| The black-footed ferret (Mustela nigripes) is an endangered North American carnivore. This species has relied on assisted reproductive techniques, like artificial insemination (AI), to propagate genetically valuable animals that failed to breed naturally. Artificial insemination that utilizes fresh semen has a 60% pregnancy rate, but only has 22% success when frozen-thawed semen is used. This decreased efficiency is due to a sub-optimum cryopreservation method resulting in reduced sperm viability. This project was designed to develop an improved sperm cryopreservation technique for the black-footed ferret by determining the (1) semen osmolality and the effect of media osmolality, (2) effect of various cooling rates (ultra-rapid to ultra-slow), (3) influence of culture medium (Ham's), egg yolk (TEST Yolk Buffer, TYB) and glycerol (TYB with 4% glycerol), (4) influence of different cryomethods (pellets versus straws) and (5) effect of various temperatures (5°C versus 25°C) and rates (1-step versus 3-steps) of glycerol addition on post-thaw sperm viability. Results demonstrated that black-footed ferret semen has a high osmolality (∼500 mOsm; range, 366-791 mOsm) and that ferret spermatozoa, particularly sperm motility, were sensitive to hyperosmolality. Sperm viability was not affected by seminal plasma, and did improve over time (P < 0.001) when maintained at 25°C versus 37°C. Although TYB maintained better sperm motility and forward progression (P=0.005), more NAR were retained in Ham's (P < 0.001). Slow cooling (0.2°C/min) was the best method (P<0.001), whereas ultra-rapid cooling (9.0°C/min) was the most detrimental treatment based on sperm viability (P<0.01). The pellet and 2-step straw methods were better freezing methods than the 1-step straw method (P = 0.002). Spermatozoa from black-footed ferrets were more sensitive to cryopreservation damage than from domestic ferrets. Glycerol did not have a negative effect on black-footed ferret spermatozoa, and can be added safely at 25°C or 5°C in 1-step or 3-steps. Acrosomal integrity was better maintained after thawing at 50°C versus 37°C (P = 0.02). Overall, acrosomal integrity was more negatively affected by freeze-thawing than sperm motility and forward progression. These findings will guide the development of improved assisted breeding protocols contributing to the genetic management of this rare species. |
