Cryopreservation of Boar Semen Studies on sperm viability in vitro and fertility

Eriksson, B. 2000. Cryopresewation of boar semen. Studies on sperm viability in vitro and fertility. Doctoral thesis. ISSN 1401-6257. ISBN 91-576-5903-6 The world’s pig population is consistently being upgraded through the international trade of superior genetics. The two major systems that are used for this purpose are the transport of live animals and the export of frozen boar semen. The main limiting factors for a wider use of frozen-thawed (FT) boar semen are low fertility levels of FT in comparison with liquid semen, and between-boar variation in freezing success. Consequently, there is a need for improved boar semen freezing methods. The main objective of this thesis was to develop a method for commercial freezing of boar semen, and the study of the effect of different factors on boar sperm survival in vitro and fertility after freezing-thawing in large, one-AI-dose containers. Semen was split-sample frozen in 5 mL Maxi-straws and in Cochette plastic bags. A computer-assisted sperm analyser (CASA) was used to evaluate sperm motility, while plasma membrane integrity (PMI) was assessed with fluorescent dyes. The fertilising capacity of the semen frozen in the two containers was assessed by inseminating (AI) gilts. The Cochettes yielded a significantly ( W 0 . 0 5 ) higher motility post-thaw, but the opposite was observed for PMI. No difference in fertilising capacity (pregnancy rate and number of viable embryos) could be seen. It was concluded that though proven feasible, the Cochette is not a suitable container for the commercial freezing of boar semen. The effects of different freezing and thawing rates on the post-thaw motility and PMI of boar spermatozoa, processed in either Maxi-straws or flat plastic packages called FlatPacks were studied. Values for percentage motile spermatozoa, sperm velocity and lateral head displacement, were significantly higher for samples frozen in FlatPacks than for those frozen in Maxi-straws (P<0.05). The best postthaw motility was obtained in semen frozen in FlatPacks at 50°C/min and thawed for 13 sec in a 50°C water-bath. No significant effect on post-thaw PMI was seen for the different freezing and thawing rates as well as package types used. Along with the freezing and thawing rates, extenders, packages and boars used in the present study, thawing rate had the greatest influence on post-thaw sperm survival followed by boar and freezing rate. The effect of holding time (HT) during cooling on PMI and motility before and after freezing as well as the in vitro penetration ability of boar spermatozoa post-thaw were investigated. Before freezing, the HT used had no significant ( P 0 . 0 5 ) effect on either PMI or percentage of motile spermatozoa. Post-thaw, PMI was significantly higher for a 10 h and a 20 h HT than for 3h, while the percentage of motile spermatozoa decreased significantly with a 20 h HT, as opposed to a HT of 3 h and 10 h (P<0.05). In terms of in vitro penetration ability, there was no significant difference among HTs. Based on these results, there is no reason to change the current protocol of a 3 h HT. However, a prolonged HT may be used if needed for practical reasons (e.g. sperm transport after collection for freezing, and more convenient time schedules, etc). In an insemination trial using boar semen frozen in FlatPacks and exported for artificial insemination (AI) to overseas nucleus herds, a mean farrowing rate (FR) of 73% (308 litters from 421 inseminations) and a mean total number of piglets born (TNB) of 10.7 were obtained. The results for A1 with FT vs. liquid semen, in a within-sow analysis of purebred Landrace (L) and Yorkshire (Y) animals, showed a lower FR (-6.5%) and TNB (-0.3 piglets) for the FT semen (both differences non-significant). Although a significant boar effect on the in vitro sperm parameters could be seen (P<0.05), the between-boar variations in post-thaw sperm quality were relatively small. These are encouraging results, especially considering the fact that they were achieved without any pre-selection, based on semen freezability, of boars or ejaculates. Collectively, the results show that the present freezing-thawing protocol and the FlatPacks maintain high sperm viability and fertility post-thaw, indicating they are a reliable alternative for freezing of boar semen under commercial A1 conditions.