| The preservation and utilization of animal genetic resources is essential for the high-quality development of the livestock industry.The cryopreservation of gametes,embryos and somatic cells are key methods to achieve the long-term preservation and inter-temporal utilization of animal genetic martials,which can accelerate the breeding and population improvement of livestock.However,problems such as oxidative damage,lipid peroxidation,structural damage and membrane damage in embryos caused by vitrification have also limited the further development of this method.While the health of the offspring from frozen embryo has raised great concerns.Melatonin is well known antioxidant and free radical scavenger.Numerous studies have found that melatonin can improve the quality of animal embryos,and enhances embryo development and transfer efficiency.Melatonin has the potential to become a novel protection agent for vitrified embryos.Currently,there is a lack of studies on the supplementation of melatonin to vitrification and warming solution to reduce embryo cryoprotection damage.In this study,we investigated the effect of melatonin on the cryopreservation of mouse and sheep embryos.The following studies were carried out:1.The effect of melatonin addition to vitrification and warming solution on the vitrification of mouse morulae were investigated.The development of mouse in vitro morulae after treatment with vitrification and warming solution supplemented with different concentrations(0,10-3,10-5 and 10-7 mol/L)of melatonin were explored.It was found that 10-5 mol/L melatonin was the optimal concentration to promote embryo development.The developmental efficiency,level of oxidative apoptosis,and mitochondrial function of morulae were compared in Vitrification（V）,vitrification and warming solution with 10-5 mol/L melatonin as a protective agent（V-MT）,and fresh（F）mouse morulae.The results showed that the addition of 10-5 mol/L melatonin to vitrification and warming solution significantly increased the cell number of hatched blastocyst and the proportion of inner cell mass after thawing of frozen embryos（94.93±2.13 vs.83.50±4.09,28.51±0.94%vs.24.75±1.03%,P<0.05）,the blastocyst rate and hatched blastocyst rate（96.3%vs 80.0%,74.10%vs 60.0%,P>0.05）were increased,but there were no significant difference between the group F（96.30%vs 97.60%,74.10%vs 87.80%,P>0.05）.After transferring the blastocysts of morulae that had developed for 12 h,the average litter size and weaning number of the group V-MT were significantly higher than those of group V（6.22±0.68 vs.3.89±0.37,and 6.22±0.68vs.3.78±0.36,P<0.05）,and there was a tendency to increase the live birth rate（44.44%vs 32.14%,P>0.05）,which was no significant difference from group F（44.44%vs 52.14%,P>0.05）.Compared with group F,the mitochondrial membrane potential and ATP level significantly decreased and the reactive oxygen species（ROS）level significantly increased after freeze-thawing of embryos in group V（P<0.05）.But melatonin significantly increased the mitochondrial membrane potential and ATP level,and significantly decreased the ROS level in vitrified-warmed embryos（P<0.05）.Therefore,these results suggest that melatonin improve the mitochondrial function of vitrified embryos by exerting an antioxidant effect thereby reducing cryoinjury.2.In order to investigate the mechanism of melatonin in improving the mouse embryos vitrification,Single embryo RNA-Seq were performed on blastocysts developed from morulae by vitrification（V）,vitrification and warming solution supplemented with 10-5 mol/L MT（V-MT）,and fresh（Fresh）in vivo in mouse.The results showed that vitrification mainly affected amino acids,fatty acid degradation,pyruvate metabolism and glucose metabolism pathways in blastocysts,which suggest that vitrification affects the developmental potential of embryos by altering their metabolic patterns.Melatonin alleviated the metabolic impairment of embryos,especially nucleotide synthesis,by reducing the freezing-induced overexpression of Rela and Nfkb1 and thereby inhibiting the over-activation of NF-κB signaling.Melatonin alleviated the freezing-induced decrease in the expression of the genes regulating nucleotide synthesis,Ctps2,Nme4,Gmps,Nudt2,Patat,Impdh2,which were conducive to cell division and proliferation.Melatonin also restored the expression of the functional mitochondrial genes Sucla2 and Timm17a in frozen embryos.This suggests that melatonin can alleviate freezing injury by improving cell metabolism,mitochondrial function,macromolecular synthesis and nucleotide synthesis in vitrified embryos.3.Differences in different embryo freezing methods and the mechanism by which melatonin improves the vitrification of embryo were investigated.Firstly,by comparing the transfer efficiency of vitrified embryos,programmed frozen embryos and fresh morulae in sheep,it was found that the pregnancy and lambing rates after transfer of vitrified embryos were significantly higher than programmed freezing（54.20%vs.44.00%;52.10%vs.42.60%,P<0.05）.Subsequently,Single embryo RNA-Seq was performed on 11 fresh sheep morulae（fresh）,9 vitrified morulae（frozen）and 7 frozen morulae with10-5 mol/L MT added to the vitrification and warming solution.The results showed that differentially expressed genes（DEGs）were mainly enriched in protein kinase activity,adhesion process,calcium signaling pathway,and Wnt,PI3K/AKT,Ras,Erb B,and MAPK signaling pathways in vitrified embryos compared to fresh.In contrast,melatonin upregulated the expression of key pathways such as kinase activity,Erb B and PI3K/Akt signaling,thereby enhancing the resistance of morulae to vitrification injury.Melatonin also exerts a protective effect on DNA repair in vitrified embryos by upregulating XPA and HIF1A.4.In order to evaluate the effect of melatonin on the offspring of vitrified embryos,the pregnancy and birth,growth and development of the offspring,major organ lesions,and blood physiological and biochemical levels of the offspring were compared among fresh embryo transfers（F,blastocysts of in vivo morulae developed for 12 h in vitro）,vitrified embryo transfers（V）,and melatonin-added vitrified embryo transfer（V-MT）,as well as those of naturally fertilized female mouse（C）.The results showed that there were no significant difference in pregnancy rate and gestation period among the four groups of mouse recipients（P>0.05）.The first birth weight of the offspring in the three embryo transfer groups（F,V,and V-MT）was significantly higher than those in group C（P<0.05）.During the 3-week postnatal lactation period,the rate of weight gain of the embryo transfer offspring was significantly higher than that of the group C（P<0.05）,especially the fastest weight gain of the offspring females in the group V.However,after weaning,the weight gain rate of the offspring of group V decreased significantly（P<0.05）,whereas the weight gain rate of the offspring of V-MT were corrected（P<0.05）,and were no significant difference with that of the offspring of group F or group C（P>0.05）.The liver coefficients of females and epididymal coefficients of males in the 11-week-old vitrified embryo transfer offspring were significant decreased（P<0.05）,while those of V-MT offspring returned to normal.There was no significant difference in the hematological parameters of the 11-week-old offspring between the groups（P>0.05）,and there were no obvious pathological changes in the tissue sections.The liver coefficients of male and female mouses in the 20-week-old offspring of each group did not change significantly（P>0.05）,but the serum levels of ALT,TP,GLB,and A/G of male mouse in the offspring of the vitrified embryo transplantation were abnormal,suggesting a possible impairment of liver function.This indicates that the addition of melatonin to the vitrification warming solution improved the abnormalities appearing in the vitrified embryo offspring to a certain extent.In conclusion,the supplementation of melatonin to embryo vitrification warming solutions can improve the quality and developmental competence of embryos after freeze-thawing in mouse and sheep.And melatonin can partially correct the developmental abnormalities of the offspring born from vitrificated embryo.Melatonin exerts its cryoprotective effects by promoting DNA repair and nucleotide synthesis,which together maintain nucleic acid stability in vitrified embryos. |
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