Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year

Abstract

The roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P<0.05) values for post-thaw acrosome integrity (43.3% vs. 25.0%) and straight-line velocity (19 μm/s vs. 4 μm/s). For both freezing methods, sperm heads were smaller post-thawing than pre-freezing (P<0.001). In conclusion, both the pre-rutting and rutting season are suitable periods for freezing roe deer sperm. Ultra-rapid freezing did not provide suitable results.

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Acknowledgements

We thank the following hunting reserves for providing biological samples: Coto Regional de Caza de Morcín, Coto Regional de Caza Cabranes, Coto Regional de Caza del Cordal de Peon, Coto Regional de Caza de Valdedios, Coto Regional de Caza Sierra de Pulide, Coto Regional de Caza de Nalón, Reserva Regional de Caza de Caso, Reserva Regional de Caza de Aller, and Reserva Regional de Caza de Somiedo.

Funding

This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. P. Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was the recipient of a research fellowship from the PASPA-DGAPA-UNAM (México). V.N. Flores-Gil was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).

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Santiago-Moreno, J., Castaño, C., Bóveda, P. et al. Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year. Eur J Wildl Res 67, 24 (2021). https://doi.org/10.1007/s10344-021-01468-4

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Keywords

  • Cryopreservation
  • Spermatozoa
  • Ultra-rapid freezing
  • Wild ruminant