Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes
ABSTRACT Exposure of porcine oocytes to increased concentrations of NaCl prior to manipulation has been reported not only to increase cryotolerance after vitrification, but also to improve developmental competence after somatic cell nuclear transfer (SCNT). In the present study we compared the effects of NaCl with those of concentrated solutions of two non-permeable osmotic agents, namely sucrose and trehalose, on the cryotolerance and developmental competence of porcine oocytes. In Experiment 1, porcine in vitro-matured cumulus-oocyte complexes (COCs; n = 1200) were exposed to 588 mOsmol NaCl, sucrose or trehalose solutions for 1 h, allowed to recover for a further 1 h, vitrified, warmed and subjected to parthenogenetic activation. Both Day 2 (where Day 0 is the day of activation) cleavage and Day 7 blastocyst rates were significantly increased after NaCl, sucrose and trehalose osmotic treatments compared with untreated controls (cleavage: 46 +/- 5%, 44 +/- 7%, 45 +/- 4% and 26 +/- 6%, respectively; expanded blastocyst rate: 6 +/- 1%, 6 +/- 2%, 7 +/- 2% and 1 +/- 1%, respectively). In Experiment 2, COCs (n = 2000) were treated with 588 mOsmol NaCl, sucrose or trehalose, then used as recipients for SCNT (Day 0). Cleavage rates on Day 1 did not differ between the NaCl-, sucrose-, trehalose-treated and the untreated control groups (92 +/- 3%, 95 +/- 3%, 92 +/- 2% and 94 +/- 2%, respectively), but blastocyst rates on Day 6 were higher in all treated groups compared with control (64 +/- 2%, 69 +/- 5%, 65 +/- 3% and 47 +/- 4%, respectively). Cell numbers of Day 6 blastocysts were higher in the control and NaCl-treated groups compared with the sucrose- and trehalose-treated groups. In conclusion, treatment of porcine oocytes with osmotic stress improved developmental competence after vitrification combined with parthenogenetic activation, as well as after SCNT.
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- "For instance it was observed that placing the cells in controlled stress conditions could enhance the consequent stress tolerance during future stresses like cryopreservation . So far, several stressors have been applied to induce mild stress including hydrostatic pressure  , osmotic stress , mechanical stress , and ethanol challenge . In all of these studies the purpose was to initiate a protective mechanism in cells. "
ABSTRACT: This study was performed to investigate the effect of sub-lethal exposure of bull semen to ethanol on the post-thaw spermatozoa quality. Semen samples (n=24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled. Pooled samples were divided into 4 equal parts and each part was frozen after being diluted with Optidyl(®) extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9) and 0.15 (O-E15)% (v/v) absolute ethanol. Sperm motility and velocity, plasma membrane integrity and functionality, mitochondrial activity, malondialdehyde concentration, and apoptosis status were evaluated after thawing. A higher percentage of total motility was observed in the O-E9 group as compared to the O-E0, O-E3 and O-E15 groups (p<0.05). Also, plasma membrane integrity was higher (p<0.05) in the O-E9 group compared to the O-E3, and O-E15 groups. However, the difference between the O-E9 and O-E0 groups was not significant (p>0.05). In terms of the proportion of sperm abnormality and plasma membrane functionality no differences (p>0.05) were observed between the groups. Our results revealed that malondialdehyde level was lower in ethanol treated (O-E3, O-E9 O-E15) groups compared to the O-E0 group (p<0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in the O-E9 and O-E15 groups compared to the O-E0 and O-E3 groups (p<0.05). The O-E3 and O-E9 groups resulted in the highest and lowest percentage of apoptotic spermatozoa, respectively (p<0.05). The results of this study demonstrate that supplementation of semen extender with sub-lethal concentration of ethanol influences post-thawed bull sperm quality in a dose dependent manner. Copyright © 2015. Published by Elsevier Inc.Cryobiology 06/2015; DOI:10.1016/j.cryobiol.2015.06.008 · 1.64 Impact Factor
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- "Consistent with this, there was a positive correlation between Na and Cl in the follicular fluid in the luteal phase. Na and Cl act together in the body, and these ions (Na and Cl) are known to have a positive effect on oocyte development, oocyte cryopreservation, and developmental competence after somatic cell nuclear transfer (Oh et al., 1998; Lin et al., 2009). "
ABSTRACT: This study was designed to investigate the effects that the different stages of the estrous cycle had on the number of surface ovarian follicles and oocyte yield and quality of Anatolian water buffalo during peak breeding season. Assessments were made on the basis of ovarian morphology, serum and follicular fluid concentrations of variety of biochemical parameters. Following slaughter, blood samples were collected from each animal. The stage of estrous cycle was classified as either the luteal or follicular phase, and surface ovarian follicles were classified as small, medium, or large. The follicular fluid was aspirated, and oocytes were evaluated microscopically for classification into four categories. No statistical differences (p>0.05) were observed regarding the total number of follicles or quality of oocytes relative to the stage of the estrous cycle. Serum high-density lipoprotein cholesterol (HDL), alkaline phosphatase (ALP) and progesterone (P4) concentrations were significantly higher in the luteal phase than in the follicular phase (P<0.05). Significant correlations were observed in the luteal phase between the total number of oocytes and cholesterol (Cho), HDL, sodium (Na), chloride (Cl); A-quality oocytes and Na, Cl, Mg; C-quality oocytes and Cho, HDL, and Mg in follicular fluid. These results offer new information concerning Anatolian water buffalo reproductive physiology, which may be useful for improving oocyte quality in buffalo. This is the first study to describe the number of ovarian follicles, oocyte yield and quality, and a variety of biochemical parameters in the serum and follicular fluid of Anatolian water buffalo during peak breeding season in Turkey.Animal reproduction science 01/2013; DOI:10.1016/j.anireprosci.2012.12.004 · 1.58 Impact Factor
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- "These may be used alone or in combination with protein synthesis inhibitors such as cycloheximide (CHX) or kinase inhibitors such as 6-Dimethyl- aminopurine (6-DMAP) (Loi et al., 1998). Parthenogenetic activation of vitrified oocytes has been reported in cattle (Dinnyes et al., 2000, Hou et al., 2005, 2009; Yang et al., 2008), pigs (Du et al., 2008; Lin et al., 2009; Somfai et al., 2006), goats (Begin et al., 2003), mice (Endoh et al., 2007), humans (Morbeck et al. 2009), and sheep (Moawad et al., 2010). The use of cryopreserved oocytes as cytoplasts for nuclear transfer (NT) may also provide an additional method by which to evaluate their developmental potential. "
ABSTRACT: The development of embryos produced by somatic cell nuclear transfer (SCNT) using vitrified oocytes as cytoplast recipients has been reported in cattle but not in sheep. This study investigated the parthenogenetic development of ovine oocytes vitrified and thawed at the germinal vesicle (GV) stage, matured in vitro, and then activated using two activation protocols. The optimal activation protocol was then used to assess development when vitrified oocytes were used as cytoplast recipients for SCNT. No blastocysts were obtained from vitrified oocytes activated by CA+CHX/CB (calcium ionophore A23187 + cycloheximide, and cytochalasin B); in contrast, vitrified oocytes activated by Sr/CB (strontium chloride (SrCl(2)) + cytochalasin B) developed to blastocyst, although the number was significantly lower (p < 0.05) than in toxicity and control groups (3.8 vs. 20.0 and 27.3%, respectively). In SCNT embryos, cleavage at both 24 and 48 h postactivation (31.0 vs. 55.1% and 48.0 vs. 85.0%) was significantly lower (p < 0.05) in vitrified oocytes compared to controls. However, no significant differences were observed in the frequency of development to blastocyst (13.0 vs. 23.4%), the number of hatched blastocysts (7.0 vs. 10.3%), total cell numbers (90.3 ± 4.9 vs. 97.6 ± 4.6), number of apoptotic nuclei (13.1 ± 0.9 vs. 13.2 ± 1.4), or the proportion of diploid embryos (60.0 vs. 75.0%). This study demonstrates for the first time that ovine oocytes vitrified at the GV stage can be used successfully as recipient cytoplasts for SCNT.06/2011; 13(4):289-96. DOI:10.1089/cell.2010.0089