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        In vitro maturation of ovine oocyte in a modified granulosa cells co-culture system and alpha-tocopherol supplementation: effects on nuclear maturation and cleavage

        Adeldust, Hamideh,Zeinoaldini, Saeed,Kohram, Hamid,Roudbar, Mahmoud Amiri,Joupari, Morteza Daliri Korean Society of Animal Science and Technology 2015 한국축산학회지 Vol.57 No.8

        This study was designed to investigate the effects of ${\alpha}$-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of ${\alpha}$-tocopherol (0, 5, 10, $15{\mu}g/ml$), oocytes were incubated at $39^{\circ}C$ with 5 % $CO_2$ for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that ${\alpha}$-tocopherol had no effect on GVBD and MII as compared to control group, but when ${\alpha}$-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

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        Open Access : In vitro maturation of ovine oocyte in a modified granulosa cells co-culture system and alpha-tocopherol supplementation: effects on nuclear maturation and cleavage

        ( Hamideh Adeldust ),( Saeed Zeinoaldini ),( Hamid Kohram ),( Mahmoud Amiri Roudbar ),( Morteza Daliri Joupari ) 한국동물자원과학회(구 한국축산학회) 2015 한국축산학회지 Vol.57 No.27

        This study was designed to investigate the effects of α-tocopherol and granulosa cells monolayer on nuclear maturation and cleavage rates of ovine cumulus-oocyte complexes (COCs). The COCs (n = 2814) were matured in maturation medium supplemented with various concentration of α-tocopherol (0, 5, 10, 15 μg/ml), oocytes were incubated at 39 °C with 5 % CO2 for 24 h in three culture systems: (a) maturation medium (MM; n = 884), (b) co-cultured with granulosa cells (CG; n = 982) and (c) co-cultured with granulosa cells and cells were further cultured in MM for 12 h (CG + 12hMM; n = 948). Our results showed that α-tocopherol had no effect on GVBD and MII as compared to control group, but when α-tocopherol added to maturation medium the rate of cleavage decreased. This indicates interaction of above mentioned factors in any of the treatments showed no significant differences on the rate of maturation and cleavage stages (MII, GVBD and cleavage) (p > 0.05). The oocytes co-cultured with granulosa cells for 24 h had beneficial effects on cleavage rate. The maximum MII and cleavage rates were achieved when oocytes had extra 12 h culture in the maturation medium without granulosa cells. Results also showed our modified co-culture system (CG + 12hMM), improved rates of MII and the cleavage in comparison with other studied maturation systems.

      • Using Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality

        Nouri, Houshang,Shojaeian, Kamal,Samadian, Farhad,Lee, Soojung,Kohram, Hamid,Lee, Jeong Ik Elsevier 2018 Journal of equine veterinary science Vol.70 No.-

        <P><B>Abstract</B></P> <P>Reactive oxygen species levels in semen with low quality increased from 25% to about 40%. Resveratrol (Res) is a natural grape-derived phytoalexin, and epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (<I>Camellia sinensis</I>); both molecules are known to have high levels of antioxidant activity. The aim of this study was to investigate the effects of this antioxidant's supplementation on cryopreservation of stallion semen with poor quality. Experiment 1: Experiment design to asses stallions semen quality (n = 3). Arabian stallions were managed for breeding with fresh, extended, or chilled semen during 2 years (in two breeding season) with a resulting per cycle pregnancy rate (103/41 estrus cycle/conception) of 39.80%. A total of 81 ejaculates were collected and evaluated in fresh condition (total and progressive motility, membrane integrity, acrosome defects, morphology, and abnormal DNA condensation). Evaluation of several ejaculates revealed that sperm production and semen quality mostly did not change during the breeding season, maintaining normal sperm production with poor quality. Experiment 2: 18 ejaculates, collected with Missouri model artificial vagina from Arabian stallion (n = 3). Extended semen was centrifuged at 600 × g for 10 minutes. To eliminate individual stallion variability, the resulting sperm pellets were pooled and divided in seven different treatments in a final concentration of 100 × 10<SUP>6</SUP> sperm/mL by using semiskimmed milk extender containing no supplements (control), and the following supplements with three different concentrations: Res 5, 10, and 20 μM (Res 1, Res 2, Res 3, respectively) and EGCG 25, 50, 100 μM (EGCG 1, EGCG 2, and EGCG 3, respectively). After thawing, all samples were maintained at 37°C for 10 minutes. Evaluation of motility, plasma membrane integrity, viability, mitochondria activity, DNA fragmentation, and lipid peroxidation was determined. For all semen parameters, the lower concentration of Res (5 μM) and EGCG (25 μM) did not show significant differences compared to control (<I>P</I> > .05). Relative to the control group, Res 2 had better (<I>P</I> < .05) postthaw total and progressive motility, yet the higher concentrations (Res 3) showed a negative effect on total motility. Percentage of membrane integrity, viability, and mitochondrial activity was significantly greater in Res 2 and EGCG 2 extenders in comparison with the control group, respectively (<I>P</I> < .05). Results showed that malondialdehyde concentration was lower in Res 2 and EGCG 2 groups. In conclusion, the present study showed that the antioxidant Res prevents DNA damages in our stallions. The addition of Res and EGCG before cryopreservation can ameliorate mitochondrial activity in stallions with low-quality semen. Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damages, and preserve sperm plasma membrane integrity and mitochondrial activity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The aim of this study was to investigate the effects of resveratrol (Res) and epigallocatechin-3-gallate (EGCG) supplementation on cryopreservation of stallion (n = 3) spermatozoa with poor quality. </LI> <LI> Five micromolar of Res and 50 μM EGCG in semiskimmed milk extender before cryopreservation can ameliorate total and progressive motility, percentage of membrane integrity, and viability in stallions with low quality. </LI> <LI> In conclusion, the present study showed that the 5 μM Res could prevent DNA damages in subfertile stallions. </LI> <LI> Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damag

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