ABSTRACT
RESUMEN: El objetivo fue comparar la tasa de división (TD) y desarrollo embrionario (DE) con semen sexado X (SX) en FIV capacitado con Percoll vs. BO, y evaluar el efecto de dos concentraciones (5x106 vs. 10x106 espermatozoides/ml), capacitado con BO, comparado con el semen no-sexado (NS). Un avance importante es predeterminar el sexo en bovinos, es posible obtener mayor proporción de terneras a partir del citómetro de flujo con la capacidad de seleccionar los espermatozoides X por la diferencia del ADN (4 %, bovinos), con confiabilidad del 90 %. El SX aumenta la eficiencia reproductiva, permite la selección de hembras e incrementa la ganancia genética. Se obtuvieron complejos ovocito cúmulus (COC), de ovarios de matadero, se cultivaron para maduración en gotas de 100 µl de TCM-199 + 5 % de suero fetal bovino + 0.005 U/ml de (FSH-p) + 10 IU hCG/ml + 1µg Estradiol (E2)/ml, 15 COC/gota, cubiertas con aceite mineral, en incubadora (38,5 ºC, 5 % de CO2 y 95 % de humedad), 22 h. Posmaduración se dividieron en 4 grupos (G1, G2, G3, G4) y se realizaron dos experimentos simultáneamente: I) G1: NS a 5x106, G2: SX a 5x106, G3: SX a 10x106 espermatozoides/ml capacitados con BO. II) G2: SX a 4,5x106 espermatozoides/ml capacitados con BO y G4: SX capacitado con Percoll a 5x106 espermatozoides/ ml. Se colocaron 15 COC/gota de semen cubiertas con aceite mineral, en incubadora, 18 h. Para el desarrollo se colocaron en gotas de CR1aa, en incubadora. Se aplicó el test de χ2. La TD a las 48 h entre G1 y G3 no presentó diferencias significativas (p<0,05), sin embargo, en ambos grupos fue significativamente mayor al G2. En el DE al día 7 hubo diferencias significativas (p<0,05) a favor del G4. Se obtuvo mayor DE con el SX capacitado con Percoll respecto al BO y no hubo diferencias entre ambas concentraciones de semen.
SUMMARY: The objective of this study was to evaluate the in vitro fertility of bovine sexed semen (SX) capacitated with Percoll vs. BO. The division rate (DR), embryo development (ED) were evaluated in two concentrations 5x106 vs. 10x106 sperm/ml, capacitated with BO and compared with non-sexed semen (NS). Offspring sexing represents an important advance for livestock production. Flow cytometry separates X and Y spermatozoa by difference in DNA content (4 % greater in X) with 90 % effectiveness. The SX increases the reproductive efficiency, allows the selection of females and increases the genetic gain. Cumulusoocytes complexes (COC) were obtained from slaughterhouse ovaries. They were then cultured 22 hours for maturation in TCM199 + 5 % fetal calf serum + 0.005 IU/ml (FSH-p) + 10 IU hCG/ml + 1 mg Estradiol (E2)/ml, in 100 µl drops with mineral oil, in incubator (38.5 ºC, 5 % CO and 95 % humidity). Post maturation, 4 groups were randomly assigned (G1, G2, G3, G4) and were performed two experiments simultaneously: I) G1 was inseminated with NS at 5x106 sperm/ml, G2: SX at 5x106, G3: SX at 10x106 sperm/ml capacitated with BO. II) G2: SX at 5x106 sperm/ml capacitated with BO and G4: SX capacitated with Percoll at 5x106 sperm/ml. 15 COC/drop of capacitated semen covered with mineral oil and placed in an incubator for 18 hours. For development, they were placed in drops of CR1aa, in an incubator. Results were analyzed with the c square test. At 48 hours, there were no significant differences (p<0.05) in DR between G1 and G3, however, in both groups it was significantly greater than G2. At day 7 there were significant differences (p <0.05) in ED, greater in G4. At 48 hours, there were no significant differences (p<0.05) in DR between G1 and G3, however, in both groups it was significantly greater than G2. At day 7 there were significant differences (p <0.05) in ED, greater in G4. A higher ED was obtained with the SX capacitated with Percoll, with respect to BO and there was no difference between the two semen concentrations.
Subject(s)
Animals , Cattle , Embryonic Development , Fertilization in Vitro/veterinary , Sex Preselection/methods , Spermatozoa/physiology , Flow Cytometry , Sex Preselection/veterinary , Sperm CapacitationABSTRACT
El objetivo de este trabajo fue evaluar la fertilidad in vitro del semen bovino sexado (SX) vs. no sexado (NS) congelado-descongelado de dos toros Holstein, cada uno de la misma partida. Determinar el sexo de las crías significa un avance importante para la producción. El citómetro de flujo separa los espermatozoides X e Y por diferencia de ADN (4 % mayor en X), con 90 % de efectividad. Los complejos ovocito-cúmulus (COC) se obtuvieron de folículos de 2 a 8 mm de ovarios de frigorífico, se cultivaron para maduración 22 h en TCM-199 + 5 % de SFB + 10 % licor folicular bovino (LFB), en gotas de 100 µl, cubiertos con aceite mineral, en incubadora (38,5 C, 5 % CO2 y 95 % de humedad). Posmaduración, se formaron al azar 4 grupos de COC los cuales fueron inseminados con NS y SX de los toros 1 y 2. Los COC se incluyeron en gotas de 100 ml a razón de 10 COC por gota de semen capacitado a una concentración de 2x106 espermatozoides/ml en todos los grupos, incubados durante 6 h. Posteriormente se cultivaron en CR1aa + 5 % SFB, en incubadora. A las 48 h se evaluó el clivaje y al día 7 el desarrollo embrionario. Los resultados fueron analizados con el Test de c2. Se encontró diferencias significativas en el toro 1 en el desarrollo embrionario a favor del NS (p<0,05). En el toro 2 no se encontró diferencias significativas en el clivaje ni en el desarrollo (p<0,05).
The objective of this study was to evaluate the in vitro fertility of sexed (SX) vs. non-sexed (NS), frozen-thawed bovine semen from two Holstein bull, from the same batch each one. Offspring sexing represents an important advance for livestock production. Flow cytometry separates X and Y spermatozoa by difference in DNA (4 % greater in X) with 90 % effectiveness. Cumulus-oocytes complexes (COC) were obtained from follicles measuring between 2 and 8 mm collected from slaughterhouse ovaries; they were then cultured 22 h for maturation in TCM-199 + 5 % BFS + 10 % bovine follicular fluid (BFF) in 100 µl drops with mineral oil, in incubator (38.5 C, 5% CO2 and 95 % humidity). Postmaturation, 4 groups were randomly formed and inseminated with NS and SX of the 1 and 2 bulls, including them in 100 µl drops at 10 COC per drop of capacitated semen diluted to a concentration of 2x106 sperms/ml in all groups, incubated during 6 h. They were then cultured in CR1aa + 5% BFS in an incubator. At 48 h cleavage and at day 7 embryonic development, were assessed. Results were analyzed with c2 square Test. There were significant differences (P<0.05) in the embryonic development in bull 1, grater in NS. In bull 2 there were not significant differences in cleavage neither in embryo development.