[Investigation of membrane permeability of carp spermatozoa for water molecules].

The fundamentals of a photometry method for determination of membrane permeability of some fish spermatozoa for water molecules are presented. Osmotic tolerance of carp spermatozoa membranes was studied using EPR-spectroscopy and photometric analysis methods. It was shown that carp spermatozoa look like the ideal osmometers in their reaction on media of different osmolarity. The value of membrane permeability of carp spermatozoa for water molecules was determined. Data obtained can be used in cryobiology for creating cryoprotective media and regimes of fish sperm cryopreservation.

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development.

The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10 degree C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.8 +/- 2.4% of embryos survival compared to 77.0 +/- 2.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine.

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.

Relationship between the changes in cellular volume of fish spermatozoa and their cryoresistance.

We have investigated the hypothesis that the spermatozoa of marine fish are more resistant than freshwater species to the dynamic changes in osmotic pressure that occur during the process of cryopreservation. We show that while the spermatozoa of marine fish can be successfully activated across a wide range of osmotic pressures (0-2000 mOsmol/l), those of the freshwater species only survive activation within a more restricted range (0-300 mOsm/l). After freeze-thawing, up to 30 percent of motile cells were found in silver carp samples, while up to 90 percent of motile cells were observed in samples from the haarder (Mugil soiuy B). Haarder spermatozoa showed no change of cell volume after dilution in activating or cryoprotective media, while the silver carp spermatozoa responded by swelling and eventual cell disruption. We propose that the differences in cryoresistance of silver carp (Hypophthalmichthys molitrix V.) and haarder spermatozoa may be determined by the ability to preserve cellular volume under non-isotonic conditions.

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys.

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.

The influence of cryopreservation on parameters of energetic metabolism and motility of fowl spermatozoa.

The objective of this study was to estimate the effect of cryopreservation on the main pathways of energetic metabolism and motility of fowl spermatozoa. Sperm diluted 1:5 with the cryoprotective medium containing ethylene glycol (1.4 M final concentration) was frozen at the rate of 2-3 degrees C/min to -25 degrees C with a pause on the plateau of crystallization and then at an exponentially increasing rate to -196 degrees C. The frozen sperm was thawed in two successive water baths at 0 and at 41 degrees C. After cryopreservation, the rate of radioactive glucose oxidation to 14CO2 slightly decreased, the rate of labeled glutamate oxidation remained unchanged, and the rate of labeled succinate oxidation increased two-fold. After freeze-thawing, the rates of endogenous respiration with and without 2,4-dinitrophenol decreased; the oxidation rate of exogenous succinate in the presence of 2,4-dinitrophenol, rotenone, and digitonin slightly decreased; and the rate of respiration in the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, antimycin A, 2,4-dinitrophenol, and digitonin did not differ from that seen in control. Sperm respiration was highly sensitive to rotenone; antimycin A and cyanide blocked oxygen consumption completely. Succinate, added after 2,4-dinitrophenol and rotenone, stimulated respiration of thawed spermatozoa, which indicated plasma membrane damage. The addition of exogenous malate in the presence of 2,4-dinitrophenol and digitonin restored the respiration rate of thawed spermatozoa to that of unfrozen cells. The rate of respiration of thawed spermatozoa with oligomycin was higher than that of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)