Evaluation of Fertilization Capacity of Cryopreserved Stallion Sperm, Directly After Thawing and After Cooled Storage

In the modern equine breeding industry, use of cryopreserved semen is increasing. In comparison to using fresh or cooled semen, however, costs are higher and pregnancy rates lower. This is associated with the fact that using cryopreserved sperm requires special equipment for transport, storage and handling. Furthermore, sperm viability is reduced after cryopreservation, and cryopreserved semen therefore needs to be used for insemination close to ovulation. The aim of the studies described in this thesis was to investigate if cryopreserved semen maintains its fertilizing ability after thawing and cooled overnight transport (i.e. 24 h storage at 4°C). Furthermore, it was tested if performing insemination at a defined time point after hCG application was as effective as performing insemination close to ovulation (with inspection every 6 h). These simplifications would reduce costs and make cryopreserved insemination doses available to more practitioners and breeders. This study was divided in three parts. (1) First, viability of cryopreserved stallion sperm was determined in vitro; directly after thawing, after dilution with different extenders as well as an additional 24 h cooled storage. For cooled storage, to mimic transport conditions, thawed cryopreserved semen was diluted, transferred to a syringe and placed in a polystyrene transport box with a cool pack for maintenance at 4°C. Sperm plasma- and acrosomal membrane integrity was determined using flow cytometric analysis. Motility characteristics were evaluated using computer assisted sperm analysis (CASA). Only small differences were found between percentages of membrane intact sperm directly after thawing and after an additional 24 h cooled storage, irrespective of the extender used for dilution. Furthermore, percentages of progressively motile sperm were only 3−8% decreased after 1 d cooled storage. (2) Then, for testing fertilizing potential of cryopreserved sperm in vitro, a heterologous oocyte-binding assay was used. Therefore, cryopreserved stallion sperm was co-incubated with porcine oocytes in capacitation as well as control-medium, after which nuclei were stained and the number of sperm per oocyte/zona was counted. Sperm-oocyte binding was determined to be higher in capacitation than in control medium, and if using diluted versus cryopreserved sperm. Furthermore, the number of sperm bound per oocyte was not negatively affected if cryopreserved sperm was subjected to dilution and 1 d cooled storage after thawing, suggesting that fertilizing capacity was maintained. (3) Finally, an insemination trial was performed to determine mare pregnancy rates using cryopreserved stallion sperm directly after thawing as well as after 24−36 h cooled storage after thawing. In addition, different insemination regimes were tested, including insemination after a defined duration after hCG-administration as well as maximally 6 h after ovulation (i.e. determined according to ultrasound ovulation controls). The insemination trial revealed that pregnancy rates achieved with using cryopreserved sperm after thawing and 1 d cooled storage (13/25, 52%) were not significantly different from when using freshly thawed sperm (7/10, 70%), nor were pregnancy rates negatively affected if insemination was planned according to the moment of hCG application instead of ovulation controls. Taken together, viability and fertilizing capacity of cryopreserved stallion semen is not negatively affected if stored at 4°C for up to 24−36 h after thawing. Moreover, if used for artificial insemination, mare pregnancy rates are similar as those obtained with using freshly thawed sperm.