Characteristics of fresh and frozen–thawed red wolf (Canis rufus) spermatozoa

Ejaculates of the red wolf (Canis rufus) were evaluated immediately after collection and freeze–thawing to initiate a reproductive database for this endangered species. Electroejaculates from 13 adult red wolves collected during the breeding season (February–March; n=25; 1–3 collections/male) had a mean volume of 4.7±0.7 ml, 146.5±25.7×106 spermatozoa/ml and 71.2% motile spermatozoa. The mean proportion of cells with normal morphology was 73.6±3.2% (range, 20.3–93.7%), with 64% of ejaculates (16/25) containing 70–90% normal spermatozoa. The four most predominant abnormalities were a coiled flagellum (8.1%), a bent flagellum (4.7%), a bent midpiece with no cytoplasmic droplet (3.3%;), and a detached head defect (6.4%). After cooling in glycerolated extender, semen was frozen using a pelleting method on dry ice before plunging into liquid nitrogen. Pellets were thawed in phosphate buffered saline and examined for % sperm motility, normal morphology, viability and intact acrosomes. There was a decline (P<0.05) in sperm motility (∼40%) and percentage of normal sperm (11.9%) after freezing, but no change in the proportion of viable cells. After freezing, there was a marked decline (P<0.05) in the proportion of intact acrosomes from 74.5% to 55.5% which was accompanied by an increased proportion (P<0.05) of partial acrosomes from 11.9% to 35.8%. These data demonstrate that, although red wolf spermatozoa can survive freeze–thawing using a technique common for domestic dog sperm, the finding of significant acrosome damage reveals (1) likely species specificity in the Canis genus and (2) the need for refining sperm cryopreservation technology for the red wolf.