Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rain bow trout (Oncorhynchus mykis) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Onco rhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane, External cryoprotectants are known to stabilize the sperm cell me mbrane against such damage, In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BS A, and a soybean-protein complex (DanPro S760) singly and in various combin ations. To assess the effect of these cryoprotectants we evaluated the perc entage of cells with progressive motility, permeability of cells to propidi um iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hy po-and isoosmotic solutions of 10 and 300mOsm, and the in vitro fertility r ate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg, So me of the tested stabilizers increased motility, increased viability, or re duced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the ext ender. The highest fertility rates were obtained with DanPro S760(R) alone or in combination with BSA; the use of BSA with egg yolk did not improve th is parameter, Our results demonstrated that some external cryoprotectants e ffectively increased membrane resistance during freezing and thawing, but s ome of the tested mixtures interfered with fertilization. Soybean protein c oncentrate provided good protection and increased fertility rates in cryopr eserved trout spermatozoa. (C) 2001 by Elsevier Science Inc.