Hypoosmotic stress test

The hypo-osmotic test may also be termed a membrane stress test and relies upon the fact that the spermatozoon membrane is semi-permeable, allowing the selective passage of water through it along an osmotic pressure gradient. If spermatozoa are placed in a hypotonic solution (a medium of less than 290 mOsm), water passes into the spermatozoa, resulting in a ballooning of the spermatozoon head and deformation of the tail. The hypo-osmotic pressure test relies upon this deformation of spermatozoa tails, apparent only in spermatozoa that were intact when placed in the hypotonic solution (Jeyendran et al., 1984). As a result, the spermatozoan tails show characteristic deformation in the form of bending and coiling. This effect is only observed in spermatozoa with intact membranes (Jeyendran et al., 1984; England and Plummer, 1993; Correa and Zavos, 1994; Kumi-Diaka and Badtram, 1994; Revell and Mrode, 1994; Correa et al., 1997). These workers showed a significant correlation between the swelling of spermatozoan heads and characteristic tail coiling and sperma-tozoan motility, and the percentage successfully penetrating an ovum, though other work by England and Plummer (1993) failed to show such correlations.

A modified hypo-osmotic test is widely used in the evaluation of bull semen, where it is reported to give a good range of results (10-60%) which are closely correlated with fertilization rates (Revell and Mrode, 1994). The test has been used in stallions with some success (Zavos and Gregory, 1987; Samper et al., 1991; Samper and Crabo, 1993) and its use as a potential commercial test is being investigated (S. Revell, Wales, 1997, personal communication). Stallion spermatozoa placed in hypo-osmotic solutions (75-129 mOsm) of either NaCl or sodium citrate for 15 min will show characteristic tail coiling (Samper et al., 1991; Samper, 1993). In a modification of the bovine test (Revell and Mrode, 1994), stallion semen post-thaw has been subjected to osmotic stress and then stained with 6-Ca D/P1 to assess the proportion of spermatozoa which remain membrane-intact. The range of results obtained was not good (0-35%). Work is currently being carried out on the development of a more suitable form of the test for stallions (S. Revell, Wales, 1999, personal communication). More recent work by Cueva et al. (1997b), using hypo-osmotic solution containing citrate (18-96 mOsm) or citrate and fructose (153mOsm), indicated a significant correlation between the incidence of swollen tails after incubation for 20-30 min, and the incidence of altered acrosomes and total spermatozoan motility. Comparative tests in humans indicate that the results obtained with the hypo-osmotic stress test show a good correlation with results from the zona-free hamster oocyte test (Jeyendran et al., 1984; Rogers and Parker, 1991).

Based on the same principles, hyperosmotic tests have been investigated more recently. However, the correlation between spermatozoon quality parameters of fresh and frozen-thawed semen with hyperosmotic stress resistance was reported to be small (Cueva et al., (1997).

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