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. 2007 Oct 1;49(1):28.
doi: 10.1186/1751-0147-49-28.

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Ulrika Hermansson  1 , Eva Axnér, Bodil Ström Holst
Affiliations

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Ulrika Hermansson et al. Acta Vet Scand. .

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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Scanning electron micrograph of fresh spermatozoa bound to a frozen-thawed ZP.
Figure 2
Figure 2
Scanning electron micrograph of the ZP of in vitro matured feline oocytes.
Figure 3
Figure 3
Scanning electron micrograph of the ZP of frozen-thawed feline oocytes. The size of the scale bar is 10 μm as shown in figures.
See this image and copyright information in PMC

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