title: Birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue.
abstract: The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 μm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm.
authors.affiliation: Department of Obstetrics, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. theerawat.t@chula.ac.th
authors.forename: T
authors.initials: T
authors.lastname: Tharasanit
authors.forename: S
authors.initials: S
authors.lastname: Buarpung
authors.forename: S
authors.initials: S
authors.lastname: Manee-In
authors.forename: C
authors.initials: C
authors.lastname: Thongkittidilok
authors.forename: N
authors.initials: N
authors.lastname: Tiptanavattana
authors.forename: P
authors.initials: P
authors.lastname: Comizzoli
authors.forename: M
authors.initials: M
authors.lastname: Techakumphu
mesh_terms: D000818:Animals; D002415:Cats / Q000196:embryology*; D015925:Cryopreservation / Q000662:veterinary; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D008297:Male; D011247:Pregnancy; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology*; D013737:Testis / Q000502:physiology*