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Felid semen has historically been frozen using an egg yolk-based cryopreservation medium. However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. In the present study, our aim was to compare a chemically-defined, soy-based medium (SOY) to a commercial egg yolk-based medium (TEY) for the cryopreservation of sperm in four imperiled small cat species. Semen was collected from adult male cats (n = 6 black-footed cats; n = 6 sand cats; n = 4 fishing cats; and n = 7 Pallas' cats) via electroejaculation, split into two aliquots, and cryopreserved in SOY or TEY. Frozen-thawed samples were evaluated for sperm motility and rate of progressive motility (up to 24 h post-thaw) and acrosome status (0 and 6 h). No difference in post-thaw traits were observed between treatments in all four species. Heterologous IVF using oocytes collected laparoscopically from domestic cats demonstrated no difference among freezing treatments in percentage of mature oocytes that cleaved or the mean number of blastomeres at 48 h post-insemination. More spermatozoa frozen with SOY were bound to the zona pellucida in the sand cat (P = 0.018), but no treatment effect was observed in the other three species. These findings collectively demonstrate that SOY may be a preferable alternative to TEY for sperm cryopreservation in these four wild felid species., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk -TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender addition nor pre-freeze cooling rate had a discernable effect on post-thaw otter sperm parameters. These findings represent the first assessment of semen cryopreservation in any otter species and may be of value as a model for development of semen cryopreservation strategies in other endangered otter species., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objective was to compare a proprietary egg yolk-based cryopreservation medium with a chemically defined soy-based medium, as well as to examine effects of temperature of glycerol addition on sperm parameters and IVF after freezing and thawing of domestic cat sperm. Semen was collected from adult cats (four males and three ejaculates per male), divided in four equal aliquots, and extended in either egg yolk with 4% glycerol added before (EYG) or after (EY) cooling to 5 °C, or soy-lecithin with 4% glycerol added before (SLG) or after (SL) cooling to 5 °C. Extended sperm were frozen in straws over liquid nitrogen vapor. Sperm progressive motility (%) and rate of progressive movement (scale of 0-5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation, acrosome integrity, and DNA integrity were assessed at 15 min post-thaw. Effects of media (EY or SL) on IVF success was also examined (three males and three ejaculates per male). Sperm motility was greater (P < 0.05) in soy-based compared with egg yolk-based media at 3, 6, and 24 h post-thaw. A higher (P < 0.05) percentage of noncapacitated sperm (pattern F) were present in soy-based (SLG, 63.7 ± 9.2%; and SL, 64.1 ± 9.2%) compared with egg yolk-based (EYG, 49.9 ± 7.9%; and EY, 52.4 ± 18.6%) cryopreservation media, regardless of temperature of glycerol addition. Addition of glycerol at 5 °C increased (P < 0.05) percentage of sperm motility at 6 h (EYG 16.3 ± 8.3% vs. EY, 24.0 ± 11.7%; SLG, 36.7 ± 6.5% vs. SL, 42.9 ± 10.1%) and 24 h (EYG, 2.1 ± 3.3% vs. EY, 8.3 ± 3.9%; SLG, 11.3 ± 8.3% vs. SL, 18.8 ± 7.4%) post-thaw in both media. There were no differences (P > 0.05) between cryodiluents in embryo cleavage, percentage of embryos reaching blastocyst, or cell number per blastocyst. The chemically defined, soy-based medium resulted in better preservation of long-term motility and capacitation status of frozen-thawed domestic cat sperm compared with a commercial egg yolk-based extender, without compromising fertilizing ability., (Copyright © 2012 Elsevier Inc. All rights reserved.)