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Erdmann, R.H.; Blank, M.H.; Ribeiro, R.N.; Oliveira, M.J.d.; Cubas, Z.S.; Pradiee, J.; Goularte, K.L.; Moreira, L.
Cryopreservation of margay (_Leopardus wiedii_) spermatozoa: effects of different extenders and frozen protocols
2019  Theriogenology (143): 27-34

Sperm cryopreservation offers many benefits to wild felids conservation programs. However, the implementation of these programs is limited by the different responses of each species to the cryopreservation protocols and extenders used, requiring the formulation of species-specific protocols. For this purpose, semen samples from 6 margays (_Leopardus wiedii_) were submitted to 2 cryopreservation protocols: 1) manual freezing (cooling rate of - 0.33øC/min at 5øC/180min and freezing rate with two steps - 9øC/min for 2min and-19.1øC/min for 2min) and 2) automatic freezing machine (cooling rate of - 0.25øC/min at 5øC/120min and freezing rate with one step-20øC/min for 8.3min) using 2 commercial extenders, an egg yolk-based (Test Yolk Buffer; TYB) and an egg yolk-free extender (AndroMed; MED). Post-thawed sperm quality was assessed at 3 time points (immediately after thawing and 1 and 2h post-thawed) by sperm motility index (SMI), plasma membrane and acrosomal integrity, and mitochondrial membrane potential (MMP). Regarding SMI, TYB yielded superior results (29.4ñ3.5%) compared to MED (11.2ñ2.8%; p<0.002) immediately after thawing until 2h after thawing (TYB 3.9ñ1.7% and MED 0.0ñ0.0%; p<0.05). Furthermore, the automated freezing method provided higher motility compared to the manual freezing procedure immediately post-thaw (25.08ñ3.66% and 15.78ñ3.29%, respectively) and 1h post-thaw (13.71ñ2.56% and 6.03ñ1.97%, respectively; p<0.05). The percentage of intact acrosomes and plasma membranes and the percentage of sperm with high MMP were superior for TYB when compared to MED regardless of cryopreservation protocol (p<0.05). Conversely, the interaction between cryopreservation protocols and extenders was observed for MMP where TYB exhibits better results compared to MED (p<0.05) in both procedures, but it was higher in automated procedures. For MED, no changes were found in MMP between procedures. Considering only TYB, samples showed higher MMP when submitted to an automated procedure (p<0.05). In conclusion, the slow cooling rates with shorter time of exposure to glycerol contributed to minimize cryo damage in the Margays' sperm. Moreover, results indicated that association between TYB and automatic freezing machine ensured the minimal quality of spermatozoa after thawing required for further use in in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

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