**2.4. Sperm: Ovum interaction and fertilization**

Given the voluminous nature of the hen's ovum and the GD relative to mammalian ova, one must assume that yet-to-be identified factors "attract" sperm to the GD. Examination of the electrophoretic profile of the GD and non-GD regions of the PL revealed no variation in pro‐ tein composition [70]. Furthermore, the abrogation of the preferential interaction of sperm and the inner PL overlying the GD *in vitro* suggests the factors underlying the preferential binding of sperm are not necessarily associated with the inner PL [70]. It is clear, however, glycoproteins play a large role in the interaction between the sperm and ova, even if not di‐ rectly involved in targeting of sperm to the GD *in vivo* [71]. Pre-treatment of either the PL or sperm with N-glycanases resulted in significantly decreased sperm-ovum interaction *in vitro* [68, 71]. Conversely, N-linked oligosaccharides released from the inner PL by N-glycosidase treatment could induce the acrosome reaction in sperm *in vitro* [72]. These findings strongly suggest N-linked glycans, most likely terminal N-acetyl glucosamine residues, have an es‐ sential role in the sperm-ovum interaction in avian species, specifically in induction of the acrosome reaction [68, 72].

Interaction between the sperm and inner PL results in induction of the acrosome reaction [73]. During the acrosome reaction, the inner and outer acrosomal membranes dehisce re‐ sulting in the release of acrosin (a trypsin-like enzyme) [21, 74]. As the result of the acro‐ some reaction, sperm hydrolyze a small hole in the inner PL (Figure 5), enabling sperm to reach the microvilli-studded surface of the ovum [21, 74]. The capacity of sperm to hydro‐ lyze and penetrate the inner PL is the biological basis for the sperm penetration assay dis‐ cussed below and next section.

reduction of available ATP and sperm motility decreases [56]. Sperm are then swept out of the SST lumen into the UVJ, where they encounter various stimuli enhancing their motility. These sperm are then transported to the infundibulum, the site of fertilization [57]. Such motilityenhancing factors may include changes in environmental pH and neuroendocrine factors such as serotonin [58-62]. Further oxidation of sperm fatty acids, possibly sequestered from the surround milieu, generates the energy required for sperm to respond to such motility-

Once sperm are deposited in the oviduct, several selection barriers must be overcome prior to ascending to the infundibulum and fertilizing an ovum. This selection occurs initially in the vagina: only highly mobile (defined as progressive movement in a viscous medium at 40o

sperm traverse the vagina [9]. While sperm mobility is a major factor in sperm selection in the vagina, sperm selection is also dependent upon the glycoprotein composition of the sperm plasma membrane. The sperm glycocalyx is highly complex and heavily sialylated and modification of the glycocalyx results in reduced fertility and failure of the sperm to enter the SSTs [64-67]. Interestingly, removal of membrane-associated carbohydrates did not affect sperm entry into SSTs if sperm were inseminated directly into the UVJ or when co-incubated with UVJ explants, suggesting the glycocalyx plays a central role in sperm transport and selection through the vagina [64, 66, 68]. Further barriers to sperm prior participating in the process of fertilization include sperm release from the SST and subsequent transport to the

Given the voluminous nature of the hen's ovum and the GD relative to mammalian ova, one must assume that yet-to-be identified factors "attract" sperm to the GD. Examination of the electrophoretic profile of the GD and non-GD regions of the PL revealed no variation in pro‐ tein composition [70]. Furthermore, the abrogation of the preferential interaction of sperm and the inner PL overlying the GD *in vitro* suggests the factors underlying the preferential binding of sperm are not necessarily associated with the inner PL [70]. It is clear, however, glycoproteins play a large role in the interaction between the sperm and ova, even if not di‐ rectly involved in targeting of sperm to the GD *in vivo* [71]. Pre-treatment of either the PL or sperm with N-glycanases resulted in significantly decreased sperm-ovum interaction *in vitro* [68, 71]. Conversely, N-linked oligosaccharides released from the inner PL by N-glycosidase treatment could induce the acrosome reaction in sperm *in vitro* [72]. These findings strongly suggest N-linked glycans, most likely terminal N-acetyl glucosamine residues, have an es‐ sential role in the sperm-ovum interaction in avian species, specifically in induction of the

Interaction between the sperm and inner PL results in induction of the acrosome reaction [73]. During the acrosome reaction, the inner and outer acrosomal membranes dehisce re‐ sulting in the release of acrosin (a trypsin-like enzyme) [21, 74]. As the result of the acro‐ some reaction, sperm hydrolyze a small hole in the inner PL (Figure 5), enabling sperm to reach the microvilli-studded surface of the ovum [21, 74]. The capacity of sperm to hydro‐ lyze and penetrate the inner PL is the biological basis for the sperm penetration assay dis‐

C)

enhancing factors and transcend the oviduct [9, 22, 55, 63].

182 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

infundibulum, and their interaction with the ovum (reviewed in [69]).

**2.4. Sperm: Ovum interaction and fertilization**

acrosome reaction [68, 72].

cussed below and next section.

**Figure 5.** In the left panel, a turkey sperm stained with Hoechst 33342 prior to insemination is observed on the surface of the inner perivitelline layer (PL). The sperm's acrosome will release a trypsin like enzyme, acrosin, and digest a hole through the inner PL. The right panel shows multiple sperm holes (white perforations) in the inner PL overlying the germinal disc (GD) of a duck ovum (polyspermy is normal in birds). Sperm hole numbers can be used to assess true fertility and the duration of the fertile period..

Unlike mammals, polyspermy is the norm in avian fertilization. The GD (3.5 mm in diameter) provides a relatively small target for fertilization in the large megalecithal ova (yolk-filled ova) of chickens and turkeys (3.5 – 4.0 mm in diameter); thus polyspermy may be an evolutionary adaptation to ensure higher rates of fertilization in such species [74]. The inner PL may be penetrated by many sperm, although only one male pronucleus will ultimately fuse (syngamy) with the female pronucleus to form the nascent embryo (reviewed in [75-77]. A single sperm hole in the inner PL does not ensure fertilization. Although turkeys show a lower number of sperm interacting with ova relative to chickens, the presence of three sperm holes in the inner PL predicts a 50% probability of fertilization, whereas, six sperm holes suggest a probability greater than 95% fertilization [78]. The outer PL is rapidly depositied around the ovum in the posterior infundibulum and proximal magnum and is impenetrable by sperm [21, 78-79] thus preventing pathological polyspermy.

Given the volume of the GD relative to a single sperm, another possible function of polyspermy may be to activate specific molecular factors in the GD cytoplasm thereby initiating the process of embryogenesis. Yet, polyspermy also results in the presence of multiple male pronuclei in the GD. To cope with this potentially harmful scenario, the mature ovum has been found to have DNase I and II endonuclease activities, both of which will degrade sperm DNA [76]. In contrast, no such DNase activity has been detected in mammalian ova that engage in mono‐ spermic fertilization, further suggesting the role of these enzymes in the avian embryo is to protect against detrimental genetic consequences of polyspermy [76].

The number of holes in the inner PL is highly positively correlated with fertility. Correlations exist between the number of sperm inseminated, the number undergoing the acrosome reaction at the inner PL [80], and the number of sperm embedded in the outer PL [81]. The number of sperm holes in the inner PL and the number sperm trapped in the outer PL may be used to estimate the duration of fertility ('fertile period') in hens. While the number of sperm penetrating the inner PL shows a decreasing logarithmic relationship over time [81-82], a positive correlation between the total number of sperm penetrating the inner PL and the number of sperm stored in the SSTs was observed [83]. Given these observations, it should not be surprising there is also a positive correlation between the number of SSTs containing sperm and the proportion of sperm that have undergone the acrosome reaction at the inner PL [82].
