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Influence of Various Permeating Cryoprotectants on Freezability of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa: Effects of Concentration and Temperature of Addition

MARÍA R. FERNÁNDEZ-SANTOS^*,

MILAGROS C. ESTESO^*,

VIDAL MONTORO

JOSÉ J. GARDE^*,

From the ^* Biology of Reproduction Group, Department of Game Resources (IDR), Castilla-La Mancha University (UCLM), Albacete, Spain; and the National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, Albacete, Spain.

Correspondence to: Dr José Julián Garde, IDR, Sección de Recursos Cinegéticos y Ganaderos (IDR), Campus Universitario, 02071, Albacete, Spain (e-mail: Julian.Garde{at}uclm.es).

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of 400 x 10⁶ spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37°C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition –22°C (ambient temperature) and 5°C2 on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22°C (81.94 ± 2.4% vs 72.38 ± 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22°C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22°C, as an alternative to the more common 3%–4% of GLY and addition at 5°C, in red deer semen freezing protocols.

     Key words: Ethylene glycol, glycerol, propylene glycol, sperm, cryopreservation