In all species, penetration of the oocyte by sperm requires motility, intact receptor proteins on the sperm to bind to the zona pellucida, and the ability to undergo an acrosome reaction and bind to the plasma membrane of the oocyte. Different in vitro penetration assays have been developed to address each of these attributes .
Zona pellucida (ZP) sperm binding
Zona penetration assays evaluate sperm motility, zona binding and penetration, sperm capacitation, and the acrosome reaction . Capacitated spermatozoa from 3 fertile and 3 subfertile stallions were incubated with frozen-thawed equine oocytes . The total number of ZP-bound spermatozoa was higher for fertile than for subfertile stallions (p .
Salt-stored equine oocytes maintain spermatozoal receptors on the ZP and can be used in sperm binding assays . When salt-stored equine oocytes were used, binding of spermatozoa from some subfertile stallions appeared to be lower than for fertile stallions, but variation was present. One of the reasons for such discrepancies might be differences in the oocytes and in their ZP . In fact, immature oocytes bind fewer spermatozoa than oocytes in metaphase stage. The final stage of oocyte maturation is accompanied by some changes in the ZP .
Hemizona assay (HZA)
In the HZA, the 2 matched zona hemispheres created by bisection are functionally equal surfaces, allowing for a controlled comparison of sperm binding. Thus, the variation in binding capacity between individual ZP is eliminated. The binding capacity of two semen samples to matching hemizonae can be compared. When semen samples from 22 stallions with known fertility data were tested on salt-stored hemizonae, there was a significant relationship (p .
In another Dutch study, ejaculates from 5 stallions were split into two samples: one was frozen and the other stored and chilled for 24 h. Equine oocytes were bisected, and one hemizona incubated with the chilled semen and the matching half incubated with frozen semen. Four oocytes were used per stallion. There was a significant difference in sperm binding between chilled and frozen-thawed samples (50 ± 4 and 41 ± 4, respectively). The extent of the difference varied markedly between stallions .
Zona-free hamster oocyte penetration test (HOPT)
In vitro penetration of zona-free hamster oocytes provides information about the capability of sperm that have already undergone the acrosome reaction to penetrate the oocyte. Multiple spermatozoa can penetrate the heterologous hamster oocyte . In a Polish study, fresh hamster oocytes were incubated for 3 to 4 h with acrosome-reacted stallion spermatozoa. No conclusive relationship was established between sperm motility and the percentage of penetrated zona-free hamster oocytes . In another study, frozen-thawed zona-free hamster oocytes (20/ejaculate) and acrosome-reacted stallion spermatozoa (fresh and cooled for 24 or 72 hours) were incubated for 8 min. The ability of sperm to penetrate zona-free hamster oocytes was shown to decrease with increased storage time of semen . Penetration of zona-free hamster oocytes by frozen boar sperm was markedly reduced compared with fresh and liquid-stored semen . Neither the percentage of penetrated zona-free hamster oocytes nor the average number of spermatozoa penetrating each hamster oocyte were correlated with fertility, when cryopre-served stallion spermatozoa were tested .
In vitro fertilization (IVF)
IVF has proven to be a reliable test for sperm quality and fertilizing capacity in human fertility clinics, but being an invasive and expensive procedure, it cannot be routinely used as a semen evaluation test . IVF has been successfully used to differentiate between frozen semen from low- and high-fertility bulls . Unfortunately, IVF-techniques are not sufficiently advanced in horses to be used for this purpose.
The disadvantages of sperm penetration techniques are the time and expense needed, and that very few sperm are actually evaluated. The in vitro conditions are likely to be quite different from the in vivo environment .