Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined...
Reexamination Certificate
2000-06-30
2002-05-28
Tate, Christopher R. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
C424S450000, C424S537000, C435S001300, C435S002000, C514S078000
Reexamination Certificate
active
06395305
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method of preserving sperm to be used for the artificial insemination of domestic animals, and especially for the artificial insemination of swine and bovine. The invention can also find application in avian, other domestic animals and human sperm.
THE PRIOR ART
The most common method of sperm preservation is cryopreservation. During the process of cryopreservation, freezing and thawing damage cell membranes. Mammalian sperm are sensitive to rapid cooling. This phenomenon, called cold shock, occurs when the environmental temperature of sperm rapidly decreases from 35-37° C. (body temperature) down to a few degrees above zero (Watson and Plummer, 1985). For boar sperm, even slow cooling below a temperature of +15° C. results in a decreased survival rate (Watson and Plummer, 1985). Cryopreserved boar semen is used on a very small scale for artificial insemination because of reduced fertility compared to fresh sperm (Johnson and Larson, 1985; Hofmo and Almlid, 1991; Bwanga, 1991). Many experiments (reviewed by Watson and Plummer, 1985) attribute this poor performance to the detrimental effect of cryopreservation on the sperm membrane.
Sperm injury is manifested as loss of selective permeability and loss of integrity of the plasma membrane, outer acrosomal membrane, and mitochondria (reviewed by Parks and Lynch, 1992). These manifestations are accompanied by loss of motility, decreased energy production, changes to membrane lipid composition (Parks and Lynch, 1992; Buhr et al., 1994) and changes to membrane dynamic behaviour (Buhr et al., 1989; 1994). In order to use sperm for artificial insemination, there is a need to prevent and repair loss of selective permeability and loss of integrity of the plasma membrane, outer acrosomal membrane, and mitochondria. There is a need to characterize the damage to sperm during cryopreservation. There is also a need to develop compositions and methods that may be used to prevent and repair the damage to sperm during cryopreservation so that survival of sperm may be increased.
Susceptibility of sperm to cryopreservation differs across species and with stage of spermatozoal maturation. It could be partially due to the different lipid composition of sperm membranes as evidenced by: 1. Sperm from different animal species with similar cold shock resistance, have rather similar lipid composition, length and saturation of fatty acid chains (Watson and Plummer, 1985; Parks and Lynch, 1992); 2. Sperm from different parts of the epididymis have different sensitivity to cold shock, which is correlated with the changes in lipid composition during sperm maturation from caput to cauda epididymis (Bwanga, 1991); 3. Some lipids are released from sperm membranes during cold shock (Darin-Benneett et al., 1973; Bwanga, 1991); 4. The main protective factor from egg-yolk is crude lipid which has been demonstrated to interact with sperm plasma membrane (Gebauer et al., 1970; Pursel et al., 1972; Watson, 1975; Foulkes, 1977); and 5. Addition of some lipids to the extender has been suggested to have beneficial effects on cold resistance (Paquignon, 1985). These compositions have not significantly improved boar sperm viability after cryopreservation.
The nature and amount of specific lipids in sperm membranes differ among animal species (Darrin-Bennett et al., 1974; Poulos et al., 1973; Parks and Lynch, 1992: Buhr et al., 1994), which is consistent with the species' differences in the susceptibility of sperm to cold shock (Watson and Morris, 1981; Watson and Plummer, 1985). Sperm from rooster (Parks and Lynch, 1992; Watson, 1981), human and monkey (Holt and North, 1985), rabbit and dog (reviewed by Watson and Plummer, 1985) are more resistant to cold shock than that from domestic animals, such as bull, ram, horse and boar, with boar sperm being the most sensititive (Watson, 1981). The results from Parks and Lynch (1992) showed that the ratio of sperm membrane proteins to phospholipids (wt:wt) was lowest for rooster at 0.46, intermediate for bull and stallion (0.80 and 0.86) and highest for boar (1.26). They also found that the ratio of cholesterol to phospholipid was close to 1 in human and monkey, but less than 0.8 in bull, ram and boar (Holt and North, 1985).
Using exogenous lipid as a cryoprotectant in semen cryopreservation has been tried by several groups (Butler and Roberts, 1975; Streiner and Graham, 1987; Wilhelm et al., 1996). However, a consistent improvement of post-thaw result has not been achieved, especially with boar semen. These problems could possibly be due a failure to meet a necessity for: 1. Lipids specific for different animal species. Experimental addition of phosphatidylcholine (PC), phosphatidylserine (PS) and/or cholesterol (Pursel et al., 1973; Paquignon, 1985) was based merely on the observation that egg-yolk lipoprotein has some beneficial effects on survival of cryopreservation. 2. Specific fatty acid chain length and saturation, and/or 3. Appropriate methodology to incorporate lipids into spermatozoa and monitor the incorporation efficiency.
Lipids considered common to the cell membrane such as phosphoglycerides, sphingomyelin (SPH) and cholesterol have been identified in the spermatozoa of a variety of species (Darin-Bennet et al., 1973, 1974; Darin-Bennet and White, 1977). Generally, PC is the predominant phospholipid in sperm membranes. Sphingomyelin and phosphtidylethanolamine (PE) are relatively high also. Phosphatidylserine and phosphatidylinositol (PI) are present at low levels. Lipid composition of whole sperm or isolated membranes has been shown to change during epididymal maturation in a variety of species (Nikolopoulou et al., 1985; Hall et al., 1991; Rana et al., 1991). Sperm lipids also change during capacitation and the interaction with ova (Nikolopoulou et al., 1986; Stojanoff et al., 1988; Seki et al., 1992).
Comparing sperm from domestic species, boar sperm membranes have a low percentage of PC and higher percentage of PE and SPH (De Leeuw et al., 1990), and the PI of boar is about 3 times of bull. Parks and Lynch (1992) found that bull and rooster sperm were characterized by a high ratio of PC to PE while boar and stallion sperm had a lower PC/PE ratio. Rooster sperm were also characterized by a higher percentage of phospholipid in the PS+PI fraction than other species (Parks and Lynch, 1992).
The composition of the acyl side chains of phospholipids also differs among species. Sperm membranes from cold-shock resistant species are characterized by a high degree of saturation in fatty acid chains (Darin-Bennet and White, 1977). Cold-shock susceptible species tend to have only very minor amounts of other aldehydes present, while resistant species contain large amounts of steraldehyde (18:0) and 16:1, 18:1, 18:2 aldehydes (Poulos et al., 1973; Darin-Bennett et al., 1974). Phosphatidylcholine from mammalian sperm is characterized by a very high proportion of docosapentanoyl and docosahexanoyl chains, with 22:5 predominant in boar and stallion sperm and 22:6 highest in bull sperm (Parks and Lynch, 1992). For PE, high proportion of long chain polyunsaturated fatty acyl groups, especially 22:5 and 22:6, were contained in sperm of bull, boar, stallion and ram while 22:4 was high in rooster sperm (Parks and Lynch, 1992). These results suggest that the lipid composition of sperm membranes contributes to the cold-shock sensitivity. There is a need to use information regarding the lipids and fatty acids which are damaged during cryopreservation in designing compositions for preserving semen. It would be helpful if a composition could be developed which protected and restored the specific kinds and ratio of lipids and fatty acids sperm membrane in order to improve cold resistance. There is currently no composition for improving sperm survival after cooling which is tailored to the kinds and ratio of lipids and fatty acids in sperm membrane.
Cryopreservation decreases the fertility of sperm, especially boar sperm, by reducing motility and damaging membrane
Buhr Mary
He Li-wei
Flood Michele C.
Synnestvedt & Lechner LLP
Tate Christopher R.
University of Guelph
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