Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: Julian Garde
  • Source
    • "This could be the reason of the poor postthaw motility observed in the present study. Various diluent components, such as the buffer system and the cryoprotectants' concentration [15] , can affect the success of cryopreservation . Glycerol is an extremely efficient cryoprotectant, whose toxicity is influenced by the concentration used, the temperature that was added to the sample, and the species studied [19,31,11]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.
    Full-text · Article · Sep 2015 · Cryobiology
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015 · Theriogenology
  • Source
    • "The ram semen freezing extender is generally composed of Tris(hydroxymethyl )-aminomethane (Tris) for acid-base buffer, citric acid for osmotic pressure balance, cryoprotective agent (CPA) for cryoinjury prevention during cryopreservation and, most importantly, saccharides (sugars) for supporting energy and protection the spermatozoa (Abdelhakeam et al., 1991;Salamon and Maxwell, 2000b;Paulenz et al., 2002;Mortimer and Maxwell, 2004;Câmara et al., 2011). Many studies reported the advanced effects of sugars in a semen freezing extender on the post-thawed viability of animal spermatozoa (Garcia and Graham, 1989;Molinia et al., 1994;Garde et al., 2008;Tonieto et al., 2010). Sugars are divided into monosaccharide, disaccharide and trisaccharide types. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Semen cryopreservation alters sperm structure and biochemical properties, and subsequently impairs fertilizing ability. A combination of two different sugar molecules revealed an enhanced beneficial effect on frozen-thawed spermatozoa of various species. However, the report in ram has been scarce. The present study investigated the effect of different combinations of sugar supplementation on post-thawed ram semen qualities in a cryopreservative extender in the presence of a single sugar or a combination of sugars. Data showed that a combination of sucrose and trehalose significantly improved cryopreserved sperm motility, viability, longevity and acrosome integrity. Fertility rates following laparoscopic artificial insemination (LAI) using frozen-thawed semen (n = 35) with the sucrose and trehalose supplementation revealed the same values as those utilizing fresh semen (n = 32) (82.9 v 84.4%, respectively). Our findings suggest that the use of a Tris-Citric extender with the addition of sucrose combined with trehalose is an effective extender for ram semen cryopreservation. The optimization of concentrations and ratios between the combination of sucrose and trehalose supplemented to ram semen freezing extender should be further examined.
    Full-text · Article · Jun 2015 · The Thai veterinary medicine
Show more