Sperm cryopreservation in the Far‐Eastern wildcat (Prionailurus bengalensis euptilurus)

The Far‐Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far‐Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far‐Eastern wildcat captive‐born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen–thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2% ± 3.0% for the domestic cat and 38.0% ± 10.2% for the Far‐Eastern wildcat. The motility of frozen–thawed spermatozoa was 30.8% ± 9.8% for the domestic cat and 33.7% ± 15.1% for the Far‐Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9 ± 5.9 for the domestic cat and 55.0% ± 6.4% for the Far‐Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2% ± 4.8% and 30.8% ± 4.4% of all reported anomalies for the domestic cat and Far‐Eastern wildcat, respectively). Domestic cat epididymal and Far‐Eastern ejaculatory spermatozoa fertilized in vitro‐matured oocytes of the domestic cat (30.0% ± 5.5% and 35.5% ± 15.0%, respectively). Taken together, these results suggest that the freezing of Far‐Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.     

Effects of slow freezing and vitrification on embryo development in domestic cat

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4–8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (–2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation.     

Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus)

Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four‐cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third‐day post coitum and frozen in 0.25‐ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non‐permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non‐frozen four‐cell embryos developed up to the morula stage in rat one‐cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24‐h period of culture. The rate of embryo's surviving the freezing–thawing procedures, as estimated by light microscopy, was 60.7–68.8%. After 24‐h culturing in R1ECM, 64.7% of frozen–thawed four‐cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM‐CSF (2 ng/ml) improved the rate of Djungarian hamster frozen–thawed embryo development: 100% of the four‐cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen–thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.