129 IMPROVEMENT OF BOAR SPERM CRYOSURVIVAL BY THE OPTIMIZATION OF CRYOPRESERVATION CONDITIONS

Abstract

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60°C min^-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200°C min^-1 (37°C water bath for 20 s) or ≈1800°C min^-1 (70°C for 8 s). The cryopreservation condition including 2% glycerol, 40°C min^-1 of cooling, and ≈1200°C min^-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800°C min^-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers.

This work was supported by CICYT (AGF2005-00760), Madrid, Spain.