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Influence of insemination time on pregnancy rates of mares inseminated with frozen semen
476
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
collected from 10 stallions, aged from 10 to 23 years. Seminal plasma was removed by centrifugation and spermatozoa were washed by two successive centrifugations in Tyrode extender. Analysis was performed by immunocytochemistry, western-blot, immunofluorescence and flow cytometry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPER identification in equine species was confirmed by RT-PCR and sequencing. Both estrogen receptors ESR1 and ESR2 are present in stallion spermatozoa. For ESR1, western-blot analysis shows a single 66kDa band corresponding to the wild-type isoform; immunofluorescence analysis showed a flagellar staining. For ESR2, western-blot analysis shows a single 61 kDa band, corresponding to the molecular weight described for the wild type form of this receptor and confocal analysis demonstrated also a flagellar localization. The rate of spermatozoa positive for the detection of both of these receptors was analyzed by flow cytometry. 98% of spermatozoa were positive for ESR1 and 94% positive for ESR2 in samples from 3 stallions collected in April. The estrogen transmembrane receptor, GPER was also identified. Genome from Equus Caballus contains a GPER-like gene but identification and expression of this gene in horses has not been reported so far. We proceeded to extract RNA from a colt testis and spermatozoa from 3 stallions and with primers designed from the DNA sequence, RT-PCR amplification was performed. The products were sequenced and allowed to describe the expression of GPER in the equine species for the first time. In conclusion, the presence of estrogen receptors in stallion spermatozoa was demonstrated suggesting that sperm are a putative estrogen target. Moreover, GPER was described for the first time in horses. This is opening new possibilities to study estradiol action in male as well as in female reproduction.
Effect of nitric oxide enzyme (NOS) inhibition and effect of nitric oxide (NO) scavenger on motility patterns and hyperactivated population of cryopreserved equine sperm R.P. Arruda 1, D.F. Silva 1, A.F.C. Andrade 1, M.C. Caldas-Bussiere 2, M.A. Alonso 1, E.C.C. Celeghini 1, K.M. Lemes 1, H.F. Carvalho 1, and S.A. Florez-Rodriguez 1 1 Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 2 Laboratory of Animal Breeding and Reproduction, Center of Agriculture and Technology, State University of North Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil The aim of this study was to determine the effect of NOS inhibition and effect of an NO scavenger on motility patterns and hyperactivated sperm in cryopreserved equine semen. Three ejaculates were collected from each of three stallions (n ¼ 9). Frozen-thawed semen was centrifuged in bovine in vitro fertilization (IVF) media supplemented with sodium bicarbonate and bovine serum albumin for sperm capacitation. The pellet was incubated in the same media with L-arginine, with or without the NOS inhibitor, Nu-nitro-L-arginine metylester (L-NAME) and in media with
L-arginine with or without the NO scavenger, methylene blue: control T1¼ (C-IVF), T2¼ L-arginine 10mM, T3¼ L-NAME 1 mM, T4¼ methylene blue 100 mM, T5¼ L-arginine (10 mM) + L-NAME (1mM) and T6¼ L-arginine (10 mM) + methylene blue (100mM) for 0, 60, 120 and 300 minutes at 38 C (5% CO2). Sperm was considered hyperactivated when VCL 180mm/s and ALH 12 mm. Data were analyzed using ANOVA and subsequently compared by Tukey test (SAS). The presence of the scavenger in the culture media paralysed equine sperm cells. Total motility, progressive motility and rapid cells differed among treatments at time 0 in groups T2 and T5 (P < 0.05), when compared with T1, T3, T4 and T6 at times 0, 60, 120 and 300 minutes. VAP was reduced (P < 0.05) when L-arginine was added. L-NAME did not reduce the effect of NO. At time 0, L-arginine addition reduced VSL (T2 51.8 2.8; T5 54.1 2.8) compared to groups T1 and T3 (T1 78.0 5.4; T3 77.2 5.6). VCL was not altered by any of the treatments in all the sampling times. There was no interaction between times and treatment for ALH. When comparing only treatment effect, groups T4 and T6 had a reduction in ALH compared to groups T1, T2, T3 and T5. Treatment effect on BCF only occurred after 120 minutes of incubation. At this time, BCF was lower in T1. There were no significant differences in STR when the treatments that do not cause sperm immobilization were analyzed. At time 0 LIN suffered a reduction in groups T2 and T5 (T2 32.3 1.9; T5 35.2 31.6). In group T5 a reduction (P < 0.05) in the percentage of hyperactivated cells was seen compared to groups T1, T2, T3, T4 and T6, at times 0, 120 and 300. Our results confirm that the presence of NO modulates sperm motility patterns although it does not induce hyperactivation when sperm are incubated with methylene blue. Acknowledgments The present study received financial support FAPESP (Grants 2009/54906-5). Influence of insemination time on pregnancy rates of mares inseminated with frozen semen B.R. Avanzi, R.S. Ramos, G.H.M. Araujo, E.G. Fioratti, G.M. Greco, L.A. Trinca, J.A. Dell'Aqua, Jr., C.M. Melo, and F.O. Papa Department of Animal Reproduction and Veterinary Radiology, UNESP Botucatu, SP, Brazil, Distrito de Rubião Junior S/N, 18618-970 One of the major limiting factors to the widespread use of frozen semen is the cost associated with the intense management of mares. It is generally recommended that frozen stallion semen should be inseminated within 12 h prior to ovulation and 6 h after ovulation. The aim of the present study was to compare pregnancy rates of mares inseminated 1) post-ovulation and 2) 40 h after ovulation induction. Semen was obtained from 2 stallions, A) a Westfalen of good fertility and B) a Mangalarga Marchador of poor fertility. Six ejaculates from each stallion were collected (n ¼ 12). The semen was diluted with Botu-Semen (1:1) and centrifuged at 600 x g for 10 minutes. The
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
supernatant was discarded and the pellet resuspended in Botucrio extender. Semen was packed into 0.5mL straws that were kept at 5oC for 20 minutes. The straws were then placed 6 cm above liquid nitrogen level for 20 minutes to freeze and finally plunged into it. Straws were thawed at 46 C for 20 seconds. The insemination dose was 400x106 total sperm. The samples were analyzed by CASA (HTM – IVOS 12), and plasma membrane integrity was evaluated by fluorescent stains (CFDA and PI). The fertility trial was carried out with 29 mares: 15 were inseminated with stallion A (30 cycles) and 14 with stallion B (28 cycles). The estrus of the mares was detected via transrectal palpation and ultrasonography. One milligram of deslorelin acetate was administered intramuscularly when a preovulatory follicle of at least 35 mm in diameter was detected. Eighteen hours after the induction of ovulation, the mares were palpated every 6 hours until ovulation was detected. For protocol (1), the mares were inseminated no more than 6 hours after ovulation. For protocol (2), the mares were inseminated 40 hours after the deslorelin acetate injection. All mares were inseminated into the tip of the ipsilateral uterine horn. Semen data were analyzed by ANOVA using Proc GLM. Pregnancy rates among stallions and insemination procedures were compared by logistic regression analysis using Proc GENMOD. Significance was set as P < 0.05. Values for total motility (77.3 3.4a and 70.5 1.3b), progressive motility (42.1 3.1a and 27.0 1.4b), % rapid cells (65.3 4.0a and 47.0 2.9b) and plasma membrane integrity (34.0 3.1 and 31.7 4.8) were obtained for stallion A and B, respectively (different letters show significant statistical difference). Pregnancy rates were 46.7% (7/15) and 35.7% (5/14) when breed with 6h after ovulation with semen from stallions A and B, respectively. As for inseminations performed 40h after ovulation induction, pregnancy rates were 66.6% (10/15) and 21.4% (3/14) for stallions A and B, respectively. Based on the results of the present study we can conclude that insemination at a fixed time after ovulation induction using equine frozen semen was efficient. Thus, the present data supports the feasibility of equine frozen semen as a technology to be widely used and spread since it reduces mare management and costs. Acknowledgments Financial support: FAPESP. Effect of sex sorting on intracellular Ca2+ and ATP content of stallion spermatozoa C. Balao-da Silva, A. Morillo-Rodriguez, C. Ortega-Ferrusola, J.M. Gallardo-Bolaños, M. Plaza-Dávila, A. Miro-Morán, I.M. Aparicio, J.A. Tapia, and F.J. Peña Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain
may damage sperm through the effects of high pressure and dilution, U.V light exposure, effect of the dye and mechanical forces. Thus, understanding the mechanisms behind the damage is required, in order to improve the technique in horses. So far, the effects of sorting on the energy status and calcium homeostasis have not been addressed. Several sperm functions require substantial amounts of ATP, such as flagellar movement and signal transduction via protein phosphorylation. Energy can be produced by oxidative phosphorylation at the mitochondria, but also by glycolysis. Calcium status has been directly related to flagellar modulation, and thereby, spermatic motility. Five pools of semen from three different stallions were made. At first, selection of spermatozoa was made through the use of a colloid with a species-specific formulation (Androcoll-E, Uppsala, Sweden). Afterwards, the sample was diluted in INRATyrodes, stained with Hoechst 33344 and incubated at 34oC. Spermatozoa were sorted in a MoFLo SX DP (Beckman Coulter, Miami, FL, USA). Kinematic parameters were estimated by CASA. Evaluation of spermatozoal status was assessed by flow cytometry: viability with the combination of probes Yo-Pro-1 and Ethidium Homodimer; mitochondrial membrane potential with JC-1 probe and Calcium detection with Fluo3. A bioluminescence assay (ATP Determination Kit A22066) was also performed, to quantify the amount of ATP present. All reagents were from Molecular Probes (Leiden, The Netherlands). After sex-sorting, total and progressive motility decreased significantly, while late apoptotic and dead spermatozoa subpopulations increased. Live cells also decreased significantly. This occurred in parallel to a slight decrease in the intracellular percentage of Ca2+ in living cells of non-sorted (44.9 9.51) and sorted (40.7 9.51) samples. Regarding ATP detection, a decrease of approximately 40% between the pre-sorted and sorted sample was detected (measured in pM/100mg protein). Association of ATP levels with sperm kinematic parameters (already made in our laboratory) are corroborated by this study, since both ATP and motility values decreased. As mitochondrial membrane potential was maintained, we could relate this parameter with other cell maintenance processes, rather than flagellar modulation. On the other hand, ATP could still be produced by oxidative phosphorylation, and this loss can be explained by the opening of some membrane pores. This might be coincident with the increase in late apoptotic spermatozoa, and therefore, plasma membrane permeabilization. In summary, the intracellular ATP decrease in stallion spermatozoa is related to kinematic parameters such as total and progressive motility. Also, apoptotic and dead cells are associated with a decrease in ATP content. Calcium may also play a role on this kinematic decrease.
Acknowledgments Sex sorting flow cytometry allows the selection of X and/ or Y chromosome bearing spermatozoa. This procedure
477
Funded by: AGL 2010-20758 (GAN).
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
collected from 10 stallions, aged from 10 to 23 years. Seminal plasma was removed by centrifugation and spermatozoa were washed by two successive centrifugations in Tyrode extender. Analysis was performed by immunocytochemistry, western-blot, immunofluorescence and flow cytometry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPER identification in equine species was confirmed by RT-PCR and sequencing. Both estrogen receptors ESR1 and ESR2 are present in stallion spermatozoa. For ESR1, western-blot analysis shows a single 66kDa band corresponding to the wild-type isoform; immunofluorescence analysis showed a flagellar staining. For ESR2, western-blot analysis shows a single 61 kDa band, corresponding to the molecular weight described for the wild type form of this receptor and confocal analysis demonstrated also a flagellar localization. The rate of spermatozoa positive for the detection of both of these receptors was analyzed by flow cytometry. 98% of spermatozoa were positive for ESR1 and 94% positive for ESR2 in samples from 3 stallions collected in April. The estrogen transmembrane receptor, GPER was also identified. Genome from Equus Caballus contains a GPER-like gene but identification and expression of this gene in horses has not been reported so far. We proceeded to extract RNA from a colt testis and spermatozoa from 3 stallions and with primers designed from the DNA sequence, RT-PCR amplification was performed. The products were sequenced and allowed to describe the expression of GPER in the equine species for the first time. In conclusion, the presence of estrogen receptors in stallion spermatozoa was demonstrated suggesting that sperm are a putative estrogen target. Moreover, GPER was described for the first time in horses. This is opening new possibilities to study estradiol action in male as well as in female reproduction.
Effect of nitric oxide enzyme (NOS) inhibition and effect of nitric oxide (NO) scavenger on motility patterns and hyperactivated population of cryopreserved equine sperm R.P. Arruda 1, D.F. Silva 1, A.F.C. Andrade 1, M.C. Caldas-Bussiere 2, M.A. Alonso 1, E.C.C. Celeghini 1, K.M. Lemes 1, H.F. Carvalho 1, and S.A. Florez-Rodriguez 1 1 Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 2 Laboratory of Animal Breeding and Reproduction, Center of Agriculture and Technology, State University of North Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil The aim of this study was to determine the effect of NOS inhibition and effect of an NO scavenger on motility patterns and hyperactivated sperm in cryopreserved equine semen. Three ejaculates were collected from each of three stallions (n ¼ 9). Frozen-thawed semen was centrifuged in bovine in vitro fertilization (IVF) media supplemented with sodium bicarbonate and bovine serum albumin for sperm capacitation. The pellet was incubated in the same media with L-arginine, with or without the NOS inhibitor, Nu-nitro-L-arginine metylester (L-NAME) and in media with
L-arginine with or without the NO scavenger, methylene blue: control T1¼ (C-IVF), T2¼ L-arginine 10mM, T3¼ L-NAME 1 mM, T4¼ methylene blue 100 mM, T5¼ L-arginine (10 mM) + L-NAME (1mM) and T6¼ L-arginine (10 mM) + methylene blue (100mM) for 0, 60, 120 and 300 minutes at 38 C (5% CO2). Sperm was considered hyperactivated when VCL 180mm/s and ALH 12 mm. Data were analyzed using ANOVA and subsequently compared by Tukey test (SAS). The presence of the scavenger in the culture media paralysed equine sperm cells. Total motility, progressive motility and rapid cells differed among treatments at time 0 in groups T2 and T5 (P < 0.05), when compared with T1, T3, T4 and T6 at times 0, 60, 120 and 300 minutes. VAP was reduced (P < 0.05) when L-arginine was added. L-NAME did not reduce the effect of NO. At time 0, L-arginine addition reduced VSL (T2 51.8 2.8; T5 54.1 2.8) compared to groups T1 and T3 (T1 78.0 5.4; T3 77.2 5.6). VCL was not altered by any of the treatments in all the sampling times. There was no interaction between times and treatment for ALH. When comparing only treatment effect, groups T4 and T6 had a reduction in ALH compared to groups T1, T2, T3 and T5. Treatment effect on BCF only occurred after 120 minutes of incubation. At this time, BCF was lower in T1. There were no significant differences in STR when the treatments that do not cause sperm immobilization were analyzed. At time 0 LIN suffered a reduction in groups T2 and T5 (T2 32.3 1.9; T5 35.2 31.6). In group T5 a reduction (P < 0.05) in the percentage of hyperactivated cells was seen compared to groups T1, T2, T3, T4 and T6, at times 0, 120 and 300. Our results confirm that the presence of NO modulates sperm motility patterns although it does not induce hyperactivation when sperm are incubated with methylene blue. Acknowledgments The present study received financial support FAPESP (Grants 2009/54906-5). Influence of insemination time on pregnancy rates of mares inseminated with frozen semen B.R. Avanzi, R.S. Ramos, G.H.M. Araujo, E.G. Fioratti, G.M. Greco, L.A. Trinca, J.A. Dell'Aqua, Jr., C.M. Melo, and F.O. Papa Department of Animal Reproduction and Veterinary Radiology, UNESP Botucatu, SP, Brazil, Distrito de Rubião Junior S/N, 18618-970 One of the major limiting factors to the widespread use of frozen semen is the cost associated with the intense management of mares. It is generally recommended that frozen stallion semen should be inseminated within 12 h prior to ovulation and 6 h after ovulation. The aim of the present study was to compare pregnancy rates of mares inseminated 1) post-ovulation and 2) 40 h after ovulation induction. Semen was obtained from 2 stallions, A) a Westfalen of good fertility and B) a Mangalarga Marchador of poor fertility. Six ejaculates from each stallion were collected (n ¼ 12). The semen was diluted with Botu-Semen (1:1) and centrifuged at 600 x g for 10 minutes. The
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
supernatant was discarded and the pellet resuspended in Botucrio extender. Semen was packed into 0.5mL straws that were kept at 5oC for 20 minutes. The straws were then placed 6 cm above liquid nitrogen level for 20 minutes to freeze and finally plunged into it. Straws were thawed at 46 C for 20 seconds. The insemination dose was 400x106 total sperm. The samples were analyzed by CASA (HTM – IVOS 12), and plasma membrane integrity was evaluated by fluorescent stains (CFDA and PI). The fertility trial was carried out with 29 mares: 15 were inseminated with stallion A (30 cycles) and 14 with stallion B (28 cycles). The estrus of the mares was detected via transrectal palpation and ultrasonography. One milligram of deslorelin acetate was administered intramuscularly when a preovulatory follicle of at least 35 mm in diameter was detected. Eighteen hours after the induction of ovulation, the mares were palpated every 6 hours until ovulation was detected. For protocol (1), the mares were inseminated no more than 6 hours after ovulation. For protocol (2), the mares were inseminated 40 hours after the deslorelin acetate injection. All mares were inseminated into the tip of the ipsilateral uterine horn. Semen data were analyzed by ANOVA using Proc GLM. Pregnancy rates among stallions and insemination procedures were compared by logistic regression analysis using Proc GENMOD. Significance was set as P < 0.05. Values for total motility (77.3 3.4a and 70.5 1.3b), progressive motility (42.1 3.1a and 27.0 1.4b), % rapid cells (65.3 4.0a and 47.0 2.9b) and plasma membrane integrity (34.0 3.1 and 31.7 4.8) were obtained for stallion A and B, respectively (different letters show significant statistical difference). Pregnancy rates were 46.7% (7/15) and 35.7% (5/14) when breed with 6h after ovulation with semen from stallions A and B, respectively. As for inseminations performed 40h after ovulation induction, pregnancy rates were 66.6% (10/15) and 21.4% (3/14) for stallions A and B, respectively. Based on the results of the present study we can conclude that insemination at a fixed time after ovulation induction using equine frozen semen was efficient. Thus, the present data supports the feasibility of equine frozen semen as a technology to be widely used and spread since it reduces mare management and costs. Acknowledgments Financial support: FAPESP. Effect of sex sorting on intracellular Ca2+ and ATP content of stallion spermatozoa C. Balao-da Silva, A. Morillo-Rodriguez, C. Ortega-Ferrusola, J.M. Gallardo-Bolaños, M. Plaza-Dávila, A. Miro-Morán, I.M. Aparicio, J.A. Tapia, and F.J. Peña Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain
may damage sperm through the effects of high pressure and dilution, U.V light exposure, effect of the dye and mechanical forces. Thus, understanding the mechanisms behind the damage is required, in order to improve the technique in horses. So far, the effects of sorting on the energy status and calcium homeostasis have not been addressed. Several sperm functions require substantial amounts of ATP, such as flagellar movement and signal transduction via protein phosphorylation. Energy can be produced by oxidative phosphorylation at the mitochondria, but also by glycolysis. Calcium status has been directly related to flagellar modulation, and thereby, spermatic motility. Five pools of semen from three different stallions were made. At first, selection of spermatozoa was made through the use of a colloid with a species-specific formulation (Androcoll-E, Uppsala, Sweden). Afterwards, the sample was diluted in INRATyrodes, stained with Hoechst 33344 and incubated at 34oC. Spermatozoa were sorted in a MoFLo SX DP (Beckman Coulter, Miami, FL, USA). Kinematic parameters were estimated by CASA. Evaluation of spermatozoal status was assessed by flow cytometry: viability with the combination of probes Yo-Pro-1 and Ethidium Homodimer; mitochondrial membrane potential with JC-1 probe and Calcium detection with Fluo3. A bioluminescence assay (ATP Determination Kit A22066) was also performed, to quantify the amount of ATP present. All reagents were from Molecular Probes (Leiden, The Netherlands). After sex-sorting, total and progressive motility decreased significantly, while late apoptotic and dead spermatozoa subpopulations increased. Live cells also decreased significantly. This occurred in parallel to a slight decrease in the intracellular percentage of Ca2+ in living cells of non-sorted (44.9 9.51) and sorted (40.7 9.51) samples. Regarding ATP detection, a decrease of approximately 40% between the pre-sorted and sorted sample was detected (measured in pM/100mg protein). Association of ATP levels with sperm kinematic parameters (already made in our laboratory) are corroborated by this study, since both ATP and motility values decreased. As mitochondrial membrane potential was maintained, we could relate this parameter with other cell maintenance processes, rather than flagellar modulation. On the other hand, ATP could still be produced by oxidative phosphorylation, and this loss can be explained by the opening of some membrane pores. This might be coincident with the increase in late apoptotic spermatozoa, and therefore, plasma membrane permeabilization. In summary, the intracellular ATP decrease in stallion spermatozoa is related to kinematic parameters such as total and progressive motility. Also, apoptotic and dead cells are associated with a decrease in ATP content. Calcium may also play a role on this kinematic decrease.
Acknowledgments Sex sorting flow cytometry allows the selection of X and/ or Y chromosome bearing spermatozoa. This procedure
477
Funded by: AGL 2010-20758 (GAN).