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

Date Published: October 1, 2007

Publisher: BioMed Central

Author(s): Ulrika Hermansson, Eva Axnér, Bodil Ström Holst.


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.

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°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°C and 100% humidity in the presence of 5 × 106 fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

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. In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.

Partial Text

A combination of various in vitro tests is better at predicting fertility than is a single test as they can be combined to measure different attributes needed for fertilisation and early embryonic development [1]. Some laboratory tests, such as the zona pellucida binding assay (ZBA), together with tests used to investigate the penetration of the zona pellucida (ZP) and in vitro fertilisation (IVF), are able to test for several parameters and interpret the interaction between spermatozoa and the oocyte (for a review, see [2]). The approach of using such functional tests gives a better estimation of sperm fertilising capacity. Zona pellucida binding assays have been used to predict fertility in humans [3] and animals, for example cattle [4], dogs [5-8] and cats [9-11]. The ZBA has also been used to study how different sperm abnormalities influence fertility (for review, see [12,13]). Abnormal cat spermatozoa are capable of binding to and entering the outer ZP but are usually compromised in their ability to penetrate the inner zona and reach the perivitelline space to fertilise the oocyte [14,15].

Both fresh and FT spermatozoa bound to the ZP of in vitro matured oocytes (Table 1, Figure 1). The percentage of binding of electroejaculated spermatozoa was higher in in vitro matured ZP than in the immature, FT ZP (P < 0.05). Frozen-thawed epididymal spermatozoa bound to in vitro matured ZP but not to FT ZP (P < 0.001). Also, comparatively more fresh, electroejaculated than FT spermatozoa bound to the ZP (P < 0.001), with barely one fresh spermatozoon bound per FT ZP. Overall, binding was significantly different between in vitro matured and immature, FT ZP (P < 0.05). The results reveal that under the conditions of the present study, FT, immature ZP (oocytes) are less suitable for use in feline ZBA. Only a very low number of fresh spermatozoa bound to FT ZP, compared with their fresh, in vitro matured counterparts. With FT spermatozoa tested on FT ZP, there was no sperm-zona binding at all. This may partly be due to changes in the capacity of spermatozoa to maintain a normal plasma membrane surface that could bind to the specific receptors present in the ZP. The hypothesis that cat spermatozoa, either fresh or FT, could bind to homologous ZP of oocytes retrieved from FT queen ovaries to a similar extent as to the ZP of fresh, in vitro matured oocytes, proved false. Whatever the cause, under the conditions of the present study, ZP from immature oocytes from FT ovaries are less suitable for use in feline ZBA, in contrast to the situation in other species. Further studies are needed to explore the causes of our results. The author(s) declare that they have no competing interests. All authors participated in the design of the study. UH and EA collected the samples, and UH performed the ZBAs. UH drafted the manuscript. EA and BSH participated in the coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.   Source:


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