Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus)

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon)

Spermatozoa must undergo the process of capacitation to fertilize the egg which involves a cell destabilizing process. Capacitation-like changes such as protein tyrosine phosphorylation (PTP) are associated with cryopreservation. The aim of this study was to compare the cryoresistance and capacitation response of epididymal and ejaculated sperm of European mouflon (Ovis musimon). Post-thaw sperm parameters were analysed from epididymal and ejaculated samples cryopreserved by slow-freezing or ultrarapid-freezing for comparison. Sperm capacitation status was assessed by the semiquantification of PTP levels, cell localization of PTP and kinematic clustering. Epididymal sperm had higher cryoresistance than ejaculated sperm in both freezing techniques, and slow-freezing rendered better results than ultrarapid-freezing in both sperm samples. Ejaculated sperm had higher PTP levels than epididymal sperm and, additionally, ejaculated sperm showed higher phosphorylation in capacitating (CA) than in non-capacitating (NCA) conditions while there was no effect of medium in epididymal sperm. There was a higher tail PTP in CA than in NCA conditions in both types of sperm. Kinematic analysis revealed that the cluster associated with hyperactivated movement increased in ejaculated sperm incubated in CA whereas no effect of medium was observed in epididymal sperm clusters. In conclusion, epididymal sperm showed better freezability and lower capacitation status compared to ejaculated sperm.

100 A COMPARISON OF GLYCEROL AND EGTA FOR ULTRARAPID FREEZING OF BULL EPIDIDYMAL SPERM

The ultrarapid freezing technique was developed as an alternative to slow conventional freezing to avoid formation of ice crystals (Tucker MJ and Liebermann J 2007, Vitrification in Assisted Reproduction, 87-92). The recent use of ethylene glycol tetraacetic acid (EGTA) instead of glycerol as a cryoprotectant for sperm is a result of efforts to reduce osmotic and cytotoxic effects. Accordingly, the objective of the present study was to compare the cryoprotective effects of EGTA v. glycerol on bovine sperm frozen using an ultrarapid method. A pool of epididymal sperm collected from 5 bulls was maintained in Botusui extender (Biotech, Botucatu, Brazil) containing 5% BSA, either EGTA (5%) or glycerol (5%), and 0 or 2 mg mL of polyvinyl alcohol (PVA). All samples were loaded into 0.25-mL French straws and either placed into a freezing machine (TK 4000 compacta, TK equipamentos para reproducao, Uberaba, Brazil) and cooled at a rate of 50°C/min, or exposed to liquid nitrogen (LN2) vapor for 5 to 15 min before plunging into LN2. After overnight storage in LN2, straws were thawed and examined using CASA (HTM IVOS 12, Hamilton Thorne Research, Beverly, MA, USA) for total motility (TM), progressive motility (PM), path velocity (VAP), progressive velocity (VSL), and track speed (VCL). Acridine Orange (AO) was used to check DNA integrity of sperm cells (Chirinea VH et al. 2006 Ciencia Animal Brasileira 7, 407-415). Our results indicate that, despite the potentially damaging effects of the extreme temperature changes, epididymal sperm showed 95% of DNA integrity and, therefore, should be able to participate in the fertilization process. Addition of PVA had a negative effect on sperm motility characteristics. CASA revealed that glycerol was a more suitable cryoprotectant for ultrarapid freezing of bull epididymal spermatozoa than was EGTA (see Table 1). Table 1. CAPES.