Effects Of Flaxseed Oil And Vitamin E On Semen Quality Of Bulls

Flaxseed oil(FO)contains more than 50% polyunsaturated fatty acids(PUFA).Adding an appropriate amount of PUFA to rooster diet or semen extender plays a very important role in maintaining sperm structure and resisting sperm freezing injury.The combined of antioxidants and PUFA can avoid the oxidative stress damage caused by PUFA to sperm.As an important antioxidant,vitamin E(VE)has many functions such as promoting animal growth,improving reproductive ability and immunity.The addition of VE to boar semen extender can improve the antioxidant capacity of frozen-thawed sperm,while the addition of VE to sheep diet can protect sperm from oxidative stress damage and improve the quality of fresh and frozen-thawed semen.However,there is no relevant report on the effect of the combined addition of FO and VE to the semen extender and diet of bulls on semen quality.Therefore,in this study,FO and VE were added to the semen extender and diet respectively,and the effects on semen quality were investigated by in vitro fertilization,metabolomics and transcriptomics.Different concentrations of FO(0,10,100,and 1000 ng/m L)or VE(0.05,0.1,and 0.2mg/m L)were added to the bull semen extender to detect fresh and frozen-thawed sperm viability,motility,kinematic parameters,plasma membrane integrity,acrosome integrity and oxidative stress indicators of sperm after freeze-thaw.The results showed that the addition of 10ng/m L FO or 0.1 mg/m L VE alone significantly improved sperm viability,motility parameters,endogenous antioxidant enzyme activities,and plasma membrane integrity after freeze-thaw(P< 0.05).Therefore,in the subsequent experiments,in addition to the control group(Control group),10 ng/m L FO(FO group),0.1 mg/m L VE(VE group)and 10 ng/m L FO + 0.1 mg/m L VE(FOVE group)were added to the semen extender,respectively.Sperm viability,motility,kinematic parameters,plasma membrane integrity,acrosome integrity rate,and oxidative stress indicators of sperm after freezing and thawing were detected.The results showed that the addition of FO and VE to the semen extender alone could significantly improve sperm motility,motility parameters,endogenous antioxidant enzyme activities of seminal plasma after freezing-thawing(P < 0.05),but there was no interaction between FO and VE(P > 0.05).Based on the results that the combined addition of FO and VE in the semen extender could improve the semen quality without interaction(P > 0.05),this experiment was designed to study the effect of adding FO and VE in the diet on the semen quality of bulls,and to observe whether there is a interaction.16 Simmental bulls in good breeding condition were selected and randomly divided into 4 groups(n=4),and given the following diets respectively,1)Control group: basal diet;2)FO group: basal diet + 24g/ Kg FO(concentrate supplement weight);3)VE group: basal diet + 150mg/Kg(concentrate supplement weight)VE;4)FOVE group: basal diet+ 150mg/Kg VE + 24g/Kg FO,The trial period lasted 10 weeks.The results showed that the addition of 24 g/Kg FO to the diet alone could significantly increase the ejaculate volume,sperm concentration,kinematic parameters of frozen-thawed sperm,the activities of CAT and GSH-Px in seminal plasma,and the levels of T and E2 in seminal plasma(P < 0.05),significantly decreased the levels of MDA and ROS in seminal plasma(P < 0.05).Supplementation of 150 mg/Kg VE alone in the diet could significantly improve the sperm viability,motility parameters,CAT and SOD activities in seminal plasma after freezing-thawing,and significantly reduce the levels of MDA and ROS in seminal plasma(P < 0.05).The combined addition of FO and VE in the diet had an interactive effect on improving sperm motility and motility parameters(P < 0.05).Therefore,semen samples from the Control group and the FOVE group were selected for in vitro fertilization experiments.The results showed that the addition of FO and VE to the diet of bull sperm could significantly improve the in vitro fertilization rate,early embryonic development ability,ATP content in the cytoplasm at the8-cell stage,The content of CAT in the embryo medium,and the expression levels of blastocyst antioxidant genes and pluripotency genes(P < 0.05),significantly decreased the level of ROS in the cytoplasm of 8-cell stage embryos(P < 0.05).In order to clarify the specific mechanism of the combined addition of FO and VE in the diet to improve the semen quality and in vitro fertilization ability of bulls,we carried out metabolomic and transcriptomic studies on seminal plasma and sperm,respectively.The results of seminal plasma metabolome showed that the combined addition of FO and VE in the diet can improve the in vitro fertilization ability of sperm by increasing the content of lanosterol in seminal plasma,and improve the antioxidant capacity of sperm by increasing the content of riboflavin.At the same time,by increasing the content of lactic acid,palmitic acid and aconitic acid,the citric acid(TCA)cycle of sperm is promoted to improve the sperm motility of bulls.Sperm transcriptome results showed that dietary supplementation of FO and VE could improve sperm motility by regulating ABAT and HADHA genes in the propionic acid metabolic pathway,thereby improving semen quality and subsequent early embryonic development of bulls.Taken together,the results of this study suggest that the addition of 10 ng/m L FO or 0.1mg/m L VE alone to the semen extender can improve the viability,kinematic parameters,endogenous antioxidant enzyme activity,and plasma membrane integrity of frozen-thawed sperm.Based on the combined analysis of transcriptome and metabolome,it was found that the combined addition of 150 mg/Kg VE and 24 g/Kg FO in the diet improve semen quality and embryo development may be related to increased aconitic acid via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway.