Effects of cryoprotectant and plunging temperature in liquid nitrogen on the in vitro and in vivo development of murine morulae

The effects of plunging temperature in liquid nitrogen and cryoprotectant dilution methods were evaluated for compacted mouse morulae frozen in 1.5 M ethylene-glycol (E), 1.5M propylene-glycol (P) or 1.4M glycerol (G). Morulae were equilibrated for 10 minutes in cryoprotectant solution and loaded into 0.25 ml straws with cryoprotectant solution in 3 columns (groups E1, P1, G1) or cryoprotectant in the center and PBS in the lateral columns (E2, P2). Straws were cooled at 0.5§C/min to -25 or -30§C and plunged into liquid nitrogen. Straws were thawed in water at 22§C for 20 seconds. Cryoprotectant was diluted in 3 steps for group G1 and in one step for groups E1 and P1 (direct transfer to PBS + 10 por cento FCS) and E2 and P2 (shaken to mix the 3 columns before transferring to PBS+ 10 por cento FCS). Plunging temperature had no significant effect on the proportion of morulae developed to blastocysts in vitro; this proportion was higher (p < 0.0001) in E1 (69.2 por cento) than in E2 (60.3 por cento), G1 (51.9 por cento) and combined for P1 and P2 (46.9 por cento). In second experiment, the proportion of transferred morulae that developed to viable fetuses was lower (p < 0.07) for E1-25 than for E1-30, G1-30, E2-25 or unfrozen (control) embryos (8.7, 20.0, 20.0, 17.4 and 19.8 por cento, respectively). In conclusion, the ethylene glycol diluted directly in PBS (E1) exhibit the highest rate of in vitro embryos development, but based on in vivo embryos development was more efficacious in plunging temperature at -30§C (E1-30)

Cryopresevation of mouse morulae in glycerol, sucrose and honeybee royal jelly

Compacted mouse morulae were frozen at 0.3§C/min. or 0.5§C/min. from -6§C to -24§C or -32§C in 10 por cento of glycerol plus different sucrose concentrations with or without 0.1 por cento of honeybee royal jelly. Embryos were thawed in water bath at 22§C for 20 seconds and cryoprotectant dilution was done in three steps. Embryos were cultured in Whitten's medium for 24, 48 and 72 hours at 37§C, 5 por cento of CO2 and 100por cento of humidity. The in vitro development ranged from 56.6 por cento to 100 por cento after 72 hours. Expanded blastocysts were transferred to pseudopregnant recipients on the third day of the estrous cycle. Viable fetuses rates for embryos frozen to -24 or -32§C at 0.3§C/minute in 10 por cento glycerol + 10 por cento sucrose, 10 por cento glycerol + 10 por cento sucrose + 0.1 por cento honeybee royal jelly, 10 por cento glycerol + 0.1 por cento honeybee royal jelly or 10 por cento glycerol were respectively: 28.1 por cento and 13.6 por cento, 48.7 por cento and 31.9 por cento, 28.6 por cento and 13.2 por cento, 20.0 por cento and 42.4 por cento. Viable fetuses for embryos frozen to -24§C or -32§C at 0.5§C/minute in 10 por cento glycerol + 10 por cento sucrose or 10 por cento glycerol + 10 por cento sucrose + 0.1 por cento honeybee royal jelly were respectively 29.0 por cento and 15.3 por cento, 48.8 por cento and 32.0 por cento. We can conclude that addition of 10 por cento sucrose to 10 por cento glycerol was efficient for embryo freezing at 0.3 or 0.5§C/minute and plunged in liquid nitrogen at -24§C. The honeybee royal jelly addition provided higher viable fetuses rates when embryos were cooled at 0.3 or 0.5§C/minute and plunged in liquid nitrogen at -24§C

Effects of cryoprotectant and plunging temperature in liquid nitrogen on the in vitro and in vivo development of murine morulae / Efeitos do crioprotetor e da temperatura de imersão em nitrogênio líquido sobre o desenvolvimento in vivo e in vitro de mórulas de camundongos

The effects of plunging temperature in liquid nitrogen and cryoprotectant dilution methods were evaluated for compacted mouse morulae frozen in 1.5 M ethylene-glycol (E), 1.5M propylene-glycol (P) or 1.4M glycerol (G). Morulae were equilibrated for 10 minutes in cryoprotectant solution and loaded into 0.25 ml straws with cryoprotectant solution in 3 columns (groups E1, P1, G1) or cryoprotectant in the center and PBS in the lateral columns (E2, P2). Straws were cooled at 0.5ºC/min to -25 or -30ºC and plunged into liquid nitrogen. Straws were thawed in water at 22ºC for 20 seconds. Cryoprotectant was diluted in 3 steps for group G1 and in one step for groups E1 and P1 (direct transfer to PBS + 10% FCS) and E2 and P2 (shaken to mix the 3 columns before transferring to PBS+ 10% FCS). Plunging temperature had no significant effect on the proportion of morulae developed to blastocysts in vitro; this proportion was higher (p 0.0001) in E1 (69.2%) than in E2 (60.3%), G1 (51.9%) and combined for P1 and P2 (46.9%). In second experiment, the proportion of transferred morulae that developed to viable fetuses was lower (p 0.07) for E1-25 than for E1-30, G1-30, E2-25 or unfrozen (control) embryos (8.7, 20.0, 20.0, 17.4 and 19.8%, respectively). In conclusion, the ethylene glycol diluted directly in PBS (E1) exhibit the highest rate of in vitro embryos development, but based on in v

Os efeitos das temperaturas de imersão em nitrogênio líquido e dos métodos de remoção dos crioprotetores foram avaliados em mórulas de camundongos congeladas em etilenoglicol (E), propilenoglicol (P) e glicerol (G). Os embriões foram equilibrados em E (1,5M), P (1,5M) ou G (1,4M) por 10 minutos e envasados em palhetas de 0,25 ml com crioprotetor nas três colunas (E1, P1 G1) ou PBS nas colunas das extremidades (E2, P2). As palhetas foram resfriadas a 0,5ºC/minuto até -25 ou -30ºC e imersas em nitrogênio líquido. A descongelação dos embriões foi feita em água a 22ºC por 20 segundos. O crioprotetor dos embriões congelados em glicerol (Gl) foi removido em 3 etapas, dos congelados em etilenoglicol e propilenoglicol com crioprotetor nas 3 colunas (El e Pl) removido diretamente em PBS e dos congelados em etilenoglicol e propilenoglicol com PBS nas colunas das extremidades (E2, P2), após mistura das três colunas dentro da palheta, em PBS. Não houve influência da temperatura de imersão sobre o desenvolvimento embrionário in vitro, observando maior taxa (p 0,0001) para El (69,2%) que para E2 (60,3%), Gl (51,9%) e a combinação P1 e P2 (46,9%). Para o desenvolvimento in vivo, a taxa de fetos foi menor (p 0,07) para o grupo E1-25 do que para o Controle; Gl-30ºC; El-30ºC e E2-25ºC. Pode-se concluir que in vitro o melhor crioprotetor foi o etilenoglicol com remoção direta em PBS e q

Cryopreservation of mouse morulae in glycerol, sucrose and honeybee royal jelly / Criopreservação de mórulas de camundongos em glicerol, sacarose e geléia real

Compacted mouse morulae were frozen at 0.3ºC/min. or 0.5ºC/min. from -6ºC to -24ºC or -32ºC in 10% of glycerol plus different sucrose concentrations with or without 0.1% of honeybee royal jelly. Embryos were thawed in water bath at 22ºC for 20 seconds and cryoprotectant dilution was done in three steps. Embryos were cultured in Whittens medium for 24, 48 and 72 hours at 37ºC, 5% of CO2 and 100% of humidity. The in vitro development ranged from 56.6% to 100% after 72 hours. Expanded blastocysts were transferred to pseudopregnant recipients on the third day of the estrous cycle. Viable fetuses rates for embryos frozen to -24 or -32ºC at 0.3ºC/minute in 10% glycerol + 10% sucrose, 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly, 10% glycerol + 0.1% honeybee royal jelly or 10% glycerol were respectively: 28.1% and 13.6%, 48.7% and 31.9%, 28.6% and 13.2%, 20.0% and 42.4%. Viable fetuses for embryos frozen to -24ºC or -32ºC at 0.5ºC/minute in 10% glycerol + 10% sucrose or 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly were respectively 29.0% and 15.3%, 48.8% and 32.0%. We can conclude that addition of 10% sucrose to 10% glycerol was efficient for embryo freezing at 0.3 or 0.5ºC/minute and plunged in liquid nitrogen at -24ºC. The honeybee royal jelly addition provided higher viable fetuses rates when embryos were cooled at 0.3 or 0.5ºC/minute and plunged in liquid nitr

Embriões de camundongos foram congelados em 10% de glicerol adicionado de várias concentrações de sacarose e/ou 0,1% de geléia real, nas velocidades de 0,3ºC ou 0,5ºC/minuto até -24ºC ou -32ºC e descongelados em água a 22ºC durante 20 segundos. A diluição dos crioprotetores foi realizada em três etapas e o cultivo dos embriões no meio de Whitten em estufa com 5% de CO2, 100% de umidade e 37ºC por 72 horas. O desenvolvimento in vitro até blastocisto variou entre 56,6% e 93,4%, mostrando que 10% de sacarose + 10% de glicerol foi eficiente na congelação. Blastocistos expandidos oriundos de embriões congelados até -24 ou -32ºC à velocidade de 0,3ºC/minuto em soluções contendo 10% de glicerol + 10% de sacarose; 10% de glicerol + 10% de sacarose + 0,1% de geléia real; 10% de glicerol + 0,1% de geléia real ou 10% de glicerol, transferidos para receptoras pseudoprenhes, apresentaram, respectivamente, taxas de fetos viáveis de 28,1% e 13,6%, 48,7% e 31,9%, 28,6% e 13,2%, 20,0% e 42,4%. Embriões congelados até -24ºC ou -32ºC a 0,5ºC/minuto nas soluções de 10% de glicerol + 10% de sacarose ou 10% de glicerol + 10% de sacarose + 0,1% de geléia real apresentaram, respectivamente, taxas de fetos viáveis de 29,0% e 15,3%, 48,8% e 32,0%. Adição de sacarose e de geléia real ao meio de congelação contendo 10% de glicerol proporcionou maiores taxas de fetos viáveis a 0,3ºC e 0,5ºC/minuto e imersã