The Incubator Temperature And PH Effects On In Vitro Fertilization And Embryo Development In Mice

Objectives To study the effects of temperature and p H effects on the in vitro fertilization and embryo development of mice.Observing and counting the fertilization rate, cleavage rate, blastocyst formation rate and blastocyst hatching rate,and using laser scanning confocal microscopy to observe the embryo spindle changes.To explore the optimum temperature and p H effects on the in vitro fertilization incubator,and provide more standardized experimental methods and data for human assisted reproductive technology.Methods Mouse oocyte and sperm were used for in vitro fertilization, and the fertilized embryos were cultured in embryo culture medium.Five temperature points(36.4℃,36.7℃,37.0℃,37.3℃,37.6℃) of the invitro fertilization incubator were set in our study.Observing and counting first day fertilization rate,second day cleavage rate,fourth day blastocyst formation rate and fifth day blastocyst hatching rate,using laser scanning confocal microscopy to observe the embryo spindle changes.Five p H points(7.05,7.20,7.35,7.50,7.65) of the invitro fertilization culture medium were set in our study.Observing and counting first day fertilization rate,second day cleavage rate,fourth day blastocyst formation rate and fifth day blastocyst hatching rate,using laser scanning confocal microscopy to observe the embryo spindle changes.Results 1 At the effect of incubator temperature on the in vitro fertilization and embryo development of mouse,the highest fertilization rate is 37.0 ℃.The fertilization rate in36.4℃comparing with 37.3℃group have no significant difference(P > 0.005), in 36.4℃comparing with 37.0 ℃ group have no significant difference(P > 0.005), in 36.4 ℃comparing with 37.6 ℃ group have statistical significance(P < 0.005).It indicate that between 36.4 ℃and 37.3℃is suitable for fertilization of gamete.The highest cleavage rate is 36.7℃. The cleavage rate in 36.4℃ comparing with 37.0℃group have no significant difference(P > 0.005), in 36.4 ℃ comparing with 37.3 ℃ group have no significant difference(P > 0.005), in 36.4℃ comparing with 37.6℃group have obviously statistical difference(P < 0.005).It indicate that between 36.4 and 37.3 ℃ is suitable for zygote cleavage.The highest blastocyst formation rate is 37.0℃.The blastocyst formation rate in36.4℃ comparing with 37.0℃ group have obviously statistical difference(P < 0.005), in36.7℃ comparing with 37.0℃ group have no significant difference(P > 0.005), in 36.7℃comparing with 37.3℃ group have no significant difference(P > 0.005). It indicate that between 36.7 ℃ and 37.0 ℃ is suitable for blastocyst formation.The highest blastocyst hatching rate is 37.0 ℃. The blastocyst hatching rate in 36.4℃comparing with 37.0℃group have statistical significance(P < 0.005), in 36.7℃ comparing with 37.0℃group have no significant difference(P > 0.005), in 36.7℃ comparing with 37.3℃ have no significant difference(P > 0.005).It indicate that between 36.7℃ and 37.0℃ is suitable for blastocysts hatched. 2 At the effect of culture medium p H on in vitro fertilizationand embryo development of mice, the highest fertilization rate is p H 7.50. The fertilization rate in 7.05 comparing with p H 7.20 group have obviously statistical difference(P < 0.005),in p H 7.20 comparing with p H 7.65 group have no significant difference(P > 0.005).It indicate that between p H 7.20 and p H 7.65 is suitable for fertilization of gamete.The highest cleavage rate is p H 7.35.The cleavage rate in p H 7.05 comparing with p H 7.20 group have statistical significance(P < 0.005), in p H 7.20 comparing with p H7.50 group have no significant difference(P > 0.005), in p H 7.50 comparing with p H 7.65 groups have obviously statistical difference(P < 0.005). It indicate that between p H 7.20 and p H 7.50 is suitable for zygote cleavage.The highest blastocyst formation rate is p H 7.50. The blastocyst formation rate in p H 7.05 comparing with p H 7.20 group have obviously statistical difference(P < 0.005), in p H 7.20 comparing with p H 7.50 group have no significant difference(P > 0.005), in p H 7.50 comparing with p H 7.65 group have obviously statistical difference(P < 0.005). It indicate that between p H 7.20 and p H 7.50 is suitable for blastocyst formation.The highest blastocyst hatching rate is p H 7.35. The blastocyst hatching rate in p H 7.05 comparing with p H 7.20 group have statistical significance(P < 0.005), in p H 7.20 comparing with p H 7.50 group have no significant difference(P > 0.005), in p H 7.50 comparing with p H 7.65 groups have obviously statistical difference(P < 0.005).It indicate that between p H 7.20 and p H 7.50 is suitable for blastocysts hatched.Conclusions 1 The incubator temperature between 36.4 ℃-37.3 ℃ is suitable for fertilization of gametes and zygote cleavage. That between 36.7 ℃-37.0 ℃ is suitable for blastocyst formation and blastocyst hatching. 2 Incubator culture between p H 7.20-7.65 is suitable for fertilization of gametes.That p H between 7.20-7.50 is suitable for zygote cleavage, blastocyst formation and blastocyst hatching.