Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm.
The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane 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, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced 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 extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.
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Department of Cell Biology, León University, Spain. dbcmho@unileon.es
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Herraéz
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D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004530:Egg Yolk / Q000502:physiology; D005260:Female; D005298:Fertility / Q000187:drug effects / Q000502:physiology; D005434:Flow Cytometry / Q000662:veterinary; D005456:Fluorescent Dyes / Q000737:chemistry; D008297:Male; D017686:Oncorhynchus mykiss / Q000502:physiology*; D011419:Propidium / Q000737:chemistry; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D030262:Soybean Proteins / Q000494:pharmacology; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa
D003451:Cryoprotective Agents; D005456:Fluorescent Dyes; D030262:Soybean Proteins; D012710:Serum Albumin, Bovine; D011419:Propidium