Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25° or 37°C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 °C and pelleted on solid CO₂ or frozen in 0.25 ml straws (20°C/min to -100°C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25°C was superior (P < 0.05) to 37°C for sustaining sperm motility, and glycerol did not influence (P > 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37°C (P < 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits). These results illustrate the sensitivity of ferret sperm motility and acrosomal integrity to different cryopreservation conditions; and demonstrate the biological competence of frozen-thawed ferret spermatozoa.