Julie A. LONG
Murray R. BAKST
Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD, U.S.A.
Introduction
Turkeys are the only commercial livestock species completely dependent upon artificial insemination (AI) for fertile egg production. Given that every breeder hen must be inseminated weekly during egg production, AI is both time and labour-intensive. Methods for the timing, frequency, semen dosage and site of semen deposition that were optimized 10-20 years ago are still in use today and yield exceptional fertility rates when used with freshly collected semen. This necessitates keeping toms on the same farm as hens and handling toms as frequently as hens for AI. The turkey industry would benefit greatly if semen could be stored for 24-48 hours without affecting fertility. Currently, extended turkey semen is stored for no longer than 6 hours, and fertility rates slowly drop after 12 weeks of egg production when stored semen is used for AI.
Our research aim is to determine how and when turkey sperm lose functional competence during semen storage, and to use this knowledge as a basis for developing successful sperm storage methods in vitro. We summarize here a series of studies characterizing biochemical and cellular changes associated with storage of turkey sperm at 4 °C.
Poultry Semen Extension
Poultry semen is viscous and highly concentrated, containing sperm concentrations of 6 (rooster) to 12 (tom) billion sperm per ml of ejaculate (Donoghue and Wishart, 2000). After collection, poultry semen must be diluted with buffered salt solutions, or extenders, to maintain the viability of sperm in vitro. Dilution of neat semen also is advantageous in that more hens can be inseminated from a single semen sample. There are many variations in extender composition; however, the basic goals are to maintain pH and osmolarity, as well as provide an energy source for metabolism.
Semen extenders being used today are modifications of formulas developed at least 20 years ago that were intended for extending fresh semen. By lowering the extender pH (Lake and Ravie, 1979; Giesen and Sexton, 1982) and providing an aerobic environment (Sexton, 1974; Wishart, 1981), most commercial extenders maintain viable turkey sperm with commercially acceptable fertility rates when stored at 4-10 °C for up to 6 hours. The fertility rates of semen stored for longer than 6 hours is not commercially acceptable.
In an interesting paradox, while turkey sperm stored in vitro for 24 hours lose functional competence, turkey sperm residing in the hen's sperm storage tubules (SST's) for as long as 16 weeks are capable of fertilizing eggs. Elucidation of the mechanism(s) by which storage is achieved in vivo may provide new insight for in vitro storage methods. For example, it is hypothesized that prolonged storage in the SST's is supported by reversible suppression of metabolism and motility and/or stabilization of the plasma membrane and maintenance of the acrosome (Bakst et al., 1994).
The key to successful long-term in vitro storage protocols may involve suppression of sperm metabolism during storage and reactivating prior to AI; however, limited information is available about the cellular and biochemical properties of poultry sperm and what deleterious physiological changes may occur during semen storage.
Poultry Sperm Physiology - Membrane Phospholipids
Lipids are a major component and integral part of sperm membranes that are involved a series of biochemical and functional changes ultimately required for fertilization, such as sperm maturation and the acrosome reaction (Brèque et al., 2003). It is known that poultry sperm contain a high proportion of polyunsaturated fatty acids (PUFAs) in the plasma membrane (Surai et al., 1998). Phospholipids in avian spermatozoa are enriched mainly with n-6 PUFAs, including arachidonic (20:4n_6) and docosatetraenoic (22:4n-6) acids. The high levels of PUFAs render them vulnerable to lipid peroxidation (Fujihara and Howarth, 1978; Cecil and Bakst, 1993), a chemical reaction occurring in the presence of oxygen radicals. Normal by-products of oxidative metabolism form free radicals of O2 and H2O2, which induce the formation of lipid peroxides that are extremely toxic to sperm (Wishart, 1984). For mammalian sperm, lipid peroxidation been linked to a decline in sperm motility and metabolism in vitro (Jones and Mann, 1976). For turkey sperm, the occurrence of lipid peroxidation was demonstrated in samples held at 4 °C by measuring malonaldehyde, a byproduct of peroxidation (Cecil and Bakst, 1993), and the degree of lipid peroxidation was shown to vary characteristically for each male (Long and Kramer 2003). Most importantly, lipid peroxidation is a major contributor to the lower fertility rates associated with stored turkey semen (Long and Kramer, 2003).
The major phospholipids class of turkey sperm is phosphatidylcholine, comprising up to 39 % of the total phospholipids content. It has been shown that the phospholipid content of turkey spermatozoa decrease by 30 % during 24 hours storage at 4 °C, with most of the loss (20 %) occurring between 1 and 4 hours (Douard et al., 2003). It also has been demonstrated that membrane-bound phospholipids, especially phosphatidylcholine, are lost during in vitro storage of poultry sperm (Bleisbois et al., 1999; Dourad et al., 2000). Inclusion of typical antioxidants such as Vitamin E in the extender has not proven to be effective in preventing lipid peroxidation of turkey sperm during semen storage (Long and Kramer 2003; Douard et al., 2004). Ongoing work in our lab has demonstrated that turkey sperm cells are able to incorporate exogenous phosphatidylcholine into the plasma membrane during in vitro semen storage in a dose-dependent manner and that fertility rates of semen stored in phosphatidylcholine-supplemented extender are greatly improved.
Poultry Sperm Physiology - Surface Carbohydrates
The sperm glycocalyx is a dense carbohydrate layer extending 20-60 nm from the cell surface (Bearer and Friend, 1990) that emanates from either plasma membrane proteins (glycoproteins) or lipids (glycolipids) and represents the primary interface between the sperm cell and its environment. In mammals, the glycocalyx is a critical component for sperm maturation, sperm transport and sperm-egg interaction (Diekman, 2003); however, the functional significance of the poultry sperm glycocalyx is not well defined. It has been shown that rooster spermatozoa acquire surface-associated glycoproteins during maturation within the male duct system that remain associated with spermatozoa in the female tract (Morris et al., 1987). Impaired fertility has been associated with alterations in the carbohydrate content of rooster spermatozoa (Froman and Engel, 1989). In particular, sialic acid is required for sperm passage through the hen's vagina (Steele and Wishart, 1996) and for sequestration in the hen's sperm storage tubules (Froman and Thursam, 1994). N-acetyl-D-glucosamine, localized on the perivitelline layer of the chicken ovum, is necessary for sperm-egg interaction (Robertson et al., 2000). Although these data pertain to fowl, the possibility exists that similar implications occur in the turkey. Further, the loss and/or alteration of surface glycoproteins during semen storage may also impact the ability of turkey spermatozoa to traverse the hen's reproductive tract and to recognize/bind to the ovum.
We recently characterized the carbohydrate composition of the turkey sperm glycocalyx in fresh semen from males with average-mobility semen, by means of flow cytometry assessment of lectin binding (Peláez and Long, 2006). The glycocalyx of turkey spermatozoa is extensively sialylated and contains residues of α-mannose/α-glucose, α- and β-galactose, α-fucose, α- and β-N-acetyl-galactosamine and N-acetyl-lactosamine, as well as monomers and dimers of N-acetyl-glucosamine in variable amounts. Our next aim was to determine if these surface carbohydrates are altered during conventional in vitro semen storage at 4 °C for 24 hours. Because the majority of terminal carbohydrate residues are masked by sialic acid, we used a neuraminidase treatment to detect differences in unmasked residues over time. We also compared the carbohydrate composition of semen from high and low sperm mobility phenotypes, as males exhibiting a high mobility index have a competitive advantage with respect to paternity (Donoghue et al., 1998).
Quantitative changes occurred in the carbohydrate content of turkey sperm surface glycocalyx during a 24-hour period of storage at 5 °C (Pelaez and Long 2006). The pattern of changes varied among sugar residues, with increased rates of lectin binding being observed during the incubation period. Of special interest was the fact that sperm glycocalyx was not considerably altered until after 6 hours of storage. Males of low-semen-mobility phenotype appeared to contain higher amounts of some sugar moieties as compared to the high-mobility phenotype, and also presented differences in the pattern of changes with regard to the latter. Increases in the rate of lectin binding revealed an augmentation of non-sialylated terminal residues that could modify the sperm antigenicity, eventually impacting fertility negatively. Ongoing studies are evaluating the effects of exogenous sialic acid on the viability, motility and fertility of 24h-stored turkey semen.
Summary
It has been long recognized that the ability to store turkey semen for 24 hours in vitro without a significant loss in fertility upon insemination would benefit the commercial turkey industry. Using a systematic approach of identifying why and how turkey sperm lose functional competence during semen storage, we have shown that both the lipid and carbohydrate content of turkey sperm membranes are altered during 24 hours of aerobic storage at 4 °C. This new knowledge has enabled the application of novel extender components that should improve the fertilizing capacity of stored turkey semen.
References are available on request
From Proceedings of the "Midwest Poultry Federation Convention", St. Paul, Minnesota, U.S.A.
