Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray (Canis lupus) and Mexican gray wolves (C. l. baileyi)

A current priority for the preservation of the endangered Mexican gray wolf (Canis lupus baileyi) is the development of a sperm-based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitüb, Tübingen, Germany) on the characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.