Effects Of Vitamin E Supplementation On Ovine Spermatogenesis And Its Regulating Mechanisms

The current research composed of five experiments were conducted to investigate the effects of different levels of vitamin E supplementation on ovine spermatogenesis and its regulatory mechanisms.Forty healthy three-month-old weaned Hu male lambs with similar body weight were randomly allocated to 4 groups with ten in each.The lambs in the control group were fed with basal diets,and the lambs in the three treatment groups were additionally supplemented with 200,400 and 2000 IU/d vitamin E,respectively.The experiment lasted for 105 days after a 15-day adaption periods.The animals were fed in individual pen with free access to water.All the lambs were slaughtered at the end of the experiment in Halal methods,and the relative samples were collected for analysis.Exp.1:Effects of dietary vitamin E supplementation on growth performance and carcass traits of Hu male lambs.The dry matter intake(DMI)was measured each day.The body size and body weight were tested in the 35,70 and 105 days of the experiment,and the average daily gain(ADG)and feed conversion rate(FCR)were determined.All the animals were slaughtered at the end of the experiment for carcass traits measurement.The results showed that dietary vitamin E supplementation tended to increase ADG of Hu male lambs(0.05 ?P<0.1),and significantly increased FCR during the whole and the first 35 days of the experiment(P<0.05).The DMI in the last 35 days were significantly increased in the vitamin E added groups(P<0.05).The lambs in the vitamin E-supplemented groups had a higher body length(0.05 ? P<0.1)and chest circumference(P<0.05).It was noted that dietary vitamin E supplementation could improve carcass weight of Hu male lambs(0.05<P<0.1),and a better meat performance was observed in the vitamin E-supplemented groups(P<0.05).The results of this experiment revealed that vitamin E was beneficial to growth and development of Hu male lambs,and also showed a obvious effect on meat performance.Vitamin E supplementation of 2000 IU/d was suitable in the first 35 days of the experiments,but the optimal dose during the whole experimental period based on this experiment was 200 to 400 IU/d.Exp.2:Effects of dietary vitamin E supplementation on spermatogenesis of Hu male lambs.The scrotum sizes were measured at the 35,70 and 105 days of the experiment.The samples of testis and epididymis were taken out after the slaughtering process for organ development measurement.The paraffin sections were made for germ cells counting.The total RNA of testis were extracted for apoptosis-related gene expression analysis.The results showed that dietary vitamin E supplementation increased scrotum length(0.05 ? P<0.1)and width(P<0.05)of Hu male lambs,and also showed the tendencies to increase testicular weight and testicular width(0.05<P<0.1).The number of spermatocytes and mature spermatozoa were significantly increased due to vitamin E supplementation,in which VE200 and VE400 groups showed the better effect(P<0.05).The apoptosis index of germ cells in the VE2000 group were obviously higher than others,as well as the BAX gene expression(P<0.05).It was concluded that dietary vitamin E supplementation could facilitate the reproductive organ development and spermatogenic process of Hu male lambs.However,the high dose of vitamin E supplementation could lead to apoptosis of germ cells,indicating it might be detrimental to ovine spermatogenesis.Exp.3:Effects of vitamin E on ovine primary testicular cell viability.The experiment were conducted three times.The freshly matured testes samples were collected for testicular primary cell isolation.The motility rate of cells was identified,followed by primary cell culture.The culture medium was added with different levels of vitamin E(50,100,200,400,800,1000,1400 and 1600 ?mol/L),and the blank control group and negative control group were also designed in this experiment.The testicular cells were cultured for 24 h,48 h,72 h and 96 h,respectively,and cell viability was detected by CCK-8 methods.The samples in the control group,1000 ?mol/L treated group and 1600 ?mol/L treated group were also selected for intracellular vitamin E concentration analysis.The results showed that the motility rate of ovine testicular primary cell was 92.3%.The primary cell viability showed similar trends in all testing time that.it was improved significantly in the vitamin E-added groups,in which 1000 ?mol/L treated group showed a better effect(P<0.05).The intracellular vitamin E concentration was not detected in the blank control group,while the concentration in vitamin E-treated groups were obviously higher than control(P<0.05),but there was no significantly difference between the treatment groups(P>0.05).The results of this experiment revealed that vitamin E could play direct roles in ovine testicular cell proliferation and development.Exp.4:Effects of dietary vitamin E supplementation on ovine serum hormone levels and gene expressions of hormone receptors.The blood of each lamb was gathered at the end of the experiment and serum was collected after centrifugation for analysis of vitamin E content and serum hormone levels.The pituitary and testis were also collected for gene expression analysis.The results showed that the serum and testicular vitamin E content were significantly increased with dietary vitamin E supplementation(P<0.05),and VE2000 group also showed higher content than those of VE200 and VE400 groups(P<0.05).There was no significant difference on growth-related hormone including growth hormone(GH),insulin-like growth factor 1(IGF-1),triiodothyronine(T3)and reproductive hormone levels including gonadotropin releasing hormone(GnRH),follicle stimulating hormone(FSH),luteinizing hormone(LH)and testosterone among groups(P>0.05),except that the serum tetraiodothyronine(T4)concentration was significantly lower in the VE2000 group(P<0.05).The gene expressions of FSHR and AR in ovine testis were downregulated in the vitamin E added groups(P<0.05),but no difference of GnRHR and LHR gene expressions was observed(P>0.05).The results of this experiment revealed that dietary vitamin E supplementation could improve vitamin E concentration in ovine serum and tissues,and showed no effect on serum hormone levels.But high dose of vitamin E supplementation might lead to the abnormal thyroid function,followed by decreased T4 concentration.The obvious downregulation of FSHR and AR gene expressions in the VE2000 group might be the cause of declined effects on spermatogenesis.Exp.5:Research on the mechanisms of ovine spermatogenesis affected by vitamin E using RNA-seq and iTRAQ.Three testis samples were randomly selected in the CK,VE200 and VE2000 groups for RNA-seq and iTRAQ analysis.The RNA-seq results showed that there were 36 significant differentially expressed genes(DEGs)between the CK and the VE200 groups,and 145 DEGs between the CK and the VE2000 groups.Some overlapping PEQs such as NDRG1,FSCN3 and CYP26B1 were considered as important factors which were involved in the ovine spermatogenic process regulated by vitamin E.The validation of qRT-PCR also revealed that dietary vitamin E supplementation could significantly downregulate NDRG1 and CYP26B1 gene expressions in ovine testis,as well as significantly upregulate FSCN3 gene expression.The correlation analysis suggested that these changes might be the reason why germ cell number differed.Furthermore,only the ovine testis in the VE2000 group had a lower gene expressions of cytoskeleton and extracellular matrix(ECM)components related genes,including TUBB,VIM and various subtypes of collagen,which played important roles in modulating spermatogenesis.The iTRAQ results also showed that the meiosis and mitotic gene conversion processes were affected in the VE200 group,but high dose of vitamin E supplementation influenced TGF-? signaling pathway,which leading to the downregulating expressions of ECM components related genes.This would result in the reduced ECM contents,leading to the detrimental effect on ovine spermatogenesis eventually.In conclusion,dietary vitamin E supplenmentation could influence ovine growth and development,as well as spermatogenesis.Vitamin E was able to improve ovine testicular viability and affected key factor expressions during the stages of spermatogonia proliferation,meiotic division of spermatocytes and maturation of spennatids,including NDRGI,FSCN3 and CYP26B1.Meanwhile,high dose vitamin E supplementation showed a detrimental effect including decreased number of germ cells and increased number of apoptotic cell numbers on ovine spermatogenesis.This probably resulted from decreased serum T4 hormone level and downregulated testicular FSHR and AR gene expressions.It was noted that TGF-P signaling pathway was obviously affected,so the ECM components related gene expressions were downregulated.This may probably reduce the ECM contents,which had a negative effect on ovine spermatogenesis eventually.