- Email: [email protected]
Comparing SSV and OPS vitrification techniques and different solutions on cryopreservation of mouse blastocysts
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Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
A5 DNA barcoding approach in diversification of species from genus Loligo Emre Keskin, Hasan Huseyin Atar Department of Fisheries and Aquaculture Engineering, Agricultural Faculty, Ankara University, Ankara, Turkey E-mail address: [email protected] (E. Keskin) Squid is used to generalize seafood products manufactured from species belonging to the families Loliginidae and Ommastrephidae. The genus Loligo is one of the most representative and widely distributed groups of myopsid squids. In this study, a DNA barcoding approach was used to clarify phylogenetic relationship among 8 members of Loliginidae family. Muscle tissue samples of each species (Loligo bleekeri, L. forbesii, L. gahi, L. opalescens, L. pealei, L. plei, L. subulata and L. vulgaris) were taken from each specimen and stored at −20◦ C until DNA extraction. Mitochondrial COI gene was amplified using polymerase chain reaction with specific primers designed for Loligo genus. Amplified DNA’s were purified and DNA sequencing reactions were conducted. Alignment of sequences and phylogenetic analyses were conducted using MEGA 5. Tamura-Nei model was used in computing pair-wise distances. Maximum likelihood method was used in construction of phylogenetic tree. Including all species studied, 67.98% of amplified region was found to be conserved among species. Rate of transitional pairs over transversional pairs (R) was calculated as 1.30. Average distance of evolutionary divergence between sequences was 0.178. In conclusion, DNA barcoding approach was successful in determining the phylogenetic relationship among members of genus Loligo at species level. doi:10.1016/j.copbio.2011.05.138
A6 Effect of lutein on swine oocyte viability, cumulus expansion and embryo culture Ileana Miclea, Andrea Hettig, Marius Zahan, Iulian Roman, Vasile Miclea University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Husbandry and Biotechnology, Cluj-Napoca, Romania E-mail address: [email protected] (I. Miclea) The goal of this research was to establish the influence of several lutein concentrations on swine oocyte viability, cumulus cell function, maturation and embryo development. Pig oocytes were maturated for 45 hours in M199 supplemented with lutein (2.5, 5, 8, 10 m) and cumulus oophorus expansion was examined. Viability and the presence of the first polar body were assessed by fluorescent staining with FDA, PI and Hoechst 33258. Mature oocytes were fertilized in TALP and then embryos were cultured for 48 hours in NCSU-37 supplemented with the same lutein concentrations. Oocytes matured in lutein medium supplemented with 8 m had fully expanded cumulus oophorus (86.73%, P < 0.05, LSD test). Viability was improved by the addition of lutein: 2.5 m (98.29%, P < 0.05) and 5 m (97.66%, P < 0.05). After fertilization, significantly more embryos reached the morula stage (26.87%, P < 0.05) for the 2.5 m lutein concentration. Selected lutein concentrations have a beneficial influence on cumulus expansion and oocyte viability, therefore improving maturation. Embryo developmental potential to the morula stage was also increased. This is because the antiox-
idant lutein protects lipid components in oocytes and embryos against oxidative injury. doi:10.1016/j.copbio.2011.05.139
A7 Comparing SSV and OPS vitrification techniques and different solutions on cryopreservation of mouse blastocysts Tolga Akkoc 1 , Ali Cihan Taskin 1 , Arzu Tas Caputcu 1 , Sezen Arat 1 , Haydar Bagis 2 1 TUBITAK MRC, Genetic Engineering and Biotechnology Institute (GEBI), Kocaeli, Turkey 2 Department of Medical Genetics, Faculty of Medicine, Adiyaman University, Adiyaman, Turkey
E-mail address: [email protected] (T. Akkoc) Cryopreservation of mammalian embryos is important in transgenic technology, cloning, embryo shipping and conservation of genetic resources. In this study we vitrified blastocysts stage mouse embryos with Solid Surface Vitrification (SSV)and Open Pulled Vitrification (OPS). The aim of this study was to compare both techniques and solutions each other. In the study, blastocysts were divided in five different groups, they were vitrified-warmed than cultured in embryo culture medium for one day to investigate expandation rates and total nucleus number. 1. group (SSV technique using SSV solutions), 2. group (SSV technique using OPS solutions), 3. group (OPS technique using OPS solutions), 4. group (OPS technique using SSV solutions)and non-vitrified PN embryos were used as a control group. As a result re-expandation rates of control and experimental groups 1, 2, 3 and 4 were 34/35 (%97), 53/58 (%91), 53/54 (%98), 43/49 (%88), 14/35 (%42) respectively, total cell numbers were 93.2, 83.6, 85.5, 75.2, and 40, respectively. It is observed that both re-expandation rates and total nucleus number of vitrified-warmed blastocysts using SSV technique with OPS solutions was higher than the other groups. doi:10.1016/j.copbio.2011.05.140
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
A5 DNA barcoding approach in diversification of species from genus Loligo Emre Keskin, Hasan Huseyin Atar Department of Fisheries and Aquaculture Engineering, Agricultural Faculty, Ankara University, Ankara, Turkey E-mail address: [email protected] (E. Keskin) Squid is used to generalize seafood products manufactured from species belonging to the families Loliginidae and Ommastrephidae. The genus Loligo is one of the most representative and widely distributed groups of myopsid squids. In this study, a DNA barcoding approach was used to clarify phylogenetic relationship among 8 members of Loliginidae family. Muscle tissue samples of each species (Loligo bleekeri, L. forbesii, L. gahi, L. opalescens, L. pealei, L. plei, L. subulata and L. vulgaris) were taken from each specimen and stored at −20◦ C until DNA extraction. Mitochondrial COI gene was amplified using polymerase chain reaction with specific primers designed for Loligo genus. Amplified DNA’s were purified and DNA sequencing reactions were conducted. Alignment of sequences and phylogenetic analyses were conducted using MEGA 5. Tamura-Nei model was used in computing pair-wise distances. Maximum likelihood method was used in construction of phylogenetic tree. Including all species studied, 67.98% of amplified region was found to be conserved among species. Rate of transitional pairs over transversional pairs (R) was calculated as 1.30. Average distance of evolutionary divergence between sequences was 0.178. In conclusion, DNA barcoding approach was successful in determining the phylogenetic relationship among members of genus Loligo at species level. doi:10.1016/j.copbio.2011.05.138
A6 Effect of lutein on swine oocyte viability, cumulus expansion and embryo culture Ileana Miclea, Andrea Hettig, Marius Zahan, Iulian Roman, Vasile Miclea University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Husbandry and Biotechnology, Cluj-Napoca, Romania E-mail address: [email protected] (I. Miclea) The goal of this research was to establish the influence of several lutein concentrations on swine oocyte viability, cumulus cell function, maturation and embryo development. Pig oocytes were maturated for 45 hours in M199 supplemented with lutein (2.5, 5, 8, 10 m) and cumulus oophorus expansion was examined. Viability and the presence of the first polar body were assessed by fluorescent staining with FDA, PI and Hoechst 33258. Mature oocytes were fertilized in TALP and then embryos were cultured for 48 hours in NCSU-37 supplemented with the same lutein concentrations. Oocytes matured in lutein medium supplemented with 8 m had fully expanded cumulus oophorus (86.73%, P < 0.05, LSD test). Viability was improved by the addition of lutein: 2.5 m (98.29%, P < 0.05) and 5 m (97.66%, P < 0.05). After fertilization, significantly more embryos reached the morula stage (26.87%, P < 0.05) for the 2.5 m lutein concentration. Selected lutein concentrations have a beneficial influence on cumulus expansion and oocyte viability, therefore improving maturation. Embryo developmental potential to the morula stage was also increased. This is because the antiox-
idant lutein protects lipid components in oocytes and embryos against oxidative injury. doi:10.1016/j.copbio.2011.05.139
A7 Comparing SSV and OPS vitrification techniques and different solutions on cryopreservation of mouse blastocysts Tolga Akkoc 1 , Ali Cihan Taskin 1 , Arzu Tas Caputcu 1 , Sezen Arat 1 , Haydar Bagis 2 1 TUBITAK MRC, Genetic Engineering and Biotechnology Institute (GEBI), Kocaeli, Turkey 2 Department of Medical Genetics, Faculty of Medicine, Adiyaman University, Adiyaman, Turkey
E-mail address: [email protected] (T. Akkoc) Cryopreservation of mammalian embryos is important in transgenic technology, cloning, embryo shipping and conservation of genetic resources. In this study we vitrified blastocysts stage mouse embryos with Solid Surface Vitrification (SSV)and Open Pulled Vitrification (OPS). The aim of this study was to compare both techniques and solutions each other. In the study, blastocysts were divided in five different groups, they were vitrified-warmed than cultured in embryo culture medium for one day to investigate expandation rates and total nucleus number. 1. group (SSV technique using SSV solutions), 2. group (SSV technique using OPS solutions), 3. group (OPS technique using OPS solutions), 4. group (OPS technique using SSV solutions)and non-vitrified PN embryos were used as a control group. As a result re-expandation rates of control and experimental groups 1, 2, 3 and 4 were 34/35 (%97), 53/58 (%91), 53/54 (%98), 43/49 (%88), 14/35 (%42) respectively, total cell numbers were 93.2, 83.6, 85.5, 75.2, and 40, respectively. It is observed that both re-expandation rates and total nucleus number of vitrified-warmed blastocysts using SSV technique with OPS solutions was higher than the other groups. doi:10.1016/j.copbio.2011.05.140