrupt female cyclicity and may induce a sub-fertile state; however limited data exists on the amount of phytoestrogens in equine feeds and ultimately, the circulating concentrations within the mare. The objectives of this study were to: 1) quantify specific phytoestrogens in commonly fed concentrate ingredients, hay and legumes in the equine diet and 2) quantify serum phytoestrogen concentrations in broodmares. Feed samples analyzed in this study included alfalfa hay, perennial peanut hay, bahiagrass pasture, Coastal bermudagrass hay, soybean meal, soy hulls, and grain concentrate. Monthly venous blood samples were collected from 8 (mean ± SE, 530 ± 19 kg) mature non-pregnant stock breed mares for 3 mo (JanuaryeMarch). Basal diets of the mares included grain concentrate and ad libitum bahiagrass pasture and Coastal bermudagrass hay. All feeds and serum samples were analyzed for phytoestrogen composition using targeted high performance liquid chromatography-multiple reaction monitoring mass spectrometry to determine genistein, daidzein, genistin, daidzin, glycitin, formononetin, biochanin A, and coumestrol concentrations. Data were analyzed using one-way ANOVA or a mixed model ANOVA with repeated measures. All phytoestrogens evaluated were detected in soy products, but only coumestrol, genistein, and genistin were detected in legume and grass samples. Due to low concentrations in feeds, phytoestrogens were combined to compare total phytoestrogen concentration between feeds. Soybean meal was highest (P < 0.0001) in total phytoestrogens followed by soy hulls, grain concentrate, alfalfa hay, bahiagrass pasture, perennial peanut hay and Coastal bermudagrass hay. Total phytoestrogen concentration was higher in soybean meal (P < 0.0001) compared with soy hulls, and higher in alfalfa (P < 0.0001) compared with perennial peanut hay and Coastal bermudagrass hay, which contained negligible amounts. Concentrations of total phytoestrogens in bahiagrass pasture peaked in February with only trace amounts detected in January and March. Most phytoestrogens evaluated were not within detectable limits in mare serum. Genistein was the predominant phytoestrogen detected in serum, and was highest (P < 0.05) in March. Results confirm that equine feeds contain phytoestrogens which are
111
reflected in serum concentrations; however, further studies are needed to link dietary phytoestrogens to the reproductive health of broodmares. Key Words: genistein, coumestrol, horse
164 Effects of combined estradiol cypionatesulpiride treatment and follicle ablation on vernal transition in mares: Evaluation of plasma and follicular fluid hormones E.L. Oberhaus*1, D.L. Thompson, Jr. 1, C.R. Pinto 2, B.A. Foster 1, N. Arana-Valencia 1 1 Louisiana State University Agricultural Center, Baton Rouge, LA, USA; 2 Louisiana State University School of Veterinary Medicine, Baton Rouge, LA, USA This experiment was designed to assess the hormonal production and secretory aspects of early-induced ovulatory-sized follicles to those naturally occurring in the spring. Seasonally anovulatory mares were treated on Jan 21 with (1) 50 mg estradiol cypionate (ECP, n ¼ 8) alone or (2) with ECP on Jan 21 followed by 2 sulpiride injections (3 g, s.q., in vegetable shortening, n ¼ 8) 5 and 12 d later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned via ultrasonography regularly until detection of a 32e35 mm follicle; follicular fluid was then recovered via aspiration and analyzed for prolactin, estradiol and progesterone. Blood was collected every 4 d to characterize prolactin, LH, FSH and estradiol. Also, on the day of aspiration and for 6 more days, blood was collected to characterize LH and progesterone. Mean date to first 35-mm follicle was advanced (P < 0.05) in sulpiride-treated mares: 5 of the 8 (63%) responded within 28 d of the first sulpiride treatment. Complete follicle ablation had no effect (P > 0.05) on the ovarian response. Plasma prolactin was stimulated (P < 0.0001) in ECP-sulpiride treated mares for 16 d. There was no difference (P > 0.05) in plasma prolactin in mares that responded versus mares that did not. Estradiol cypionate stimulated plasma LH in all mares (P < 0.05) and ablation had no effect (P > 0.05) on LH response to ECP. Plasma LH was higher (P < 0.05) in treated mares that responded compared with mares that did not respond on d 8 and 12 post sulpiride treatment. In plasma samples collected daily after aspiration, both LH and progesterone were higher (P < 0.01) in mares that were treated with ECP-sulpiride and were not ablated compared with ECP-only treated mares (both ablated and not) and to ECP-sulpiride treated mares that were ablated. Plasma estradiol was similar (P > 0.05) in all mares on day of aspiration. There was no effect (P > 0.05) of treatment or ablation on follicular fluid prolactin, estradiol or progesterone. In conclusion, combined ECP-sulpiride treatment stimulated circulating prolactin and hastened the date to first preovulatory follicle, but degree of prolactin response was not a predictor of ovarian response. Sulpiride-treated mares that were not ablated had elevated plasma progesterone and LH after aspiration. Ablation may inhibit maturity of the first preovulatory follicle based on decreased progesterone and LH production, but does not inhibit growth and development of the first preovulatory follicle. Key Words: seasonal transition, prolactin, sulpiride