The Study On REE Improve The Quality Of Bovine Frozen Sperm And The Effects Of Seasonal Factors On Bovine Spermatogenesis

Rare earth elements can promote growth,scavenge free radicals,increase cell membrane fluidity and improve sperm quality,so they have a wide range of applications on agriculture,animal husbandry and medicine.In order to investigate the influence of freezing diluents with traces of rare earth elements on frozen bovine sperm quality,and to provide a basis for improving the preservation effect of freezing diluents,we carried out the following studies.In this study,we select LaCl3,CeCl3,GdCl3 and YbCl3 as the representative of light and heavy rare earths.We added rare earth compounds with 0.01～0.3μmol/L concentration to OPTIXcellTM freezing diluents.The freezing diluents were used to freeze cells,after resuscitation the survival rate of the cells was counted in order to compare and analyze the impact of these rare earth compounds on cryopreserved cell viability.With the results of these experiments,we can select a kind of rare earth compound used for following study about bovine frozen sperm.It was found that,within a given range of rare earth concentrations,the survival rate of frozen cells was enhanced as the increase of concentrations,but the difference was not significant statistical results.To study the influence of rare earth elements on mitochondria energy supplying,CeCl3,YbCl3 and a certain concentration of BaCl2 were added to the freezing diluent,then the freezing diluent was used to freeze bovine semen.We evaluated the frozen sperm motility and deformity rate.The results showed that:adding CeCl3 and YbCl3 could improve bovine sperm motility,but adding BaCl2 with CeCl3 and YbCl3 did not improve bovine sperm motility.It means that BaCl2 may be not relevant with mitochondrial energy supplying,so it is not suitable for adding to freeze diluents.Different rare earth element compound was added to bovine semen,then we determined the sperm cell membrane fluidity using ANS and DPH as fluorescent probes.The results showed that certain concentration of LaCl3,CeCl3,GdCl3 and YbCl3 could increase the membrane fluidity,and 0.3 μmol/L CeCl3 was most helpful.Furthermore,different concentration of CeCl3 was added to the freezing diluents.Frozen-thawed semen was evaluated the sperm motility,survival time,acrosome integrity,membrane integrity and deformity rate.The results showed that freezing diluents with 0.03-3μmol/L CeCl3 increased sperm motility and survival time,and freezing diluents with 0.3 μmol/L CeCl3 significantly enhanced acrosome integrity of bovine sperm after freezing and thawing(P<0.05).Analyse comprehensive,0.3-1μmol/L of CeCl3 can improve the quality of semen,so it can be used as an additive adding to bovine semen freezing diluents.To investigate the mechanism why rare earth elements improved sperm quality,we extracted the sperm mitochondria and sperm membrane,studied the effect of rare earth elements on mitochondrial membrane fluidity and sperm membrane fluidity.The results showed that 0.1-3μmol/L CeCl3 significantly improved sperm membrane fluidity.Then we studied the effect of rare earth elements on lipid peroxidation.The results showed that MDA content in seminal plasma was not significantly affected by 0.3μmol/L CeCl3.These results suggest that the mechanism why trace CeCl3 improves frozen sperm acrosome integrity may be related with increasing the sperm membrane fluidity.In recent years,as the greenhouse gas emissions increases year by year,global warming is also growing,the development of livestock industry is seriously affected.Especially,bovine sperm quality is influenced by the seasons,the sperm quality always decreases in summer.To investigate the impact of seasonal factors on bovine spermatogenesis,in this study we selected bovine testicular tissue samples of spring,summer and autumn as research material.By HE staining and observation,it was found that the spermatogenic cells of bovine testicular seminiferous tubules in spring divided strong,while the division of spermatogenic cell in summer was blocked,and the division of spermatogenic cells in autumn recovered partly,but they divided not as strong as the division in spring.After the analysis of Jinan area’s temperature in spring,summer and autumn,we considered the high temperature of Jinan area in summer to be the mine resean that affected the process of bovine spermatogenesis.To study the mechanism why seasonal factors influence the bovine spermatogenesis,We examined the expression levels of LC3 which is a autophagy-related protein in the bovine testicular tissues derived from spring and summer,the results showed that the expression of LC3 had little difference in the bovine testicular tissues derived from spring and summer.It means that the autophagic activity of spermatogenic cells was so weak that it was unable to resist cell damage caused by high temperatures.We also examined the expression levels of HSP70 and HSP90 which are heat shock proteins in the bovine testicular tissue derived from different seasons.The results showed that the expression level of HSP70 in the testicular tissue derived from summer was higher than those derived from spring and autumn,but the difference was not significant,while the expression levels of HSP90 in bovine testicular tissue derived from different seasons had little difference.We examined the expression level of Cdc2 which is a cell-cycle related protein in the bovine testicular tissues derived from different seasons.The results showed that the expression levels of Cdc2 in bovine testicular tissues derived from different seasons had little difference.We examined the apoptosis level.in the bovine testicular tissues derived from different seasons,compared the differenc.e of apoptosis levels in these tissues.The results showed that the level of spermatogenic cell apoptosis in the bovine testicular tissue derived from summer was significantly higher than those from spring and autumn.We examined the expression level of P53 and Bcl-2 which are apoptosis-related proteins in the bovine testicular tissues derived from different seasons.The results showed that,the expression level of P53 in bovine testicular tissue derived from summer was significantly higher than those from spring and autumn,while the expression level of Bcl-2 in bovine testicular tissue derived from summer was slightly higher than those from spring and autumn.It indicats that the heat weather in summer leaded to spermatogenic cell apoptosis,P53 and Bcl-2 were involved in the regulation of spermatogenic cell apoptosis in bovine testicular.