Sperm Morphology and Physiology

Morphology

Mature turkey and chicken spermatozoa exhibit similar morphological features (see extensive review by Jamieson, 2007). The spermatozoa are long and narrow with a vermiform appearance, being 0–5–0.8 mm at the widest point. The acrosome of the turkey spermatozoon is 1.8 mm long, reaching 2.5 mm for the rooster acrosome (Marquez and Ogasawara, 1975). A fibrous, actin-containing, rod-like structure, known as the perforatorium or the acrosomal spine (Etches, 1996), is located between the acrosome and the anterior pole of the nucleus in the spermatozoa of non-passerine birds; no corresponding structure is evident in the subacrosomal space of passerine birds (Pudney, 1995) or mammalian spermatozoa, except in rodents (Baccetti et al., 1980). At 1.0 μm in length, the perforatorium of the turkey and rooster is appreciably shorter than that of the guinea fowl (1.9 μm). The role of this structure in bird spermatozoa is suggested to be skeletal in nature, acting as an inner conical support for the acrosome (Baccetti et al., 1980).

The sperm nucleus consists of dense chromatin, forming a concave implantation fossa where it joins the midpiece of the tail. The turkey sperm nucleus is shorter (7–9 μm) than that of guinea fowl or chicken (10–14 μm), and the junction of the nucleus with the midpiece at the neck region is not as conspicuous as in guinea fowl spermatozoa (Thurston and Hess, 1987).

For turkey and chicken spermatozoa, the neck region of the midpiece consists of a proximal and an elongated distal centriole, while guinea fowl spermatozoa contain only a single elongated centriole and associated pericentriolar projections (Thurston and Hess, 1987). Cross sections of the centrioles have the typical ‘pinwheel’ arrangement of nine triplet microtubules embedded in a cylindrical, dense wall (Jamieson, 2007). Enveloping the distal centriole and extending to the annulus are 25–30 helically arranged mitochondria (Thurston and Hess, 1987). In contrast to the guinea fowl, the cristae of turkey and rooster sperm mitochondria are parallel to the outer membrane (Jamieson, 2007). The absolute size of the midpiece region and the proportion of the midpiece to the head are greater in turkey than in chicken spermatozoa (Lake and Wishart, 1984).

A major role of glycosylation is the formation of specific molecular surfaces that permit specific intermolecular recognition (Ivell, 1999). The sperm glycocalyx is modified extensively during sperm transport and maturation, and represents the primary interface between the male gamete and its environment. While the function of these glycoconjugates is still largely unknown, in the hen, sialic acid has been implicated for both sperm passage through the vagina (Steele and Wishart, 1996) and sperm sequestration in the sperm storage tubules (Froman and Thursam, 1994), and N-acetyl-D-glucosamine is necessary for sperm–egg interaction (Robertson et al., 2000). Long and colleagues have demonstrated that hypothermic storage of turkey semen (4°C) and chicken semen (–196°C) affects the composition of the sperm glycocalyx (Peláez and Long, 2008; Peláez et al., 2011). In particular, terminal sialic acid residues are less abundant after hypothermic storage for both species. Sialic acids act as masking agents on antigens, receptors and other recognition sites of the cell surface, and it appears that the amount of sialic acid in sperm cells is considerably higher than that in somatic cells (Diekman, 2003).

Lipids are a major component and integral part of sperm membranes, and are involved with the series of biochemical and functional changes required for fertilization, such as sperm maturation and the acrosome reaction (Bréque et al., 2003). Phospholipids represent the main lipid type found in avian sperm membranes, with phosphatidylcholine comprising ~40% of the total phospholipid content. Poultry spermatozoa also contain a high proportion of polyunsaturated fatty acids in the plasma membrane (Surai et al., 1998). Polyunsaturated fatty acids are classified as n–3 or n–6 based on the location of the last double bond relative to the terminal methyl end of the molecule. In contrast to the plasma membranes of mammalian spermatozoa, which contain mainly n-3 polyunsaturated fatty acids such as docosahexaenoic acid (22:6n-3; DHA), the phospholipids in avian spermatozoa are enriched mainly with n-6 polyunsaturated fatty acids, including arachidonic (20:4n-6; AA) and docosatetraenoic (22:4n-6; DTA) acids. It has been proposed that the functional significance of this n-6/n-3 dichotomy in the fatty acid profiles of avian and mammalian spermatozoa may represent an adaptation to temperature (Kelso et al., 1997). The higher body temperature of birds (41°C) compared with that of mammals (37°C), in tandem with the fact that avian testes are internalized, requires avian spermatozoa to develop and function in a considerably warmer environment. The major poly-unsaturated fatty acid (PUFA) of avian spermatozoa, DTA, displays the same chain length but fewer double bonds than the mammalian PUFA counterpart DHA; this difference in the degree of polyunsaturation provides a means for maintaining the appropriate biophysical properties of sperm membranes at the different body temperatures.

Sperm metabolism and motility

Although the gross morphology of turkey and chicken spermatozoa is similar, ultrastructural differences in the midpiece region appear to be indicative of the different biochemical features of spermatozoa from these two species. The main distinction in energy production is that turkey spermatozoa require oxygen to produce ATP, but chicken spermatozoa can metabolize using either aerobic or anaerobic respiration (Sexton, 1974). Both turkey and chicken spermatozoa use the Embden–Meyerhof–Parnas pathway of glycolysis and the Krebs cycle for oxidative metabolism (Kraft et al., 1978). Turkey spermatozoa have a low glycolytic capacity and require the presence of oxygen for optimal ATP production (Lake and Wishart, 1984). Under anaerobic conditions, chicken spermatozoa form lactate from glucose 13 times faster than turkey spermatozoa (Sexton, 1974; Wishart, 1982) owing, in part, to the 20-fold higher activity of lactate dehydrogenase in chicken spermatozoa (McIndoe and Lake, 1973). Profiles of glycolytic enzymes are similar for spermatozoa from both species; however, chicken sperm enzyme activities are twofold to fourfold higher than those of turkey sperm, with glycerophosphate mutase and lactate dehydrogenase activities 9.5 and 41 times greater, respectively (Wishart and Carver, 1984). Glutamate and glycine are not used by either species as oxidizable substrates, while acetate and succinate increase the rate of oxygen consumption in chicken spermatozoa only (Sexton, 1974).

Spermatozoa from some avian species exhibit a unique sperm motility phenomenon of reversible temperature-dependent immobilization (Ashizawa et al., 2010) in which, in simple salt solutions, spermatozoa become immotile at body temperature (40–41°C) but regain motility when the temperature is lowered to 30°C or by the addition of Ca²⁺ at 40°C (Munro, 1938; Wishart and Ashizawa, 1987; Ashizawa et al., 1989; Thomson and Wishart, 1991; Ashizawa et al., 1994). This pattern of temperature-dependent motility occurs in chicken, duck and copper pheasant spermatozoa, but occurs only partially in turkey and not at all in quail or Houbara bustard spermatozoa (Wishart and Wilson, 1999; Ashizawa et al., 2010).

Turkey sperm motility was only partially inhibited at 40°C, while quail and bustard spermatozoa showed no reduction in the percentage of motile spermatozoa at 40°C (Wishart and Wilson, 1999). Ca²⁺ restored the motility of chicken and duck spermatozoa at 40°C and also released the partial inhibition of turkey sperm motility; however, Ca²⁺ had no apparent effect on the proportion of motile bustard spermatozoa and completely inhibited quail sperm motility at 40°C (Wishart and Wilson, 1999). The axoneme and/or accessory cytoskeletal components appear to be directly involved in the temperature-dependent regulatory system, because the motility of de-membranated spermatozoa is similar to that of intact spermatozoa, being negligible at 40°C and restored at 30°C (Ashizawa et al., 2000). This temperaturedependent motility has been shown to be a function of intracellular Ca²⁺ content; intracellular levels of Ca²⁺ were reduced at the higher temperatures, where spermatozoa were immotile, and increased as the temperature was lowered from 40 to 30°C (Thomson and Wishart, 1991). Further, the temperaturedependent retention of Ca²⁺ was a function of the rate of Ca²⁺ efflux rather than influx, and involved a Ca²⁺ ATPase that was relatively inactive at 30°C, but active at 40°C (Thomson and Wishart, 1991). Chicken sperm motility was inhibited by loading with an intracellular Ca²⁺ chelator, and subsequently reactivated by the addition of excess Ca²⁺ to the medium (Ashizawa et al., 1994). Not all avian species demonstrate this temperature-dependent sperm motility phenomenon.

When the sperm mobility assay was applied to populations of randomly bred broiler roosters, the phenotype varied among males (Froman et al., 1997), was independent of age (Froman et al., 1999) and determined male fecundity (Froman and Feltmann, 1998). Subsequently, the heritability of this quantitative trait was estimated (Froman et al., 2002), and it was shown that mitochondrial dysfunction accounted for the phenotypic variation (Froman and Kirby, 2005; Froman et al., 2006). In the turkey, the high sperm mobility phenotype was characterized by a straightline velocity of 45.5 μm/s and an average path velocity of 60.3 μm/s, compared with 28.8 and 45.1 μm/s, respectively, in males exhibiting the low sperm mobility phenotype (Donoghue et al., 1998). Like the sperm mobility phenotype established for the chicken, that in turkeys also varies characteristically among males (King et al., 2000a), is independent of age (Holsberger et al., 1998) and influences paternity (Donoghue et al., 1999). The turkey sperm mobility phenotype does not, however, predict the outcome of sperm function after 24 h of storage at 4°C, as sperm mobility values decline to similar levels irrespective of the male phenotype (Long and Kramer, 2003). Finally, male turkeys characterized as either high or low sperm mobility phenotype exhibit striking differences in the content of carbohydrates in the glycocalyx, including mannose/glucose, N-acetyl-glucosamine and N-acetylgalactosamine (Peláez and Long, 2008).

Seminal Plasma

The components of poultry seminal plasma are thought to be derived from the extragonadal duct system within the epididymis, primarily the proximal efferent duct, which appears to be where spermatozoa are mixed with secretions as they are concentrated (Hess et al., 1976; Aire, 1979; al-Aghbari et al., 1992). During natural mating in chickens, a lymphlike, transparent fluid is mixed with the ejaculate. This transparent fluid, derived from the paracloacal vascular bodies (Fujihara, 1992), is chemically distinct from deferent duct fluid (Lake, 1966, 1984). In turkeys, a frothy fluid is secreted from the triangular fold of the cloaca and mixed with the ejaculate during copulation (Fujihara et al., 1985). The chemistry of the frothy fluid resembles that of blood plasma, rather than that of seminal plasma (Fujihara and Nishiyama, 1984). As outlined below, the biochemistry of seminal plasma differs markedly from that of blood plasma. Thus it is not surprising that prolonged exposure of turkey spermatozoa to this frothy fluid adversely affects sperm morphology (Fujihara et al., 1987).

Electrolytes

Amino acids and peptides

Glutamic acid is the principal amino acid in poultry seminal plasma, and is thought to serve as the main anion in place of Cl^– (El Jack and Lake, 1969). Lake and McIndoe (1959) reported that glutamic acid comprises 90% of the free amino acids found in chicken seminal plasma; similarly, a high proportion of glutamic acid is found in turkey seminal plasma (Ahluwalia and Graham, 1966b; Graham et al., 1971). The concentration of glutamic acid in poultry seminal plasma is ten times the total amino acid concentration in bull semen (Lake and McIndoe, 1959), and several 100 times greater than that found in blood plasma (Lake and Wishart, 1984). The functional significance of glutamate, other than in establishing osmolality, is unclear, as poultry spermatozoa lack adequate metabolic capabilities to use glutamate as an energy source (Etches, 1996).

Of the 20 recognized amino acids (excluding glutamic acid), arginine, asparagine, threonine and glycine are the most prevalent in chicken seminal plasma, whereas arginine, asparagine, aspartic acid and serine are the most prevalent in turkey seminal plasma (Ahluwalia and Graham, 1966b). Graham et al. (1971) determined that the semen collection frequency in turkeys influenced the amount of free amino acids in seminal plasma; the levels of most amino acids (except for glutamic acid) were higher when semen was collected at 2 day intervals than at 4 or 7 day intervals. It appears that the seminal plasma amino acid profile also varies among genotypes. Froman and Bernier (1987) identified a heritable reproductive disorder in the male rooster in which spermatozoa degenerate prematurely. The inheritance of this trait was attributed to a single dominant gene (Froman et al., 1990), and mutant roosters were characterized by a reduced surface-to-volume ratio within their proximal efferent ducts (Kirby et al., 1990) and a reduced level of glutamic acid in seminal plasma compared with normal roosters (al-Aghbari et al., 1992).

Carnitine plays a vital role in transporting long chain fatty acids across the inner mitochondrial membrane to produce energy through β-oxidation (Bremer, 1983), and may also function as an antioxidant to scavenge free radicals (Agarwal and Said, 2004). Both carnitine and acetyl carnitine are present in poultry seminal plasma (Golan et al., 1982; Lake and Wishart, 1984). While the levels of carnitine (3.2 mmol) and acetyl carnitine (2.1 mmol) are comparable to those reported for mammalian semen (Mann and Lutwak-Mann, 1981), turkey seminal plasma contains about half (1.7 and 0.64 mmol, respectively) of the levels found in chicken seminal plasma (Lake and Wishart, 1984).

Fructose is the principal unbound sugar of seminal plasma for a number of mammalian species (Mann and Lutwak-Mann, 1981), but only trace amounts of fructose are found in poultry seminal plasma (Kamar and Rizik, 1972; Lake and Wishart, 1984; Garner and Hafez, 1993). A few older reports document glucose levels of 80–88 mg/dl in chicken seminal plasma (Mann, 1964; Hammond et al., 1965), though only 3.1 mg/dl was detected by Ahluwalia and Graham (1966a); rather, the primary carbohydrate isolated from chicken seminal plasma in that report was inositol (20.4 mg/dl), along with low amounts of glycerol (2.8 mg/dl). Wishart (1982) concluded that chicken and turkey semen did not contain glycolytic substrates, as no lactate was produced when semen was diluted in buffer without glucose and incubated under anaerobic conditions. A more recent report documented the presence of free oligosaccharides in human seminal plasma – the first report for any species (Chalabi et al., 2002); however, the function of these oligosaccharides remains unknown.

Lipids and phosphodiesters

Two of the six non-cyclic phosphodiesters are found in semen: glycerol phosphorylcholine in mammalian seminal plasma and serine ethanolamine phosphodiester in avian seminal plasma (Burt and Ribolow, 1994). In chicken seminal plasma, serine ethanolamine phosphodiester was shown to be 23–26% of the total observable phosphate in three males, but in two other males, it was only 7–9% (Burt and Chalovich, 1978). Further, turkey semen collected in the spring showed ‘substantial levels’ of serine ethanolamine phosphodiester (Burt and Ribolow, 1994), which decreased as males began to stop semen production in late summer (Ribolow and Burt, 1987). Phosphodiesters are a class of compounds that may function as lysophospholipase inhibitors in semen, a role that would decrease the rate of membrane phospholipid turnover and lead to a net sparing of phospholipids (Burt and Ribolow, 1994).

Enzymes and other proteins

Chicken seminal plasma contains 52–77% protein (Blesbois and Hermier, 1990) or 2.0–2.4 g/dl (Harris and Sweeney, 1971; Amen and Al-Daraji, 2011), while turkey seminal plasma averages 1.8 g protein/dl (Thurston et al., 1982). Many proteins in poultry seminal plasma have been identified as enzymes. Autoproteolytic activity has been detected in the seminal plasma of the domestic chicken (Droba, 1986), and serine proteinase inhibitors are present in chicken seminal plasma, with their proposed function being the inactivation of acrosin that is released from dead or damaged spermatozoa (Lessley and Brown, 1978). Turkey seminal plasma contains a unique serine proteinase not found in the chicken or guinea fowl, and the amidase activity of turkey seminal plasma is 23–28 times greater than for chicken or guinea fowl (Thurston et al., 1993). The turkey seminal plasma serine proteases were found to be distinct from the sperm enzyme acrosin (Thurston et al., 1993). The biological roles of these enzymes in birds are unknown as, unlike in mammals, turkey seminal plasma serine proteases cannot participate in semen liquefaction or coagulation because these processes do not occur in birds (Holsberger et al., 2002). More recent research has shown that turkey seminal plasma contains at least three trypsin inhibitors, providing further evidence for increased activities of serine proteinases as a unique characteristic of turkey seminal plasma (Kotłowska et al., 2005).

A number of glycosidases are present in poultry seminal plasma, including β-N-acetylglucosaminidase, the α and β forms of mannosidase, galactosidase and glucosidase, and β-glucuronidase (Kannan, 1974; McIndoe and Lake, 1974; Droba and Droba, 1987; Droba and Dzugan, 1993), which hydrolyse a variety of compounds containing terminal, non-reducing carbohydrate residues. Among the glycolytic enzymes present in whole semen, β-N-acetylglucosaminidase was the most active, with α-mannosidase ranking next in activity, whereas the activities of β-mannosidase, both forms of galactosidase and glucosidase, and β-glucuronidase, were almost negligible (Kannan, 1974). The activity of acid phosphatases, which catalyse the hydrolysis of phosphate esters, is distinctively high in human and poultry seminal plasma (Wilcox, 1961; Bell and Lake, 1962; Mclndoe and Lake, 1974; Hess and Thurston, 1984; Dumitru and Dinischiotu, 1994). The presence of several phospholipases have been confirmed in turkey seminal plasma, including phospholipase A2, phospholipase A1 and lysophospholipase; these have been linked with sperm membrane phospholipid loss during hypothermic semen storage (Douard et al., 2004).

Transport proteins, such as albumin, comprise the majority of the protein content of poultry seminal plasma (Blesbois and Caffin, 1992). For example, over 60% of the protein spots from 2D SDS-PAGE gels for chicken and turkey seminal plasma have been identified as serum albumin precursor, ovalbumin or ovotransferrin (J.A. Long, unpublished data). Ovotransferrin is mainly known as an iron-binding glycoprotein found in the egg white (Kurokawa et al., 1999), but it has recently been shown to be a multifunctional protein with a major role in avian natural immunity (Giansanti et al., 2012). Ovalbumin also has been traditionally associated with the egg white and development of fertilized eggs (Smith and Back, 1962; Sugimoto et al., 1999). Serum albumin is the main protein of blood plasma, in which a high binding capacity for water, Ca²⁺, Na⁺, K⁺ and fatty acids contributes to the main function of regulating the colloidal osmotic pressure of blood. It seems highly likely that serum albumin precursor has a similar role in seminal plasma.