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1.
Anim. Reprod. (Online) ; 11(4): 543-548, Oct.-Dec.2014. tab
Artigo em Inglês | VETINDEX | ID: biblio-1461134

Resumo

Studies were conducted to compare viability of immature buffalo oocytes vitrified in different types of cryoprotectants on the post-thaw morphological appearance, the in vitro maturation and developmental competence of buffalo oocytes. The vitrification solution (VS) consisted of Dulbecco’s phosphate buffered saline (DPBS) supplemented with 0.5 M sucrose, 0.4% bovine serum albumin (BSA) and different types of molar (M) concentrations of the cryoprotectants which were composed of either glycerol (G), ethylene glycol (EG) or dimethyl sulfoxide (DMSO) in order to determine the best type of vitrification cryoprotectants. The concentrations tested were 4 M, 7 M and 7M concentration of G, EG and DMSO, respectively. Cumulus oocyte complexes (COCs) were obtained from slaughterhouse ovaries. OocytesAnd their cumulus cells were vitrified immediately after collection .The COCs were pre-equilibrated in 50% of the VS for 3-5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 ml straws for 7-10 days of storage in liquid nitrogen. The straws were thawed in warm water at 37°C for 10 s. The COCs were equilibrated in 0.5 M sucrose in modified phosphate buffer (M-PBS) for 5 min and then washed in washing medium (TCM-199 plus 10% FCS). Oocyte-cumulus cells were evaluated for morphological damage. Morphologically normal COCs (Oocyte-cumulus cells) were cultured in vitro and evaluated for maturation. Oocytes were fertilized with frozen-thawed semen capacitated in Brackett and Oliphant (BO) medium containing heparin and caffeine and were evaluated for cleavage and embryonic development. The results revealed that the proportion of buffalo oocytes found to be morphologically normal was significantly (P < 0.05) higher in EG and DMSO than those obtained in G (85.0 and 83.33 vs. 65.0%, respectively). Among the damaged oocytes, cracking of zona pellucida was the most frequent abnormality observed.


Assuntos
Feminino , Animais , Búfalos , Crioprotetores/uso terapêutico , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Dimetil Sulfóxido , Etilenoglicol , Glicerol , Vitrificação
2.
Anim. Reprod. ; 11(4): 543-548, Oct.-Dec.2014. tab
Artigo em Inglês | VETINDEX | ID: vti-27033

Resumo

Studies were conducted to compare viability of immature buffalo oocytes vitrified in different types of cryoprotectants on the post-thaw morphological appearance, the in vitro maturation and developmental competence of buffalo oocytes. The vitrification solution (VS) consisted of Dulbeccos phosphate buffered saline (DPBS) supplemented with 0.5 M sucrose, 0.4% bovine serum albumin (BSA) and different types of molar (M) concentrations of the cryoprotectants which were composed of either glycerol (G), ethylene glycol (EG) or dimethyl sulfoxide (DMSO) in order to determine the best type of vitrification cryoprotectants. The concentrations tested were 4 M, 7 M and 7M concentration of G, EG and DMSO, respectively. Cumulus oocyte complexes (COCs) were obtained from slaughterhouse ovaries. OocytesAnd their cumulus cells were vitrified immediately after collection .The COCs were pre-equilibrated in 50% of the VS for 3-5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 ml straws for 7-10 days of storage in liquid nitrogen. The straws were thawed in warm water at 37°C for 10 s. The COCs were equilibrated in 0.5 M sucrose in modified phosphate buffer (M-PBS) for 5 min and then washed in washing medium (TCM-199 plus 10% FCS). Oocyte-cumulus cells were evaluated for morphological damage. Morphologically normal COCs (Oocyte-cumulus cells) were cultured in vitro and evaluated for maturation. Oocytes were fertilized with frozen-thawed semen capacitated in Brackett and Oliphant (BO) medium containing heparin and caffeine and were evaluated for cleavage and embryonic development. The results revealed that the proportion of buffalo oocytes found to be morphologically normal was significantly (P < 0.05) higher in EG and DMSO than those obtained in G (85.0 and 83.33 vs. 65.0%, respectively). Among the damaged oocytes, cracking of zona pellucida was the most frequent abnormality observed.(AU)


Assuntos
Animais , Feminino , Búfalos , Crioprotetores/uso terapêutico , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Vitrificação , Dimetil Sulfóxido , Etilenoglicol , Glicerol
3.
Anim. Reprod. ; 6(2): 416-421, 2009. tab
Artigo em Inglês | VETINDEX | ID: vti-9398

Resumo

The aim of the present study was to improve in vitro maturation and cleavage rates of buffalo oocytes. Good quality oocytes were divided into two experiments. In Experiment 1 oocytes were cultured for 24 h in a CO2 incubator at 38.5oC either in TCM-199, Hams F-10, MEM or FertiCult medium supplemented with either 10% FCS or 0.3% BSA. Experiment 2 was carried out to investigate the effect of different hormones (either 50 µg/ml eCG, 50 µg/ml FSH or 1 mg/ml E2) added to four of the afore mentioned media enriched with 10% FCS at the same culture conditions. Matured oocytes were fertilized in vitro using frozen thawed semen capacitated with heparin and caffeine. The sperm-oocytes were co-cultured for 22 h in BO or TALP medium. The fertilized oocytes were cultured in either BO or TALP medium for an additional five days at the same culture conditions and checked daily for cleavage. Addition of FCS to all media led to a higher maturation rate, without any significant variation, than BSA did (75.6 vs. 71.3%). Although no influence on the maturation rate was observed, addition of eCG to TCM-199, Hams F-10 or MEM media resulted in a non-significant increase of in vitro maturation rate of buffalo oocytes compared to other hormonal additives to the same media. Furthermore, supplementation of maturation media with eCG resulted in a non-significant higher in vitro maturation rate of buffalo oocytes compared to FSH and E2 (80.4% for eCG supplemented media vs. 74.0% and 73.0% for FSH and E2, respectively). There was a non-significant difference in the cleavage rate of buffalo oocytes matured in TCM-199 supplemented with either sera or hormones and fertilized either in BO or TALP medium. However, the highest non-significant cleavage rate was achieved when oocytes were matured in TCM-199 supplemented with eCG and fertilized in TALP medium (50%). The overall cleavage rate was not significantly greater in TALP than in BO medium (33.7 vs. 15.5%). It could be concluded that supplementation of maturation media with FCS and/or eCG could successfully improve IVM rate of buffalo oocytes. Furthermore, high cleavage rate could be achieved when oocytes were matured in TCM-199 supplemented with FCS and eCG and fertilized in TALP medium.(AU)


Assuntos
Clivagem do DNA , Oócitos/citologia , Búfalos , Técnicas de Maturação in Vitro de Oócitos
4.
Anim. Reprod. (Online) ; 6(2): 416-421, 2009. tab
Artigo em Inglês | VETINDEX | ID: biblio-1461596

Resumo

The aim of the present study was to improve in vitro maturation and cleavage rates of buffalo oocytes. Good quality oocytes were divided into two experiments. In Experiment 1 oocytes were cultured for 24 h in a CO2 incubator at 38.5oC either in TCM-199, Ham’s F-10, MEM or FertiCult medium supplemented with either 10% FCS or 0.3% BSA. Experiment 2 was carried out to investigate the effect of different hormones (either 50 µg/ml eCG, 50 µg/ml FSH or 1 mg/ml E2) added to four of the afore mentioned media enriched with 10% FCS at the same culture conditions. Matured oocytes were fertilized in vitro using frozen thawed semen capacitated with heparin and caffeine. The sperm-oocytes were co-cultured for 22 h in BO or TALP medium. The fertilized oocytes were cultured in either BO or TALP medium for an additional five days at the same culture conditions and checked daily for cleavage. Addition of FCS to all media led to a higher maturation rate, without any significant variation, than BSA did (75.6 vs. 71.3%). Although no influence on the maturation rate was observed, addition of eCG to TCM-199, Ham’s F-10 or MEM media resulted in a non-significant increase of in vitro maturation rate of buffalo oocytes compared to other hormonal additives to the same media. Furthermore, supplementation of maturation media with eCG resulted in a non-significant higher in vitro maturation rate of buffalo oocytes compared to FSH and E2 (80.4% for eCG supplemented media vs. 74.0% and 73.0% for FSH and E2, respectively). There was a non-significant difference in the cleavage rate of buffalo oocytes matured in TCM-199 supplemented with either sera or hormones and fertilized either in BO or TALP medium. However, the highest non-significant cleavage rate was achieved when oocytes were matured in TCM-199 supplemented with eCG and fertilized in TALP medium (50%). The overall cleavage rate was not significantly greater in TALP than in BO medium (33.7 vs. 15.5%). It could be concluded that supplementation of maturation media with FCS and/or eCG could successfully improve IVM rate of buffalo oocytes. Furthermore, high cleavage rate could be achieved when oocytes were matured in TCM-199 supplemented with FCS and eCG and fertilized in TALP medium.


Assuntos
Clivagem do DNA , Oócitos/citologia , Búfalos , Técnicas de Maturação in Vitro de Oócitos
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