Effect of Season on Sperm Membrane Protein 22 and Selected mRNAs in Fresh and Cryopreserved Stallion Sperm


Abstract Wrench, Nicola. Effect of season on SP22 protein and selected mRNAs in fresh and cryopreserved stallion sperm. (Under the direction of Drs. Charlotte E. Farin and Carlos R.F. Pinto). The objective of this study was to determine if season or semen cryopreservation had an effect on the expression of fertility-related protein SP22 and selected mRNA transcripts in stallion sperm. Six stallions were collected in June 2005, September 2005, December 2005 and March 2006. Each ejaculate was partitioned for evaluation of sperm parameters in fresh and cryopreserved samples. The percentages of normal morphology, primary abnormalities, secondary abnormalities, membrane integrity, viability, total motility, progressive motility and acrosome integrity were recorded. In addition, aliquots of fresh and cryopreserved ejaculates were analyzed for SP22 protein expression and expression of mRNA transcripts. For SP22 immunocytochemistry, samples were stained using a sheep anti-rat recombinant SP22 primary antibody and a FITC-conjugated secondary antibody. At least 200 stained sperm were counted using a fluorescent microscope and categorized into one of two patterns: Pattern 1, overlying the equatorial region (ER) only; Pattern 2, overlying the acrosomal and equatorial region (AER), neck (N) and tail (T). For mRNA transcript expression, sperm were washed with a hypoosmotic solution to induce somatic cell lysis. RNA was extracted from sperm samples using Tri-Reagent and cDNA was synthesized. PCR was performed using the cDNA to assess mRNA expression. Data were analyzed for all six stallions as well as for the subset of four stallions whose collections were repeated every season (subset stallions). Data were analyzed using general linear model procedures (SAS Institute Inc., Cary, NC, USA). The process of cryopreservation significantly (P<0.05) affected all sperm parameters. In general, freezing decreased the proportion of sperm exhibiting normal morphology as well as the proportions of viable and motile sperm. In contrast, freezing was associated with an increased incidence of primary and secondary morphology abnormalities. 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. However, there was no effect of season on total motility or progressive motility. Significant (P<0.05) season by freezing treatment interactions were found for progressive motility and intact acrosomes for all 6 stallions. For the 4 subset stallions a significant (P<0.05) season by treatment interaction was found for total motility, progressive motility and secondary abnormalities. There were no significant stallion by treatment interactions. The proportion of sperm stained for SP22 was significantly (P<0.05) affected by season, stallion and freezing treatment. A significant (P<0.05) season by freezing treatment interaction was also present. A tendency (P<0.08) for the intensity of staining for SP22 to differ among stallions was noted for the 4 subset stallions. For cryopreserved sperm, the proportion of sperm staining for SP22 on the equatorial segment was affected differently by stallion or season depending on the SP22 antibody used. RNA yield from sperm was not affected by season, stallion, cryopreservation or their interactions. There was no effect of season or freezing treatment on relative quantity of mRNAs for PGK2, TPX1, TIMP3 or ACTB. Also, no season by freezing treatment or stallion by freezing treatment interactions were found. 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 a stallion effect on ACTB mRNA content. Sperm parameters and proportion of sperm staining for SP22 were affected by season, stallion and freezing treatment. Certain mRNA transcripts (PGK2 and ACTB) were also affected by stallion but season and freezing treatment do not appear to have an effect on any mRNA transcripts. Understanding these differences that exist in sperm from different seasons and stallions may prove to be beneficial to determine the best stallions and the best time to collect semen for cryopreservation.



Stallion, SP22, mRNA, Cryopreservation





Animal Science