- Email: [email protected]
Effect of season on sperm membrane protein 22 and selected mRNAs in fresh and cryopreserved stallion sperm
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Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
55 Effect of season on sperm membrane protein 22 and selected mRNAs in fresh and cryopreserved stallion sperm N. Wrench a,∗ , C.R.F. Pinto b , G.R. Klinefelter c , D.J. Dix c , W.L. Flowers a , C.E. Farin a a
Department of Animal Science, North Carolina State University, Raleigh, NC 27695, USA of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA c US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27711, USA b Department
E-mail address: carlos [email protected] (N. Wrench). The objective of this study was to determine if either season or semen cryopreservation would have an effect on the expression of fertility-related protein SP22 and selected mRNA transcripts in stallion sperm. Six stallions, with age varying from 7 to 18 years and history of normal fertility were collected in June, September, December and March in the Northern Hemisphere (longitude: 75◦ 30 W to 84◦ 15 W; latitude: 34◦ N to 36◦ 21 N). Each ejaculate was partitioned for evaluation of sperm parameters (normal morphology, primary and secondary abnormalities, membrane integrity, viability, total motility, progressive motility and acrosome integrity), SP22 protein expression and expression of mRNA transcripts [beta actin (ACTB), tissue inhibitor of metalloproteinase 3 (TIMP3), testis specific protein 1 (TPX1) and phosphoglycerate kinase 2 (PGK2)] in fresh and cryopreserved samples. For SP22 immunocytochemistry, samples were stained using a sheep anti-rat recombinant SP22 primary antibody and a FITC-conjugated secondary antibody and analyzed for staining pattern. For mRNA transcript expression, sperm were washed with a hypoosmotic solution to induce somatic cell lysis. RNA was extracted from sperm samples and cDNA was synthesized. PCR was performed using the cDNA to assess mRNA expression. Data were analyzed for all six stallions and for the subset of four stallions whose collections were repeated every season (subset stallions) using general linear model procedures (SAS Institute Inc., Cary, NC, USA). The process of cryopreservation significantly (P < 0.05) affected all sperm parameters. A significant (P < 0.05) effect of season was noted for normal morphology, primary abnormalities, total motility and progressive motility for all 6 stallions. In general, the same seasonal effects were present for the 4 subset stallions, except for the absence of effect of season on total and progressive sperm motility. RNA yield from sperm was not affected by season, stallion, cryopreservation or their interactions. There was no effect of season or cryopreservation on the relative quantity of mRNAs for PGK2, TPX1, TIMP3 or ACTB. However, differences between stallions (n = 6) were apparent for PGK2 (P = 0.08) and ACTB (P = 0.01) content. For the 4 subset stallions there was a tendency (P = 0.1) for an effect of stallion on ACTB mRNA content. The percentage of sperm staining for SP22 on the equatorial segment was affected by cryopreservation, season and stallion, and by season × cryopreservation interaction (P < 0.05). Understanding differences that exist in sperm across seasons and stallions should enable us to identify the best time to collect semen for cryopreservation. Based on data presented, we recommend collecting semen for cryopreservation between March and June in the Northern hemisphere (spring and early summer). doi:10.1016/j.anireprosci.2008.05.134
ANIREP 3632 1–59
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
55 Effect of season on sperm membrane protein 22 and selected mRNAs in fresh and cryopreserved stallion sperm N. Wrench a,∗ , C.R.F. Pinto b , G.R. Klinefelter c , D.J. Dix c , W.L. Flowers a , C.E. Farin a a
Department of Animal Science, North Carolina State University, Raleigh, NC 27695, USA of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA c US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27711, USA b Department
E-mail address: carlos [email protected] (N. Wrench). The objective of this study was to determine if either season or semen cryopreservation would have an effect on the expression of fertility-related protein SP22 and selected mRNA transcripts in stallion sperm. Six stallions, with age varying from 7 to 18 years and history of normal fertility were collected in June, September, December and March in the Northern Hemisphere (longitude: 75◦ 30 W to 84◦ 15 W; latitude: 34◦ N to 36◦ 21 N). Each ejaculate was partitioned for evaluation of sperm parameters (normal morphology, primary and secondary abnormalities, membrane integrity, viability, total motility, progressive motility and acrosome integrity), SP22 protein expression and expression of mRNA transcripts [beta actin (ACTB), tissue inhibitor of metalloproteinase 3 (TIMP3), testis specific protein 1 (TPX1) and phosphoglycerate kinase 2 (PGK2)] in fresh and cryopreserved samples. For SP22 immunocytochemistry, samples were stained using a sheep anti-rat recombinant SP22 primary antibody and a FITC-conjugated secondary antibody and analyzed for staining pattern. For mRNA transcript expression, sperm were washed with a hypoosmotic solution to induce somatic cell lysis. RNA was extracted from sperm samples and cDNA was synthesized. PCR was performed using the cDNA to assess mRNA expression. Data were analyzed for all six stallions and for the subset of four stallions whose collections were repeated every season (subset stallions) using general linear model procedures (SAS Institute Inc., Cary, NC, USA). The process of cryopreservation significantly (P < 0.05) affected all sperm parameters. A significant (P < 0.05) effect of season was noted for normal morphology, primary abnormalities, total motility and progressive motility for all 6 stallions. In general, the same seasonal effects were present for the 4 subset stallions, except for the absence of effect of season on total and progressive sperm motility. RNA yield from sperm was not affected by season, stallion, cryopreservation or their interactions. There was no effect of season or cryopreservation on the relative quantity of mRNAs for PGK2, TPX1, TIMP3 or ACTB. However, differences between stallions (n = 6) were apparent for PGK2 (P = 0.08) and ACTB (P = 0.01) content. For the 4 subset stallions there was a tendency (P = 0.1) for an effect of stallion on ACTB mRNA content. The percentage of sperm staining for SP22 on the equatorial segment was affected by cryopreservation, season and stallion, and by season × cryopreservation interaction (P < 0.05). Understanding differences that exist in sperm across seasons and stallions should enable us to identify the best time to collect semen for cryopreservation. Based on data presented, we recommend collecting semen for cryopreservation between March and June in the Northern hemisphere (spring and early summer). doi:10.1016/j.anireprosci.2008.05.134
ANIREP 3632 1–59