**2.2. Oviduct**

In the following chapter we will review the basics of AI and fertility evaluation in poultry. To better appreciate the biological basis of these techniques, an overview of the reproductive biology of poultry is provided. Detailed descriptions of techniques for the collection, evalua‐ tion, dilution, and storage of poultry semen are available in a recent publication by Bakst and Long [4]. Earlier comprehensive reviews include Lake and Stewart [5], Bakst and Wishart [6],

This section will introduce to some and review for others the strategy of avian reproduction with emphasis on the hen. For more comprehensive reviews on reproduction in the avian male

The goal of AI is to produce a succession of fertilized eggs between successive inseminations. To accomplish this, weekly inseminations must replenish the sperm population in the uterovaginal junction (UVJ) sperm storage tubules (SSTs). Birds do not have an estrous cycle that synchronizes copulation with ovulation. Alternatively, about 7-10 days before their first ovulation, hens mate, sperm ascend the vagina and then enter the SSTs. At the onset of egg production, individual sperm are slowly released from the SSTs, transported to the anterior end of the oviduct, and interact with the surface of the ovum (see [9-10] for recent reviews). Whether fertilized or not, over the next 24-26 hr the ovum is transported though the oviduct, accruing the outer perivitelline layer (PL) in the infundibulum, the albumen in the magnum, the shell membrane in the isthmus, and the hard shell in the uterus (also referred to as the shell gland) before oviposition. If fertilized, the blastoderm in the first laid egg consists of

Ovary: In the hen only the left ovary and oviduct become functional organs. About 2-3 wk before the onset of lay, small (less than 1 mm in diameter) white-yolk follicles begin to accumulate yellow yolk with some being recruited into a hierarchy of maturing yellow-yolk follicles (Figure 1). At the time of ovulation, the largest follicle, designated as F1, is ovulated. About 17 days were necessary for the 1 mm diameter white yolk follicle to mature to a preovulatory 40 mm diameter yellow yolk follicle [11]. After the F1 follicle is ovulated, the next largest follicle, formerly designated F2, becomes the F1 follicle and will ovulate at the beginning

The follicular sheath surrounding the maturing oocyte consists of histologically distinct con‐ centric layers of cells: the outer serosa (germinal epithelium); the theca externa, which forms the greatest portion of the follicle wall, provides structural support to the follicle and has steriodo‐ genic cells; the theca interna, a highly vascularized layer, which like the theca externa has ste‐ roid-producing cells (both thecal layers synthesize androgens and estrogens); and, the granulosa cell layer, enveloping the oocyte, which is responsible for progesterone secretion and the synthesis of the inner PL. The inner PL is homologous to the mammalian zona pellucida and

40,000-60,000 cells in the turkey and 80,000-100,000 cells in the chicken.

of the next daily "ovulatory cycle" in 24-26 hr.

and Bakst and Cecil [7].

and female see Jamieson [8].

**2.1. Overview**

**2. Reproductive biology of poultry**

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

The mature oviduct consists of five anatomically and functionally distinct segments (Figures 1 and 2): the infundibulum, which secretes an albumen-like product that forms the outer PL and prevents pathological polyspermy; the magnum, responsible for deposition of the albumen proteins; the isthmus, which forms the shell membrane; the uterus (also referred to as the shell gland), a pocket-like structure that elaborates the hard-shell; and, the vagina, which is a conduit between the uterus and cloaca for the egg-mass at oviposition and is responsible for sperm selection and storage following semen transfer. Interestingly, when the vagina and uterus are excised and fixed *in toto* and the connective tissue surrounding the vagina subse‐ quently removed, the vagina appears as a coiled segment (Figure 3) [10]. This anatomy explains the resistance one feels when performing a vaginal insemination with a straw regardless of the presence or absence of an egg mass in the uterus. If inseminating a hen within 30 min after oviposition, the connective tissue around the vagina and the smooth muscle composing the vaginal wall are flaccid. Venting (exteriorizing the vagina for placement of the inseminating straw) at this time may induce a partial prolapse leading to a deep insemination (closer to the UVJ) and the forfeiture of sperm selection by the vagina. Such deep inseminations are associated with high embryo mortality, possibly due to pathological polyspermy.

only 5,000-13,500 [29-30]. Additionally, after several generations of selection for high fertility, chicken hens possessed increased numbers of SSTs when compared to non-selected control hens, suggesting the number of SSTs may be positively correlated with fertility [31]. In contrast, under commercial conditions, different broiler strains exhibiting different fertility levels

Artificial Insemination in Poultry http://dx.doi.org/10.5772/54918 179

**Figure 2.** The segments of the turkey oviduct with a hard-shelled egg in the uterus are observed. Sperm transferred into the vagina undergo an intense selection process before reaching the sperm storage tubules (SSTs) localized in the utero-vaginal junction. Sperm are slowly released from the SSTs and ascend to the infundibulum, the site of fertiliza‐

tion. In this photograph, the vagina is enveloped by connective tissue.

revealed similar numbers of SSTs [29].

The surface mucosa of each segment of the oviduct is lined with parallel, gently spiraling folds along the longitudinal axis. The surface epithelium lining the luminal mucosa contains varying proportions of secretory and ciliated cells. All segments except the fimbriated region of the infundibulum and the vagina possess sub-epithelial tubular glands that secrete components used in egg formation [17]. However, the anterior 2-3 cm of the vagina, an area referred to as the UVJ (Figure 3), contains the SSTs, the primary sites of sperm storage [10] (Figure 4).

At ovulation, the ovum is grasped by the fimbriated region of the infundibulum and, if sperm are present, the ovum may be fertilized within a 10-15 min interval [18]. Thereafter, infundib‐ ular secretions accrue around the ovum, forming the outer PL, which acts as a barrier to further sperm penetration. Birkhead [19] observed that the number of sperm trapped in the outer PL is positively correlated with the size of the ovum and is likewise correlated with the number of sperm that have penetrated the inner PL. Interestingly, the sperm trapped in the outer PL retain an intact acrosome [20-21]. If fertilized, the first cleavage furrow in the GD appears 7-8 hr post-ovulation, while the egg-mass is in the isthmus.

### **2.3. Oviductal sperm selection, transport, and storage**

Following deposition in the oviduct, sperm are transported to UVJ by a combination of their intrinsic motility and cilia beat activity [9-10, 22-23]. Within the SST lumen, sperm are either widely spaced or oriented parallel with their heads toward the distal end of the SST (Figure 4). Sperm are apposed to, but not directly contacting the apical microvilli of the SST epithelial cells. This spatial relationship may facilitate lipid transfer between the resident sperm and the SST epithelial cells [24-25]. Interestingly, alkaline phosphatase, known to play a role in lipid transfer, has been histochemically localized in the apical region of the SST epithelium [26].

The duration of sperm storage in the SSTs is species-dependent. Chickens can store sperm for up to three weeks, whereas turkeys can maintain sperm for 10 weeks in the SST and still lay a fertilized ovum [27-28]. This may be related to number of SSTs present in the UVJ; turkeys have been reported to have 20,000-30,000 SSTs, while chickens have been estimated to have only 5,000-13,500 [29-30]. Additionally, after several generations of selection for high fertility, chicken hens possessed increased numbers of SSTs when compared to non-selected control hens, suggesting the number of SSTs may be positively correlated with fertility [31]. In contrast, under commercial conditions, different broiler strains exhibiting different fertility levels revealed similar numbers of SSTs [29].

albumen proteins; the isthmus, which forms the shell membrane; the uterus (also referred to as the shell gland), a pocket-like structure that elaborates the hard-shell; and, the vagina, which is a conduit between the uterus and cloaca for the egg-mass at oviposition and is responsible for sperm selection and storage following semen transfer. Interestingly, when the vagina and uterus are excised and fixed *in toto* and the connective tissue surrounding the vagina subse‐ quently removed, the vagina appears as a coiled segment (Figure 3) [10]. This anatomy explains the resistance one feels when performing a vaginal insemination with a straw regardless of the presence or absence of an egg mass in the uterus. If inseminating a hen within 30 min after oviposition, the connective tissue around the vagina and the smooth muscle composing the vaginal wall are flaccid. Venting (exteriorizing the vagina for placement of the inseminating straw) at this time may induce a partial prolapse leading to a deep insemination (closer to the UVJ) and the forfeiture of sperm selection by the vagina. Such deep inseminations are

associated with high embryo mortality, possibly due to pathological polyspermy.

hr post-ovulation, while the egg-mass is in the isthmus.

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

**2.3. Oviductal sperm selection, transport, and storage**

The surface mucosa of each segment of the oviduct is lined with parallel, gently spiraling folds along the longitudinal axis. The surface epithelium lining the luminal mucosa contains varying proportions of secretory and ciliated cells. All segments except the fimbriated region of the infundibulum and the vagina possess sub-epithelial tubular glands that secrete components used in egg formation [17]. However, the anterior 2-3 cm of the vagina, an area referred to as the UVJ (Figure 3), contains the SSTs, the primary sites of sperm storage [10] (Figure 4).

At ovulation, the ovum is grasped by the fimbriated region of the infundibulum and, if sperm are present, the ovum may be fertilized within a 10-15 min interval [18]. Thereafter, infundib‐ ular secretions accrue around the ovum, forming the outer PL, which acts as a barrier to further sperm penetration. Birkhead [19] observed that the number of sperm trapped in the outer PL is positively correlated with the size of the ovum and is likewise correlated with the number of sperm that have penetrated the inner PL. Interestingly, the sperm trapped in the outer PL retain an intact acrosome [20-21]. If fertilized, the first cleavage furrow in the GD appears 7-8

Following deposition in the oviduct, sperm are transported to UVJ by a combination of their intrinsic motility and cilia beat activity [9-10, 22-23]. Within the SST lumen, sperm are either widely spaced or oriented parallel with their heads toward the distal end of the SST (Figure 4). Sperm are apposed to, but not directly contacting the apical microvilli of the SST epithelial cells. This spatial relationship may facilitate lipid transfer between the resident sperm and the SST epithelial cells [24-25]. Interestingly, alkaline phosphatase, known to play a role in lipid transfer, has been histochemically localized in the apical region of the SST epithelium [26].

The duration of sperm storage in the SSTs is species-dependent. Chickens can store sperm for up to three weeks, whereas turkeys can maintain sperm for 10 weeks in the SST and still lay a fertilized ovum [27-28]. This may be related to number of SSTs present in the UVJ; turkeys have been reported to have 20,000-30,000 SSTs, while chickens have been estimated to have

**Figure 2.** The segments of the turkey oviduct with a hard-shelled egg in the uterus are observed. Sperm transferred into the vagina undergo an intense selection process before reaching the sperm storage tubules (SSTs) localized in the utero-vaginal junction. Sperm are slowly released from the SSTs and ascend to the infundibulum, the site of fertiliza‐ tion. In this photograph, the vagina is enveloped by connective tissue.

**Figure 3.** Following fixation in neutral-buffered formalin and the removal of the surrounding connective tissue, the coiled morphology of the turkey vagina is revealed. When inseminating a hen, one should insert the straw with the semen until resistance is felt, then release the semen as the straw is withdrawn. As observed here the resistance is due to the coiled vaginal and not an egg mass in the uterus.

**Figure 4.** Three views of the turkey's sperm-storage tubules (SSTs) are observed. The left panel is a stereoscope image showing the pleomorphic appearance of the SSTs. The length of the SST can be as long as 300μm. In the right panel a hen was inseminated with sperm stained with Hoechst 33342, a nuclear fluorescent dye, the UVJ mucosa containing SSTs was isolated, and an unfixed squash preparation was observed by dual interference contrast and fluorescence microscopy. Sperm with fluorescing nuclei are observed in the two SST lumina. The lower-middle panel shows a histo‐ logical section of a portion of a SST containing sperm (the dense rod-like structures in the lumen are sperm nuclei. The arrow indicates the transition between the pseudo-stratified columnar ciliated epithelium of the uterovaginal junction and the simple columnar epithelium of the SST that is characterized in histological preparations by the supra-nuclear

Artificial Insemination in Poultry http://dx.doi.org/10.5772/54918 181

Sperm exit the SSTs in a slow, continuous stream [46-49]; however, a stimulus cuing the egress of resident sperm from the SSTs has yet to be identified. The observations that receptors for estrogen and progesterone exist in the SSTs has led to the suggestion that these compounds may trigger release of resident sperm, possibly in response to hormonal cues over the course of the ovulatory cycle[50-52]. However, an alternate theory suggests the inherent mobility of the sperm plays a larger role than hormonal induction in egress of sperm from SSTs [9]. Resident sperm exhibit a slow, synchronized oscillatory movement in the lumen of SSTs, suggesting the presence of a fluid current through the SST lumen [23-24]. The identification of water channels, known as aquaporins, in the apical epithelium of SSTs lends credence to a model wherein motile sperm maintain their residence in the SST lumen by swimming against the fluid current generated via the aquaporins [53-56]. In the SST lumen, sperm retain their motility by fatty acid oxidation. It has been suggested the sperm membrane is the source of this fatty acid and that as the quality of the sperm membrane gradually decreases there is a

vacuole.

Little is known concerning the cellular and molecular mechanisms that sustain sperm within the SST lumen for prolonged periods of storage. These mechanisms likely involve the rever‐ sible suppression of sperm motility and metabolism, protection and repair of the sperm plasma membrane, uptake and storage of molecules to sustain sperm metabolism, and maintenance of the SST lumen by removing by-products of sperm metabolism and degraded sperm [32-33]. It is clear the SSTs generate a discrete environment to maintain sperm viability via the influx and efflux of compounds critical for sperm survival [25, 34). While ultrastructural analysis has revealed only limited evidence of secretory activity [25], the identification of membrane-bound vesicles released from the apical tips of the SST epithelial cell microvilli suggests a role in the maintenance of resident sperm through lipid transfer [22, 25, 26, 32, 35, 36]. A large proportion of the sperm plasma membrane is composed of polyunsaturated fatty acids [37] that are highly susceptible to damage induced by lipid peroxidation [37]. The peroxidation of these fatty acids results in increased damage to and permeability of the sperm plasma membrane [39, 40]. A complex system of anti-oxidation enzymes are present in the SST epithelial cells and presum‐ ably interact with luminal sperm to minimize damage due to lipid peroxidation and maintain sperm membrane integrity [41]. While many metabolites required by sperm in the SSTs have yet to be identified, increased avidin expression is apparent in SSTs relative to surrounding UVJ epithelial tissue possibly providing a means of sequestering biotin and other vitamins for use by the SSTs or resident sperm [42-43]. Interestingly, progesterone has been shown to induce expression of avidin in the oviduct, providing a potential link between progesterone fluctua‐ tion and sperm storage in and release from the SSTs [42, 44, 45].

**Figure 3.** Following fixation in neutral-buffered formalin and the removal of the surrounding connective tissue, the coiled morphology of the turkey vagina is revealed. When inseminating a hen, one should insert the straw with the semen until resistance is felt, then release the semen as the straw is withdrawn. As observed here the resistance is due

Little is known concerning the cellular and molecular mechanisms that sustain sperm within the SST lumen for prolonged periods of storage. These mechanisms likely involve the rever‐ sible suppression of sperm motility and metabolism, protection and repair of the sperm plasma membrane, uptake and storage of molecules to sustain sperm metabolism, and maintenance of the SST lumen by removing by-products of sperm metabolism and degraded sperm [32-33]. It is clear the SSTs generate a discrete environment to maintain sperm viability via the influx and efflux of compounds critical for sperm survival [25, 34). While ultrastructural analysis has revealed only limited evidence of secretory activity [25], the identification of membrane-bound vesicles released from the apical tips of the SST epithelial cell microvilli suggests a role in the maintenance of resident sperm through lipid transfer [22, 25, 26, 32, 35, 36]. A large proportion of the sperm plasma membrane is composed of polyunsaturated fatty acids [37] that are highly susceptible to damage induced by lipid peroxidation [37]. The peroxidation of these fatty acids results in increased damage to and permeability of the sperm plasma membrane [39, 40]. A complex system of anti-oxidation enzymes are present in the SST epithelial cells and presum‐ ably interact with luminal sperm to minimize damage due to lipid peroxidation and maintain sperm membrane integrity [41]. While many metabolites required by sperm in the SSTs have yet to be identified, increased avidin expression is apparent in SSTs relative to surrounding UVJ epithelial tissue possibly providing a means of sequestering biotin and other vitamins for use by the SSTs or resident sperm [42-43]. Interestingly, progesterone has been shown to induce expression of avidin in the oviduct, providing a potential link between progesterone fluctua‐

to the coiled vaginal and not an egg mass in the uterus.

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

tion and sperm storage in and release from the SSTs [42, 44, 45].

**Figure 4.** Three views of the turkey's sperm-storage tubules (SSTs) are observed. The left panel is a stereoscope image showing the pleomorphic appearance of the SSTs. The length of the SST can be as long as 300μm. In the right panel a hen was inseminated with sperm stained with Hoechst 33342, a nuclear fluorescent dye, the UVJ mucosa containing SSTs was isolated, and an unfixed squash preparation was observed by dual interference contrast and fluorescence microscopy. Sperm with fluorescing nuclei are observed in the two SST lumina. The lower-middle panel shows a histo‐ logical section of a portion of a SST containing sperm (the dense rod-like structures in the lumen are sperm nuclei. The arrow indicates the transition between the pseudo-stratified columnar ciliated epithelium of the uterovaginal junction and the simple columnar epithelium of the SST that is characterized in histological preparations by the supra-nuclear vacuole.

Sperm exit the SSTs in a slow, continuous stream [46-49]; however, a stimulus cuing the egress of resident sperm from the SSTs has yet to be identified. The observations that receptors for estrogen and progesterone exist in the SSTs has led to the suggestion that these compounds may trigger release of resident sperm, possibly in response to hormonal cues over the course of the ovulatory cycle[50-52]. However, an alternate theory suggests the inherent mobility of the sperm plays a larger role than hormonal induction in egress of sperm from SSTs [9]. Resident sperm exhibit a slow, synchronized oscillatory movement in the lumen of SSTs, suggesting the presence of a fluid current through the SST lumen [23-24]. The identification of water channels, known as aquaporins, in the apical epithelium of SSTs lends credence to a model wherein motile sperm maintain their residence in the SST lumen by swimming against the fluid current generated via the aquaporins [53-56]. In the SST lumen, sperm retain their motility by fatty acid oxidation. It has been suggested the sperm membrane is the source of this fatty acid and that as the quality of the sperm membrane gradually decreases there is a 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 motilityenhancing factors and transcend the oviduct [9, 22, 55, 63].

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

**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

Artificial Insemination in Poultry http://dx.doi.org/10.5772/54918 183

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

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

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].

protect against detrimental genetic consequences of polyspermy [76].

fertility and the duration of the fertile period..

preventing pathological polyspermy.
