Fertility of frozen ram semen under field conditions : with special reference to influence of extenders and freezing procedures

A freezing method for ram semen is currently applied in Scandinavia, with good fertility using cervical artificial insemination (AI). The method consists of a two-step extension procedure in a milk-egg yolk extender (7% glycerol), followed by centrifugation to concentrate the extended semen before packaging in 0.25 ml straws and freezing in a programmable freezer. However, this method is tedious and includes steps considered stressful for spermatozoa, such as centrifugation. Moreover, the extender used contains components of animal origin, a potential source of undesired contamination. If genetic improvement of current sheep flocks in developing countries is to be done using frozenthawed (FT) semen for AI, it is imperative to use cheap, uncomplicated freezing procedures and extenders free from potential contamination. The FT-semen must be easily inseminated (using cervical sperm deposition) and acceptable fertility levels after AI should be reached under prevailing conditions of animal husbandry.The overall aim of this thesis was, therefore, to simplify current procedures to freeze ram semen in mini straws (0.25 mL), including the evaluation of several extenders and freezing protocols using a split-sample design. The procedures should result in acceptable post-thaw sperm survival with potential fertilizing capacity when tested in vitro, as well as in reasonable fertility after single cervical AI of extensively managed Corriedale ewes during spontaneous oestrus in Uruguay, Thus, the studies comprised trials performed in Sweden (in vitro) and in Uruguay (in vitro and in vivo).The in vitro studies included alternative extenders and elimination of centrifugation during handling. Semen frozen in a programmable freezing chamber (studies in Sweden) or frozen in an isotherm box, 4 cm above the surface of the liquid nitrogen (LN2) for 10 min (studies in Uruguay), was analyzed for several sperm parameters post-thaw. These were subjective motility (SM), computer assisted sperm-motility analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (CTC). The in vivo studies were done under commercial-like conditions in Uruguay during two consecutive breeding seasons (April 2000 and April 2001), where a total of 1,270 Corriedale ewes were cervically inseminated, once, during spontaneous estrus. Fertility after AI was recorded as non-retum rates at 21 days (NRR-21); non-retum rates at 36 days (NRR-36); pregnancy rates (PR-US, ultrasonography at 50 days) and lambing rates (LR).Replacement of the centrifugation by an adjusted semen extension, yielded post-thaw significantly higher numbers of spermatozoa with desirable characteristics per straw, compared to controls (centrifugation). The use of a clarified milk-based extender resulted in slightly better (NS) post-thaw sperm quality compared to the non-clarified milk extender, and significantly better if compared to a conventional TRIS-citrate-fructose extender (SM: 57.5% vs. 43.8%; linear motile spermatozoa [CASA]: 46.5% vs. 33.7%, uncapacitated spermatozoa: 49.7% vs. 34.4%). Increasing the concentration of egg yolk in the milk-extender above 5-10% did not have any beneficial effects on post-thaw sperm parameters. Furthermore, post-thaw sperm quality was, generally, better preserved when the second fraction with glycerol was added at 5°C rather than at 15°C. For further studies, a two-step (two-fractions) freezing protocol using a clarified milk extender with 5% egg yolk and 7% glycerol (added in the second fraction at 5°C) was used as control and compared in vitro to Bioexcel1* (IMV, a commercial extender free from animal-derived components available for bull semen) containing either 3.2% or 6.4% glycerol. No significant differences in sperm characteristics post-thaw were seen between the milk-egg yolk extender (control) and Bioexcell® 6.4% glycerol. Both gave significantly higher (PFertility after AI was tested, in two field trials, by single cervical deposition of FTsemen (200 x 106 total spermatozoa/straw, frozen in an isotherm box) in ewes during spontaneous estrus. The two above mentioned procedures of adjusting sperm numbers prior to freezing, tested in 300 Corriedale ewes (April 2000) did not differ significantly (for either NRR-21 [30.8% vs. 29.7%], NRR-36 [28.5% vs. 27.8%] or LR [21.9% vs. 21.4%], respectively). This indicated that the simplified protocol without centrifugation not only resulted in higher numbers of viable spermatozoa, but also seemed to be a suitable procedure for freezing ram semen under field conditions. The fertility results after AI in 970 Corriedale ewes (April 2001) with FT semen extended in Bioexcell® 6.4% glycerol or milk-5% egg yolk extender did not differ significantly for NRR-21 (36% vs. 33%), NRR36 (35% vs. 33%) or PR (28% vs. 27%).It was concluded that a simple two-step protocol using adjusted extension to achieve the final sperm concentration (200 x 106 total spermatozoa/straw) and avoiding centrifugation, could be used to freeze ram semen, also when freezing in static LN2 vapors. This freezing method appeared as suitable for AI of large flocks in developing countries since it did not require any expensive equipment, yielded a good post-thaw sperm survival, and resulted in fairly acceptable fertility after a single cervical AI with FT-semen of ewes in spontaneous estrus under conditions of extensive animal husbandry. Similar fertility results could be reached with ram semen frozen either in milk-based extenders or in Bioexcell* (6.4% glycerol), the latter being a safer alternative compared to current extenders when considering introduction of genetic material