Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Reproductive Biology Group, Instituto de Investigación en Recursos Cinegéticos (CSIC-UCLM-JCCM), 02071 Albacete, Spain.
Animal reproduction science (Impact Factor: 1.51). 11/2007; 108(3-4):384-401. DOI: 10.1016/j.anireprosci.2007.09.010
Source: PubMed


Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 degrees C over 1.5h (-0.16 degrees C/min), equilibrated at that temperature for 2h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration-equilibration, after freezing and thawing, and 2h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.

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    • "Lower concentrations (100 mM) were therefore chosen for cryopreserving at the ultrarapid cooling rate. The positive influence of this sugar on sperm cryopreservation is explained in that nonpermeable saccharides contribute to cell dehydration before freezing [55]. Cell dehydration, and the maintenance of the osmotic pressure of the diluents, reduces ice crystal formation in the spermatozoa, and allows vitrification by depressing the membrane phase transition temperature of the lipids present [50] [56] [57]. "
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    • "These may, in part, explain why the absolute levels of post-thaw semen quality in this study were low, despite using general techniques that had yielded better mean results in a previous study [45]. Differences in post-thaw semen quality between males and between ejaculates from a specific male have been reported for many species [12] [25] [16] [29]. The dairy cattle industry has selected bulls for AI that deliver spermatozoa able to withstand standard cryopreservation protocols well. "
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    • "Sugars such as raffinose (trisaccharide), trehalose (disaccharide ) and fructose (monosaccharide) play a role in the protection of post-thaw sperm viability by providing an energy source, promoting the excretion of water out of the cell so as to decrease intracellular ice crystal formation, and maintaining the osmotic pressure of the extender. However, their protective effects are dependent on storage temperature, molecular weight, and the particular buffer used in the extender [1] [13] [14]. The beneficial antioxidative and protective effects of sugars against the cold shock and freeze–thaw damage of sperm have been reported in many studies [21] [43]. "
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