title: Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection.
abstract: Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%-1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos.
authors.affiliation: Department of Obstetrics, Gynaecology and Reproduction, Chulalongkorn University, Faculty of Veterinary Science, Bangkok, Thailand.
authors.forename: S
authors.initials: S
authors.lastname: Buarpung
authors.forename: T
authors.initials: T
authors.lastname: Tharasanit
authors.forename: P
authors.initials: P
authors.lastname: Comizzoli
authors.forename: M
authors.initials: M
authors.lastname: Techakumphu
mesh_terms: D000818:Animals; D002415:Cats; D002469:Cell Separation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D005298:Fertility / Q000502:physiology; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D053625:Sperm Retrieval / Q000662:veterinary; D013094:Spermatozoa / Q000166:cytology*; D013737:Testis / Q000166:cytology*