- Email: [email protected]
Factors impacting freezability of stallion sperm
372
Abstracts / Journal of Equine Veterinary Science 33 (2013) 321-399
viability and improve embryo quality and survival in many species of livestock. We hypothesized that docosahexaenoic acid (DHA) supplementation would hasten uterine involution, enhance folliculogenesis, and increase blood flow to reproductive tissues in the postpartum mare. Twenty stock breed mares (mean SE, 624 10 kg) in their third trimester of gestation were randomly assigned to one of two dietary treatments: an n-3-rich fat supplement containing an algae source of DHA (n¼10; ReleiraÒ, Arenus, St. Charles, MO) or a placebo fat supplement formulated to mimic the n-6:n-3 FA ratio (10:1) of the basal grain concentrate (n¼10). Supplements were color-coded, thus blinding researchers to treatment and fed from 90 d prior to expected foaling through the first postpartum ovulation. On average, the DHA supplement provided 18.6, 10.5, and 2 g/d of fat, total n-3 FA, and DHA, respectively. The basal diet included grain concentrate, bahiagrass pasture and Coastal bermudagrass hay. Mares were observed during parturition to document labor-related events and examined daily thereafter by transrectal Doppler ultrasonography to measure uterine fluid clearance and involution, folliculogenesis, ovarian and uterine arterial blood flow, and blood perfusion to the dominant follicle. Data were analyzed using a one-way ANOVA or a mixed model ANOVA with repeated measures using time, treatment and time*treatment as fixed effects and horse within treatment as a random variable. Mares supplemented with DHA had a faster rate of involution of the non-gravid uterine horn (P ¼ 0.002) and increased blood flow in the ovarian artery ipsilateral to the dominant follicle (P ¼ 0.003) leading up to the first postpartum ovulation compared to placebo mares. Dietary treatment had no effect on gestation length (P ¼ 0.84), length of labor (P ¼ 0.82), latency to placental expulsion (P ¼ 0.67), placental weight (P ¼ 0.30), interval to first postpartum ovulation (P ¼ 0.29), rate of uterine fluid clearance (P ¼ 0.79), uterine body (P ¼ 0.43) or gravid horn involution (P ¼ 0.39), number or size of follicles (P> 0.10), blood flow to the uterine arteries (P> 0.10), or blood perfusion to the dominant follicle (P ¼ 0.74). Results indicate that low-level DHA supplementation does not alter gestation length or influence folliculogenesis, but hastens uterine involution of the non-gravid horn and increases ovarian blood flow in the postpartum mare. Future research should investigate the clinical impacts of DHA supplementation on uterine health and embryo quality/ survival in broodmares. The authors would like to acknowledge Dr. Kenneth Kopp and Novus Nutrition Brands for support of this research.
Equine endometrial gene expression during maternal recognition of pregnancy K.K. Klohonatz, G.J. Bouma, and J.E. Bruemmer Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO The mechanism by which maternal recognition of pregnancy in the mare occurs is still unknown. Prostaglandin F2a (PGF) is produced by the endometrium of non-pregnant mares between days 14-16 post ovulation to initiate luteolysis. Recognition of a viable and mobile conceptus prior to this time frame is critical in order to prevent the
endometrium from producing PGF. While the signal from the conceptus to the mare remains unknown, we hypothesized gene expression within the endometrium is affected by this signal. Our objective was to profile the gene expression pattern during the time of maternal recognition. Twelve normally cycling mares were used in a cross-over design. Mares were randomly assigned to a collection day (day 12, 14, 16, or 18 post ovulation) and provided endometrial samples from a pregnant and non-pregnant (PT and NP) cycle. Endometrial biopsy samples were snap frozen and stored until total RNA was isolated and used to screen the Horse Gene Expression Microarray (Agilent Technologies). This microarray contained over 43,000 unique equine transcripts. Samples (n¼3) for each day were processed and data analyzed to describe log2 fold differences in gene expression with regard to day and pregnancy status. Ten genes were identified that were differentially expressed (P<0.001) 3 fold or greater in PT vs NP samples for day 14,16, and 18 (Table 1). For these ten genes, the directions of the fold change (up or down-regulation) were consistent across comparisons and across probes/spots representing the same gene. Expression levels and fold changes were confirmed for all with quantitative real time PCR. Table 1 The ten genes that were differentially expressed in PT vs NP samples for day 14, 16, and 18 Systemic Name
Gene ID
Gene Symbol
Regulation
XR_044580 NM_001100113 NM_001081890 NM_001081772 XM_001501324 XM_001492645 XM_001489348 XM_001503294 XM_001916739 XM_001500996
LOC100069491 LOC100037401 LOC100033995 LOC791249 LOC100071541 LOC100060363 LOC100054931 LOC100071881 LOC100066488 LOC100033857
JAZF1 SPLA2 S100G ESR1 SLC36A2 METTL7A RALDH1 DKK1 EIF2AK3 AMPP
Up Down Up Down Up Up Down Up Up Up
As a follow up, endometrial samples were also collected on day 13 post ovulation (n¼3) and processed for protein isolation and immunohistochemical analysis. Protein levels of SPLA2 and DKK1 were analyzed with Western blot, and cellular localization of SPLA2, DKK1, and ESR1 was assessed by immunocytochemistry. These are the first data to describe gene expression in the endometrium across the critical period of maternal recognition of pregnancy in the mare. The expression pattern and regulation of these genes will be useful in determining the mechanism and pathway(s) that halts PGF production in order to sustain a pregnancy.
Factors impacting freezability of stallion sperm J.J. Kalmar 1, B.A. Ball 1, M.H.T. Troedsson 1, J. Skaife 2, P.I. Loomis 2, and E.L. Squires 1 1 Gluck Equine Research Center, University of Kentucky, Lexington, Kentucky, 2 Select Breeders Services, Chesapeake City, Maryland Frozen stallion semen is becoming increasingly more prevalent in the equine industry. The many advantages to using frozen semen include ease of shipping around the globe, convenience of collecting semen from stallions during their off-season, and timing of insemination. Frozen semen can be stored indefinitely and offspring can be produced after a stallion’s death. Limited data are available regarding the effects of age, breed and season on post-thaw parameters, and existing
Abstracts / Journal of Equine Veterinary Science 33 (2013) 321-399
373
studies are limited by low numbers of stallions or ejaculates. The objective of this retrospective study was to determine the effects of age, season and breed on initial motility, post-thaw motility, and the percent change from initial motility to postthaw motility (freezability). Data were available from a commercial company (Select Breeders Services, Chesapeake City, Maryland). Data from ejaculates with complete computer-assisted semen analysis (CASA) information from 1996 to 2012 were used. Data from 850 stallions with at least three ejaculates and complete CASA data were analyzed by one-way ANOVA and Tukey’s test. Breeds were chosen that included at least seven stallions. Birthdate information was only available for 672 of these stallions. Ejaculates were collected during winter (December, January, February), spring (March, April, May), summer (June, July, August), and fall (September, October, November). Stallions were grouped into age categories of 2-5, 6-10, 11-15, 16-20, and greater than 20 years. Nine breeds (Andalusian, Appaloosa, Arabian, Friesian, Morgan, Paint, Quarter Horse, Saddlebred, Standardbred) and two breed groups (Pony, Warmblood) were represented. Initial motility (%) was lower (p<0.0001) for ejaculates collected in fall (87.770.10) than winter (88.720.10), spring (89.00.15), and summer (88.830.11). In contrast, post-thaw motility (%) was highest in winter (64.850.20) compared to other seasons (p<0.0001). The percent change from initial to post-thaw motility was lowest (p<0.0001) for ejaculates frozen in winter (26.990.21) versus spring (28.300.35) and summer (28.080.25). As anticipated, ejaculates frozen from older stallions resulted in poorer initial and post-thaw motility (p<0.0001). Percent change between initial and postthaw motility was greatest (p<0.0001) for stallions older than 20 years of age (33.750.95). Stallions between six and ten years of age had ejaculates with the lowest (p<0.0001) percent change (24.980.21). Initial motility and post-thaw motility (%) were found to be significantly different (p<0.0001) and were lowest for Andalusians (84.981.03, 56.772.18) and Friesians (87.200.61, 49.370.92). Percent change was also found to be significantly different (p<0.0001) and was lowest for Paints (23.180.46), Quarter Horses (23.750.17) and Appaloosas (25.321.02). Based on our findings, the ideal time to freeze a stallion appears to be when he is between six and ten years of age in the fall or winter. Stallion breed plays a role in semen freezability; stock-type horses had ejaculates that were consistently higher in initial and post-thaw motility and had the smallest percent changes compared to other breeds.
identify the location of PRL receptors (PRLr) on follicles and corpus luteum (CL) and to determine if immunoreactive PRLr (staining intensity) signal differed by follicular size (primordial, preantral, antral) or between ovaries from anestrous, winter cycling and summer cycling mares. One ovary from 9 mares was collected in February and ovarian state classified as winter cycling (follicles > 25 mm and/or luteal tissue) or anestrus (follicles <25mm, no CL). One ovary from 11 cycling mares was collected in June/July. PRLr were detected in anestrous (n¼3), winter cycling (n¼2), and summer follicular (n¼3) and luteal phase (n¼3) ovaries using an anti-prolactin monoclonal mouse antibody (U5). A single observer assessed immunoreactivity level on a 4-point scale. Staining intensity was compared between follicle sizes as well as between reproductive states using a two-way ANOVA. PRLr were detected in follicles of all stages, inCL and on oocytes. PRLr staining intensity was not different (P > .05) between primordial and preantral follicles, but higher (P < .001) in antral follicles than in primordial or preantral follicles. Heavier PRLr staining (P < .001) occurred in winter cycling primordial follicles (1.58 0.09) compared with anestrous (0.67 0.10) and summer luteal phase primordial follicles (1.16 0.12; P < .01), but not in primordial follicles during the summer follicular phase (P > .05). PRLr density in preantral follicles during anestrus was lower (P < .05) than for all other reproductive states. Winter cycling and summer luteal phase preantral follicles stained most intensely and both had higher PRLr staining than preantral follicles from anestrous ovaries (P < .001) or summer follicular phase ovaries (P < .001). PRLr staining of antral follicles was the most intense of all follicular sizes and did not vary (P > .10) between reproductive states. PRLr staining was also detected in luteal tissue of both summer cycling and winter cycling ovaries, in oocytes located in all follicle stages, and in the ovulation fossa. In conclusion, PRLr were detected in all stages of follicular growth, and concentrations were highest in large antral follicles. This may indicate a mechanistic role for PRL during late stage follicular growth and perhaps ovulation.
Immunohistochemical localization of prolactin receptors within the equine ovary
Accumulation of fluid in the uterus can negatively impact mare reproductive performance by reducing conception rates and increasing early embryonic loss. The amino acid arginine (Arg) is a component of most proteins, serves as a substrate for nitric oxide and polyamine production, and is a potent secretagogue for insulin and growth hormone. Oral L-Arg supplementation has been shown to improve reproductive performance in many species. In post-partum mares, L-arginine supplementation increased uterine arterial blood flow, hastened uterine involution, and resulted in the presence of less fluid in the uterus. To determine whether oral L-Arg could aid open mares with delayed uterine fluid clearance, 12 non-pregnant light breed mares (mean SEM, 540 56 kg) were used in a 3 X
E.L. Oberhaus, S.S. King, and K.L. Jones Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, 62901 Prolactin (PRL) appears to play a stimulatory role in ovarian follicle development in the mare. In previousexperiments, exogenouslyadministered and endogenously stimulated PRL induced follicular growth during deep anestrus and shortened the time to first seasonal ovulation. To stimulate this growth, PRL most likely exerts a physiological action at the ovary. This study’s aim was to immunohistochemically
Effect of oral L-arginine supplementation on uterine blood flow and fluid clearance in mares during estrus R.D. Jacobs, L.K. Warren, and C.J. Mortensen Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
Abstracts / Journal of Equine Veterinary Science 33 (2013) 321-399
viability and improve embryo quality and survival in many species of livestock. We hypothesized that docosahexaenoic acid (DHA) supplementation would hasten uterine involution, enhance folliculogenesis, and increase blood flow to reproductive tissues in the postpartum mare. Twenty stock breed mares (mean SE, 624 10 kg) in their third trimester of gestation were randomly assigned to one of two dietary treatments: an n-3-rich fat supplement containing an algae source of DHA (n¼10; ReleiraÒ, Arenus, St. Charles, MO) or a placebo fat supplement formulated to mimic the n-6:n-3 FA ratio (10:1) of the basal grain concentrate (n¼10). Supplements were color-coded, thus blinding researchers to treatment and fed from 90 d prior to expected foaling through the first postpartum ovulation. On average, the DHA supplement provided 18.6, 10.5, and 2 g/d of fat, total n-3 FA, and DHA, respectively. The basal diet included grain concentrate, bahiagrass pasture and Coastal bermudagrass hay. Mares were observed during parturition to document labor-related events and examined daily thereafter by transrectal Doppler ultrasonography to measure uterine fluid clearance and involution, folliculogenesis, ovarian and uterine arterial blood flow, and blood perfusion to the dominant follicle. Data were analyzed using a one-way ANOVA or a mixed model ANOVA with repeated measures using time, treatment and time*treatment as fixed effects and horse within treatment as a random variable. Mares supplemented with DHA had a faster rate of involution of the non-gravid uterine horn (P ¼ 0.002) and increased blood flow in the ovarian artery ipsilateral to the dominant follicle (P ¼ 0.003) leading up to the first postpartum ovulation compared to placebo mares. Dietary treatment had no effect on gestation length (P ¼ 0.84), length of labor (P ¼ 0.82), latency to placental expulsion (P ¼ 0.67), placental weight (P ¼ 0.30), interval to first postpartum ovulation (P ¼ 0.29), rate of uterine fluid clearance (P ¼ 0.79), uterine body (P ¼ 0.43) or gravid horn involution (P ¼ 0.39), number or size of follicles (P> 0.10), blood flow to the uterine arteries (P> 0.10), or blood perfusion to the dominant follicle (P ¼ 0.74). Results indicate that low-level DHA supplementation does not alter gestation length or influence folliculogenesis, but hastens uterine involution of the non-gravid horn and increases ovarian blood flow in the postpartum mare. Future research should investigate the clinical impacts of DHA supplementation on uterine health and embryo quality/ survival in broodmares. The authors would like to acknowledge Dr. Kenneth Kopp and Novus Nutrition Brands for support of this research.
Equine endometrial gene expression during maternal recognition of pregnancy K.K. Klohonatz, G.J. Bouma, and J.E. Bruemmer Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO The mechanism by which maternal recognition of pregnancy in the mare occurs is still unknown. Prostaglandin F2a (PGF) is produced by the endometrium of non-pregnant mares between days 14-16 post ovulation to initiate luteolysis. Recognition of a viable and mobile conceptus prior to this time frame is critical in order to prevent the
endometrium from producing PGF. While the signal from the conceptus to the mare remains unknown, we hypothesized gene expression within the endometrium is affected by this signal. Our objective was to profile the gene expression pattern during the time of maternal recognition. Twelve normally cycling mares were used in a cross-over design. Mares were randomly assigned to a collection day (day 12, 14, 16, or 18 post ovulation) and provided endometrial samples from a pregnant and non-pregnant (PT and NP) cycle. Endometrial biopsy samples were snap frozen and stored until total RNA was isolated and used to screen the Horse Gene Expression Microarray (Agilent Technologies). This microarray contained over 43,000 unique equine transcripts. Samples (n¼3) for each day were processed and data analyzed to describe log2 fold differences in gene expression with regard to day and pregnancy status. Ten genes were identified that were differentially expressed (P<0.001) 3 fold or greater in PT vs NP samples for day 14,16, and 18 (Table 1). For these ten genes, the directions of the fold change (up or down-regulation) were consistent across comparisons and across probes/spots representing the same gene. Expression levels and fold changes were confirmed for all with quantitative real time PCR. Table 1 The ten genes that were differentially expressed in PT vs NP samples for day 14, 16, and 18 Systemic Name
Gene ID
Gene Symbol
Regulation
XR_044580 NM_001100113 NM_001081890 NM_001081772 XM_001501324 XM_001492645 XM_001489348 XM_001503294 XM_001916739 XM_001500996
LOC100069491 LOC100037401 LOC100033995 LOC791249 LOC100071541 LOC100060363 LOC100054931 LOC100071881 LOC100066488 LOC100033857
JAZF1 SPLA2 S100G ESR1 SLC36A2 METTL7A RALDH1 DKK1 EIF2AK3 AMPP
Up Down Up Down Up Up Down Up Up Up
As a follow up, endometrial samples were also collected on day 13 post ovulation (n¼3) and processed for protein isolation and immunohistochemical analysis. Protein levels of SPLA2 and DKK1 were analyzed with Western blot, and cellular localization of SPLA2, DKK1, and ESR1 was assessed by immunocytochemistry. These are the first data to describe gene expression in the endometrium across the critical period of maternal recognition of pregnancy in the mare. The expression pattern and regulation of these genes will be useful in determining the mechanism and pathway(s) that halts PGF production in order to sustain a pregnancy.
Factors impacting freezability of stallion sperm J.J. Kalmar 1, B.A. Ball 1, M.H.T. Troedsson 1, J. Skaife 2, P.I. Loomis 2, and E.L. Squires 1 1 Gluck Equine Research Center, University of Kentucky, Lexington, Kentucky, 2 Select Breeders Services, Chesapeake City, Maryland Frozen stallion semen is becoming increasingly more prevalent in the equine industry. The many advantages to using frozen semen include ease of shipping around the globe, convenience of collecting semen from stallions during their off-season, and timing of insemination. Frozen semen can be stored indefinitely and offspring can be produced after a stallion’s death. Limited data are available regarding the effects of age, breed and season on post-thaw parameters, and existing
Abstracts / Journal of Equine Veterinary Science 33 (2013) 321-399
373
studies are limited by low numbers of stallions or ejaculates. The objective of this retrospective study was to determine the effects of age, season and breed on initial motility, post-thaw motility, and the percent change from initial motility to postthaw motility (freezability). Data were available from a commercial company (Select Breeders Services, Chesapeake City, Maryland). Data from ejaculates with complete computer-assisted semen analysis (CASA) information from 1996 to 2012 were used. Data from 850 stallions with at least three ejaculates and complete CASA data were analyzed by one-way ANOVA and Tukey’s test. Breeds were chosen that included at least seven stallions. Birthdate information was only available for 672 of these stallions. Ejaculates were collected during winter (December, January, February), spring (March, April, May), summer (June, July, August), and fall (September, October, November). Stallions were grouped into age categories of 2-5, 6-10, 11-15, 16-20, and greater than 20 years. Nine breeds (Andalusian, Appaloosa, Arabian, Friesian, Morgan, Paint, Quarter Horse, Saddlebred, Standardbred) and two breed groups (Pony, Warmblood) were represented. Initial motility (%) was lower (p<0.0001) for ejaculates collected in fall (87.770.10) than winter (88.720.10), spring (89.00.15), and summer (88.830.11). In contrast, post-thaw motility (%) was highest in winter (64.850.20) compared to other seasons (p<0.0001). The percent change from initial to post-thaw motility was lowest (p<0.0001) for ejaculates frozen in winter (26.990.21) versus spring (28.300.35) and summer (28.080.25). As anticipated, ejaculates frozen from older stallions resulted in poorer initial and post-thaw motility (p<0.0001). Percent change between initial and postthaw motility was greatest (p<0.0001) for stallions older than 20 years of age (33.750.95). Stallions between six and ten years of age had ejaculates with the lowest (p<0.0001) percent change (24.980.21). Initial motility and post-thaw motility (%) were found to be significantly different (p<0.0001) and were lowest for Andalusians (84.981.03, 56.772.18) and Friesians (87.200.61, 49.370.92). Percent change was also found to be significantly different (p<0.0001) and was lowest for Paints (23.180.46), Quarter Horses (23.750.17) and Appaloosas (25.321.02). Based on our findings, the ideal time to freeze a stallion appears to be when he is between six and ten years of age in the fall or winter. Stallion breed plays a role in semen freezability; stock-type horses had ejaculates that were consistently higher in initial and post-thaw motility and had the smallest percent changes compared to other breeds.
identify the location of PRL receptors (PRLr) on follicles and corpus luteum (CL) and to determine if immunoreactive PRLr (staining intensity) signal differed by follicular size (primordial, preantral, antral) or between ovaries from anestrous, winter cycling and summer cycling mares. One ovary from 9 mares was collected in February and ovarian state classified as winter cycling (follicles > 25 mm and/or luteal tissue) or anestrus (follicles <25mm, no CL). One ovary from 11 cycling mares was collected in June/July. PRLr were detected in anestrous (n¼3), winter cycling (n¼2), and summer follicular (n¼3) and luteal phase (n¼3) ovaries using an anti-prolactin monoclonal mouse antibody (U5). A single observer assessed immunoreactivity level on a 4-point scale. Staining intensity was compared between follicle sizes as well as between reproductive states using a two-way ANOVA. PRLr were detected in follicles of all stages, inCL and on oocytes. PRLr staining intensity was not different (P > .05) between primordial and preantral follicles, but higher (P < .001) in antral follicles than in primordial or preantral follicles. Heavier PRLr staining (P < .001) occurred in winter cycling primordial follicles (1.58 0.09) compared with anestrous (0.67 0.10) and summer luteal phase primordial follicles (1.16 0.12; P < .01), but not in primordial follicles during the summer follicular phase (P > .05). PRLr density in preantral follicles during anestrus was lower (P < .05) than for all other reproductive states. Winter cycling and summer luteal phase preantral follicles stained most intensely and both had higher PRLr staining than preantral follicles from anestrous ovaries (P < .001) or summer follicular phase ovaries (P < .001). PRLr staining of antral follicles was the most intense of all follicular sizes and did not vary (P > .10) between reproductive states. PRLr staining was also detected in luteal tissue of both summer cycling and winter cycling ovaries, in oocytes located in all follicle stages, and in the ovulation fossa. In conclusion, PRLr were detected in all stages of follicular growth, and concentrations were highest in large antral follicles. This may indicate a mechanistic role for PRL during late stage follicular growth and perhaps ovulation.
Immunohistochemical localization of prolactin receptors within the equine ovary
Accumulation of fluid in the uterus can negatively impact mare reproductive performance by reducing conception rates and increasing early embryonic loss. The amino acid arginine (Arg) is a component of most proteins, serves as a substrate for nitric oxide and polyamine production, and is a potent secretagogue for insulin and growth hormone. Oral L-Arg supplementation has been shown to improve reproductive performance in many species. In post-partum mares, L-arginine supplementation increased uterine arterial blood flow, hastened uterine involution, and resulted in the presence of less fluid in the uterus. To determine whether oral L-Arg could aid open mares with delayed uterine fluid clearance, 12 non-pregnant light breed mares (mean SEM, 540 56 kg) were used in a 3 X
E.L. Oberhaus, S.S. King, and K.L. Jones Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, 62901 Prolactin (PRL) appears to play a stimulatory role in ovarian follicle development in the mare. In previousexperiments, exogenouslyadministered and endogenously stimulated PRL induced follicular growth during deep anestrus and shortened the time to first seasonal ovulation. To stimulate this growth, PRL most likely exerts a physiological action at the ovary. This study’s aim was to immunohistochemically
Effect of oral L-arginine supplementation on uterine blood flow and fluid clearance in mares during estrus R.D. Jacobs, L.K. Warren, and C.J. Mortensen Department of Animal Sciences, University of Florida, Gainesville, Florida, USA