RESUMO
The aim of the present study was to evaluate the effect of brilliant cresyl blue (BCB) selection, the type of oocyte (immature or matured) and the use of hyaluronan in the vitrification solution on further embryo developmental competence. Oocytes (n = 1308) obtained from abattoir ovaries were classified by BCB stain. Control oocytes were maintained in holding media for 90 min and then subdivided to be placed into maturation media without any treatment or were vitrified. Immature or matured oocytes were vitrified by the solid-surface technique using two different vitrification solutions. VS1: composed of 10% ethylene glycol (EG) for 10 min followed by 20% EG + 0.2M trehalose for 30 sec and finally into 30% EG + 0.5M trehalose for 30 sec, or VS2 composed by 10% EG for 10 min, followed by 20% EG + 0.2M trehalose for 30 sec, and finally into 30% EG+ 0.5M trehalose + 0.1 g/ml hyaluronan for 30 sec. Oocytes were then loaded into Fyberplugs and vitrified. After one week, Fyberplugs were open and placed directly into (37°C) 0.5M sucrose solution for 5 min, then into 0.25M of sucrose for another 5 min and finally placed into maturation medium for in vitro production. Cleavage and development rates were examined on days 2 and 7 after fertilization, respectively. The blastocyst rate of vitrified oocytes selected as BCB + (5.5 ± 0.6%) were higher than those selected as BCB - (1.0 ± 0.4%) and those that were not selected by BCB (2.0 ± 1.1%; P < 0.001). Furthermore, immature vitrified oocytes had greater (P < 0.05) cleavage and blastocyst rates (44.8 ± 1.9% and 4.0 ± 0.6%) than matured vitrified oocytes (38.3 ± 2.8% and 2.5 ± 0.6%). Finally, the addition of hyaluronan to the vitrification solution had no significant effect on development rates. In conclusion, the selection of oocytes by BCB and the use of immature oocytes increase the development rates of vitrified-warmed oocytes.
Assuntos
Feminino , Animais , Bovinos , Criopreservação , Criopreservação/veterinária , Vitrificação , Bovinos/fisiologiaRESUMO
The aim of the present study was to evaluate the effect of brilliant cresyl blue (BCB) selection, the type of oocyte (immature or matured) and the use of hyaluronan in the vitrification solution on further embryo developmental competence. Oocytes (n = 1308) obtained from abattoir ovaries were classified by BCB stain. Control oocytes were maintained in holding media for 90 min and then subdivided to be placed into maturation media without any treatment or were vitrified. Immature or matured oocytes were vitrified by the solid-surface technique using two different vitrification solutions. VS1: composed of 10% ethylene glycol (EG) for 10 min followed by 20% EG + 0.2M trehalose for 30 sec and finally into 30% EG + 0.5M trehalose for 30 sec, or VS2 composed by 10% EG for 10 min, followed by 20% EG + 0.2M trehalose for 30 sec, and finally into 30% EG+ 0.5M trehalose + 0.1 g/ml hyaluronan for 30 sec. Oocytes were then loaded into Fyberplugs and vitrified. After one week, Fyberplugs were open and placed directly into (37°C) 0.5M sucrose solution for 5 min, then into 0.25M of sucrose for another 5 min and finally placed into maturation medium for in vitro production. Cleavage and development rates were examined on days 2 and 7 after fertilization, respectively. The blastocyst rate of vitrified oocytes selected as BCB + (5.5 ± 0.6%) were higher than those selected as BCB - (1.0 ± 0.4%) and those that were not selected by BCB (2.0 ± 1.1%; P < 0.001). Furthermore, immature vitrified oocytes had greater (P < 0.05) cleavage and blastocyst rates (44.8 ± 1.9% and 4.0 ± 0.6%) than matured vitrified oocytes (38.3 ± 2.8% and 2.5 ± 0.6%). Finally, the addition of hyaluronan to the vitrification solution had no significant effect on development rates. In conclusion, the selection of oocytes by BCB and the use of immature oocytes increase the development rates of vitrified-warmed oocytes.(AU)
Assuntos
Animais , Feminino , Bovinos , Vitrificação , Criopreservação , Criopreservação/veterinária , Bovinos/fisiologiaRESUMO
Four experiments were designed to evaluate the effect of different hormonal treatments on the number and quality of cumulus oocyte complexes (COCs) recovered by ovum pick-up (OPU) in beef cattle. Experiment 1 compared the synchronization of follicle wave emergence with 2 mg estradiol benzoate (EB) and 50 mg progesterone (P4) given intramuscularly (i.m.) 6 days before OPU versus a control group in which donors did not receive any treatment. Experiment 2 evaluated the use of equine chorionic gonadotropin (eCG) and prostaglandin F2α prior to OPU. Experiment 3 compared the effect of dominant follicle removal (DFR) by ultrasound guided follicle aspiration or EB+P4 to control follicular wave emergence, and treatment with eCG or FSH to superstimulate follicle growth. Experiment 4 evaluated the effect of inserting a progesterone releasing device (CIDR) during the superstimulation treatment. In experiment 1, treatment with EB+P4 resulted in more (P 0.1) the number of COCs (4.5 ± 1.0) recovered compared to the controls (4.7± 0.7). In experiment 3, there were no differences (P > 0.1) between DFR and EB+P4; however, treatment with FSH resulted in more (P < 0.05) COCs recovered than eCG (6.8 ± 0.6 vs. 3.7 ± 0.6). In experiment 4, insertion of a CIDR device did not affect the number of COCsrecovered compared to the controls (6.3 ± 0.7 vs. 5.8 ± 0.7,respectively). In conclusion, the use of treatments that synchronize follicle wave emergence, prostaglandin F2a to avoid the presence CL at the time of OPU and superstimulation with FSH were useful to improve the number of COCs recovered in beef cattle. Conversely, the insertion of a CIDR device during the superstimulation treatment prior to OPU did not improve the number of COCs recovered nor their quality.
Assuntos
Feminino , Animais , Bovinos , Bovinos/anatomia & histologia , Bovinos/fisiologia , Gonadotropinas/efeitos adversos , Sincronização do Estro/fisiologia , Sincronização do Estro/métodos , Estradiol , Hormônio Foliculoestimulante , OócitosRESUMO
Four experiments were designed to evaluate the effect of different hormonal treatments on the number and quality of cumulus oocyte complexes (COCs) recovered by ovum pick-up (OPU) in beef cattle. Experiment 1 compared the synchronization of follicle wave emergence with 2 mg estradiol benzoate (EB) and 50 mg progesterone (P4) given intramuscularly (i.m.) 6 days before OPU versus a control group in which donors did not receive any treatment. Experiment 2 evaluated the use of equine chorionic gonadotropin (eCG) and prostaglandin F2α prior to OPU. Experiment 3 compared the effect of dominant follicle removal (DFR) by ultrasound guided follicle aspiration or EB+P4 to control follicular wave emergence, and treatment with eCG or FSH to superstimulate follicle growth. Experiment 4 evaluated the effect of inserting a progesterone releasing device (CIDR) during the superstimulation treatment. In experiment 1, treatment with EB+P4 resulted in more (P < 0.05) viable COCs (5.2 ± 0.9 vs. 2.1 ± 0.4) than the controls. In experiment 2, prostaglandin F2α prior to OPU increased (P < 0.05) the number of viable COCs (7.9 ± 1.1), but treatment with eCG did not affect (P > 0.1) the number of COCs (4.5 ± 1.0) recovered compared to the controls (4.7± 0.7). In experiment 3, there were no differences (P > 0.1) between DFR and EB+P4; however, treatment with FSH resulted in more (P < 0.05) COCs recovered than eCG (6.8 ± 0.6 vs. 3.7 ± 0.6). In experiment 4, insertion of a CIDR device did not affect the number of COCsrecovered compared to the controls (6.3 ± 0.7 vs. 5.8 ± 0.7,respectively). In conclusion, the use of treatments that synchronize follicle wave emergence, prostaglandin F2a to avoid the presence CL at the time of OPU and superstimulation with FSH were useful to improve the number of COCs recovered in beef cattle. Conversely, the insertion of a CIDR device during the superstimulation treatment prior to OPU did not improve the number of COCs recovered nor their quality.(AU)
Assuntos
Animais , Feminino , Bovinos , Bovinos/anatomia & histologia , Bovinos/fisiologia , Sincronização do Estro/métodos , Sincronização do Estro/fisiologia , Gonadotropinas/efeitos adversos , Oócitos , Hormônio Foliculoestimulante , EstradiolRESUMO
Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re-expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p < 0.05) with VS3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in-straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid-surface vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos.
Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Criopreservação/veterinária , Animais , Criopreservação/instrumentação , Criopreservação/métodos , Crioprotetores , Fertilização in vitro/veterinária , Temperatura Alta , Soluções , SacaroseRESUMO
The aim of this study was to compare in vitro survival rates of in vivo and in vitro -produced bovine embryos by slow freezing or solid surface v itrification. In vivo -produced blastocysts (n = 210) and in vitro - produced blastocysts (n = 445) were randomly allocated in two cryopreservation groups. Group 1 - embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, loaded in 0.5 ml straws, frozen at -6.5ºC and seeded. After 10 min of equilibration, straws were cooled at -0.6ºC/min until - 35ºC, and then plunged into liquid nitrogen (-196ºC). Group 2 - embryos were exposed to a 15% EG + 0.25 M trehalose solution for 1 min and then a 30% EG + 1 M trehalose solution for 30 sec to be vitrified using the Cryologic Vitrification Method (CVM ® ). After at least one week of storage, embryos in the slow freezing group were thawed in a wate r bath at 30°C for 12 sec and then placed in holding medium for 5 min and transferred into SOF culture media. Vitrified embryos were placed directly into a 0.25 M sucrose solution for 5 min then cultured in SOF medium. Re-expansion and hatching rates were evaluated at 24 and 72 h, respectively. In vivo -produced embryos had higher (P < 0.01) re-expansion (179/210, 81% vs . 244/445, 54%) and hatching rates (159/210, 72% vs . 177/445, 39%) than in vitro -produced embryos, regardless of the cryopreservation method. However, re-expansion and hatching rates were higher (P < 0.01) for in vitro -produced vitrified embryos (155/223, 69% and 132/223, 59%) than in vitro -produced embryos cryopreserved by slow freezing (89/222, 40% a nd 45/222, 20%). Although similar re-expansion rates were obtained with in vivo - produced embryos cryopreserved by the two systems, hatching rates tended to be lower (P = 0.09) with in vivo -produced embryos that were vitrified as compared to slow freezing. In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro - produced embryos compared to the conventional slow freezing procedure.
Assuntos
Animais , Preservação do Sêmen/veterinária , Sacarose/análise , Bovinos/classificaçãoRESUMO
The aim of this study was to compare in vitro survival rates of in vivo and in vitro -produced bovine embryos by slow freezing or solid surface v itrification. In vivo -produced blastocysts (n = 210) and in vitro - produced blastocysts (n = 445) were randomly allocated in two cryopreservation groups. Group 1 - embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, loaded in 0.5 ml straws, frozen at -6.5ºC and seeded. After 10 min of equilibration, straws were cooled at -0.6ºC/min until - 35ºC, and then plunged into liquid nitrogen (-196ºC). Group 2 - embryos were exposed to a 15% EG + 0.25 M trehalose solution for 1 min and then a 30% EG + 1 M trehalose solution for 30 sec to be vitrified using the Cryologic Vitrification Method (CVM ® ). After at least one week of storage, embryos in the slow freezing group were thawed in a wate r bath at 30°C for 12 sec and then placed in holding medium for 5 min and transferred into SOF culture media. Vitrified embryos were placed directly into a 0.25 M sucrose solution for 5 min then cultured in SOF medium. Re-expansion and hatching rates were evaluated at 24 and 72 h, respectively. In vivo -produced embryos had higher (P < 0.01) re-expansion (179/210, 81% vs . 244/445, 54%) and hatching rates (159/210, 72% vs . 177/445, 39%) than in vitro -produced embryos, regardless of the cryopreservation method. However, re-expansion and hatching rates were higher (P < 0.01) for in vitro -produced vitrified embryos (155/223, 69% and 132/223, 59%) than in vitro -produced embryos cryopreserved by slow freezing (89/222, 40% a nd 45/222, 20%). Although similar re-expansion rates were obtained with in vivo - produced embryos cryopreserved by the two systems, hatching rates tended to be lower (P = 0.09) with in vivo -produced embryos that were vitrified as compared to slow freezing. In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro - produced embryos compared to the conventional slow freezing procedure.(AU)