title: Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes.
abstract: BACKGROUND
  Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.
  
  
  METHODS
  Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20 degrees C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO2 in air at 38 degrees C and 100% humidity in the presence of 5 x 106 fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).
  
  
  RESULTS
  Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.
  
  
  CONCLUSIONS
  In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20 degrees C are less suitable for use in feline ZBA.
authors.affiliation: Department of Clinical Sciences, Division of Diagnostic Imaging and Clinical Pathology, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-750 07 Uppsala, Sweden. ulrika.hermansson@kv.slu.se
authors.forename: Ulrika
authors.initials: U
authors.lastname: Hermansson
authors.forename: Eva
authors.initials: E
authors.lastname: Axnér
authors.forename: Bodil Ström
authors.initials: BS
authors.lastname: Holst
mesh_terms: D000818:Animals; D001681:Biological Assay; D002415:Cats; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D005260:Female; D008297:Male; D009865:Oocytes / Q000502:physiology*; D009866:Oogenesis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*; D015044:Zona Pellucida / Q000378:metabolism