Effect of cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa

DANIEL ACHA VALLS; MANUEL HIDALGO; MARIA JOSE GALVES; ISABEL ORTIZ; JOSE CARRASCO; VICTOR GOMEZ; RAFAEL CALERO; SEBASTIÁN DEMYDA PEYRÁS; JESUS DORADO

Bolonia

Congreso; 17th annual European Society for Domestic Animals Reproduction Conference; 2013

European Society of Domestic Animal Reproduction

The aim of this study was to evaluate the effect of different cooling rates on cryopreserved sperm quality in Andalusian donkeys. Eight ejaculates from four donkeys (2 ejaculates/donkey) were collected using an artificial vagina. Semen samples were pooled (2 ejaculates/pool), split, diluted in Gent B
and frozen using four different cryopreservation protocols: (i) Cooling in Equitainer
for 2 h and freezing in N2L vapour; (ii) Cooling using a programmable cooler system (the cooling rate was
2°C/min from 25 to 22°C,
0.3°C/min between 22°C and 10°C, and
0.2°C/min from 10 to 4°C) and freezing in N2L vapour; (iii) Cooling in a refrigerator at 5°C for 1 h and freezing in a programmable freezer (the freezing rate was
6°C/min from 5 to
8°C and
4°C/min between
8 and
50°C); and (iv) Ultra-rapid freezing in N2L vapour. Fresh semen and frozen-thawed samples were assessed for motility (CASA), morphology (Diff-Quik
), acrosome integrity (FITC-PNA/PI staining) and viability (Duo-Vital
kit). Data were analyzed by ANOVA and Duncan?s test. Significant (p < 0.001) differences were found between cryopreservation protocols for all sperm parameters evaluated. Semen samples frozen using protocol 2 showed significantly (p < 0.001) higher values for sperm motility, sperm viability, acrosome integrity and sperm morphology. In conclusion, our results suggest that Andalusian donkey semen can be successfully cryopreserved using slow cooling rates (particularly between 10 and 4°C) combined with freezing in N2L vapour.