| In vitro embryo production plays a crucial role in the preservation of high-quality germplasm resources,but is influenced by various factors,resulting in a low implantation rate and high abortion rate after embryo transfer.Improving in vitro maturation medium is an important method to improve in vitro production efficiency.Fatty acids,as an energy substance,can be directly utilized by oocytes,and triglycerides provide approximately 3.5times more energy than glucose.At the same time,high lipid oocytes such as pigs and cows are highly dependent on fatty acid β-oxidation during maturation.Carnitine,as a carrier,can transport fatty acids to mitochondria for oxidative decomposition,resulting in ATP production.Studies have shown that the addition of L-carnitine(LC)during in vitro maturation(IVM)can improve the quality of various animal oocytes and enhance early embryonic development potential.This study investigated the effects of LC addition during bovine oocyte IVM on nucleus,cytoplasmic maturation and lipid metabolism,and investigated the effects on early embryonic development of parthenogenetic activation(PA),in vitro fertilization(IVF)and somatic cell nuclear transfer(SCNT).It provides a theoretical basis for further improving the production efficiency of bovine embryos in vitro.The specific contents are as follows:The effect of L-carnitine on in vitro maturation of bovine oocytes:(1)This study set up a control group without adding LC and three additional groups(0.25,0.50,1.00 mg/m L)in oocyte maturation medium(OM),Compared with the control group,three additional groups had no significant effect on the excretion of the first polar body(PB1)of oocytes(P > 0.05);After counting the cleavage rate and blastocyst rate of four groups of oocytes with PB1 in PA,there was no significant difference in cleavage rate among the three added groups compared to the control group(P > 0.05);The blastocyst rate of the 0.5 mg/m L group was significantly higher than that of the control group(P < 0.05),and there was no significant difference between the 0.25 and 1.00 mg/m L groups and the control group(P > 0.05).This indicates that supplementing 0.5 mg/m L of LC during IVM can promote the early embryonic development of PA,and it is speculated that it improves the quality of oocytes to a certain extent.Subsequently,0.5 mg/m L was used as the concentration of LC supplementation;The proportion of LC group oocytes with bipolar spindle distribution and aligned chromosomes did not increase(P > 0.05);The proportion of oocytes with cortical granules(CGs)distributed in a linear pattern in the LC group was significantly increased(P < 0.05);The content of lipid droplets(LD)and fatty acids(FA)in the LC group was significantly lower than that in the control group(P < 0.05 or P < 0.01),while the ATP content was significantly higher than that in the control group(P < 0.05),indicating that LC can effectively promote cytoplasmic maturation of bovine oocytes,promote LD degradation,accelerate FA decomposition,and increase ATP content,but cannot significantly promote nuclear maturation of bovine oocytes.The effect of supplementing L-carnitine during IVM on the development of IVF embryos:The cleavage rate and blastocyst rate in the LC group were significantly increased(P < 0.01 or P < 0.05)after IVF of oocytes derived from slaughterhouses.The proportion of the number of blastocysts on the 7th and 8th days in the control group and LC group was statistically analyzed,while the development rate of the LC group derived from slaughterhouses was not significantly increased(P > 0.05);After IVF,there was no statistically significant difference in cleavage rate between the LC group and the control group(P > 0.05),but the blastocyst rate and development rate were significantly increased(P < 0.05),indicating that the addition of LC can promote early development of IVF embryos and improve development rate;There was no significant difference in the total number of blastocyst cells between the LC group and the control group(P > 0.05),but ICM/TE was significantly increased(P < 0.05)and the proportion of blastocyst apoptosis was significantly reduced(P < 0.001),indicating that the addition of LC during IVM improved the quality of IVF blastocysts;The lipid droplet content of IVF blastocysts in the LC group was significantly lower than that in the control group(P < 0.05).The effect of supplementing L-carnitine during IVM on the embryonic development of SCNT: There was no statistical difference in the cleavage rate of SCNT reconstructed embryos between the LC group and the control group(P > 0.05),but the blastocyst rate in the LC group was significantly increased(P < 0.05);There was no statistically significant difference in the total number of SCNT blastocysts and ICM/TE in the LC group(P > 0.05),but the proportion of blastocyst apoptosis was significantly reduced(P < 0.05),indicating that the quality of SCNT blastocysts was improved to some extent;The lipid droplet content of SCNT blastocysts in the LC group was significantly lower than that in the control group(P < 0.05);The pregnancy rate on the 35 th day after SCNT embryo transfer was not significantly different between the LC group and the control group(P > 0.05).The pregnancy rate on the 90 th day was 19.44% in the control group and 29.70% in the LC group.The pregnancy rate in the LC group was significantly higher than that in the control group(P < 0.05),indicating that LC promotes the stability of pregnancy after SCNT embryo transfer.In summary,0.5 mg/m L LC supplementation during bovine oocyte IVM culture can promote oocyte cytoplasmic maturation,promote lipid metabolism to produce more ATP,and improve the development potential of PA,IVF and SCNT embryos,and stabilize the pregnancy of SCNT embryos.In summary,0.5 mg/m L LC supplementation during bovine oocyte IVM culture can promote oocyte cytoplasmic maturation,promote lipid metabolism to produce more ATP,and improve the development potential of PA,IVF and SCNT embryos,and stabilize the pregnancy of SCNT embryos. |
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