Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN2 vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN2. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 M sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN2. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P > 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P > 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P > 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P < 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids.
This study was funded by the Institute of Museums and Library Services.
Oviductal Extracellular Vesicles Improve Post-Thaw Sperm Function in Red Wolves and Cheetahs
