Effects Of Dietary Choline During Gestation On Sow Reproductive Performance And Its Underlying Mechanism

Choline is a dietary component essential for normal function of all cells and plays an important role in fetal growth and development.Due to maternal weight gain,reproductive organ development and fetal growth,pregnant sows need a large amount of choline to synthesize cell membrane during gestation.The placenta and fetus cannot synthesize choline,and their needs for choline must entirely be satisfied by maternal supply.The choline requirement for gestation sows was established by NRC（1979）for the first time.Neither NRC（1998）nor NRC（2012）had revised the requirement due to lack of research data.Compared to the sows in 1970s,modern sows have a larger body weight and a higher litter size.It should be noted that the NRC（2012）is still the most important reference in the current feeding practice of pregnant sows,which putting sows at a risk for choline deficiency and acts as a constraint to their full reproductive potential.However,the effects of choline deficiency on reproductive performance of modern sows and its underlying mechanism have not been reported yet and need to be studied.Recent studies in human trophoblastic cells and mice showed that placental function and fetal growth could be regulated by choline supplementation,suggesting that increasing maternal dietary choline level may be a potential nutritional strategy to improve fetal growth and development and thus reproductive performance of sows.However,it has not been reported in pregnant sows and further research is needed.In this study,an in vivo experiment was designed to investigate the effects of dietary choline deficiency on reproductive performance of pregnant sows,and to investigate the effects of choline deficiency on physiological metabolism and placental gene expression of sows.Then,we used porcine placental trophoblast cells（p Tr）as a study object to investigate the effects of choline deficiency on the proliferation and apoptosis of p Tr cells and the expression of genes or proteins related to placental function,and to reveal the mechanism by which choline deficiency affected fetal growth.Finally,a large-scale dose-response experiment was conducted to investigate whether increasing dietary choline level in pregnant sows above NRC（2012）recommendation could improve fetal growth and reproductive performance of sows,and to explore the appropriate dietary choline level for pregnant sows.The experimental research of this paper consists of three parts:Experiment 1:Effects of dietary choline deficiency during pregnancy on reproductive performance of sowsDue to improved reproductive performance,sows may be at risk of choline deficiency in current feeding practices based on NRC（2012）recommendation,thus limiting their full reproductive potential.However,the effect of choline deficiency on reproductive performance of pregnant sows and its underlying mechanism have not been reported so far and need to be studied.Based on the reason,we carried out this experiment.A completely random（CRD）design was used in this experiment.Twenty LY sows with similar parity were randomly divided into two groups at the day of mating（day 0 of gestation）,and sows were fed choline sufficient diet（choline sufficient diet）and choline insufficient diet（choline sufficient diet）with choline design level of 1500 mg/kg and 500 mg/kg,respectively.There were 10 replicates per treatment and 1 sow per replicate.The experiment began at day 0 of gestation and ended at day 90 of gestation.At day 90 of gestation,sows were slaughtered.The number of corpus luteum,live fetuses,dead fetuses,mummified fetuses and reabsorbed fetuses were recorded,and fetal weight was weighed.Precaval vein blood of sows at days 12,30,60 and 90 of gestation,as well as uterine artery blood and uterine venous blood at day 90 of gestation were collected for P200 targeted metabolome analysis.The placental samples were collected at day 90 of gestation and the m RNA abundance of specific genes was determined.The results showed that:（1）The proportion of mummified fetus increased（P（27）0.05）and the embryo survival rate and the number of live fetus at day 90 of gestation decreased due to choline deficiency（P（27）0.05）,whereas the individual body weight of live fetus was not affected by choline deficiency（P（29）0.05）.（2）The cardiac index of foetus decreased（P（27）0.05）and the liver index of foetus decreased（P（27）0.01）due to choline deficiency.（3）P200 targeted metabolomic analysis shows that the concentrations of dimethylglycine（DMG）,betaine,deoxyxanthine,N2,N2-dimethylguanosine,xanthine,N6-methyladenosine,pseuduracil nucleoside,serine,L-anserine,L-carnosine and inositol in peripheral plasma of sows during early and middle pregnancy were significantly decreased due to choline deficiency.Similarly,the concentrations of DMG,betaine,L-carnosine,inositol and xanthine nucleoside in uterine arterial or venous plasma were significantly decreased by choline deficiency.The ifferential metabolites are involved in a series of metabolic pathways such as energy metabolism,nucleotide metabolism,amino acid metabolism and aminoacyl t RNA synthesis,as well as the body’s antioxidant capacity.（4）The m RNA abundance of VEGFA and IGF2 decreased（P（27）0.05）and the m RNA abundance of H19 increased significantly（P（27）0.05）)due to choline deficiency.The results showed that dietary choline deficiency significantly reduced fetal survival rate and increased the proportion of mummified fetuses,which may be closely related to the reduced metabolic level and nutrient supply capacity of pregnant sows,and the altered placental development and function.Experiment 2 Effects of choline deficiency on proliferation,apoptosis and cell function of porcine trophotecderm cellThe results of experiment 1 suggested that dietary choline deficiency during pregnancy may affect embryo survival and the incidence of mummified foetuses by affecting placental development and function.However,it is not clear how choline deficiency affects placental development and function of porcine placenta,so we conducted experiment 2.In this experiment,porcine trophotecderm cell（p Tr）was used as a study object The effects of choline treatment on p Tr cell proliferation,apoptosis and the gene or protein expression associated with choline metabolism,oxidative stress,inflammation,angiogenesis and nutrient transport were investigated.The cells were treated with 50μM choline（choline sufficiency）and 0μM choline（choline deficiency）respectively.There were 6 replicates per treatment.The results were as follows:（1）The m RNA abundance of KI67（P（27）0.01）and the protein abanduce of BCL-2（P（27）0.01）decreased,and the m RNA abundance of TP53 and BAX（P（27）0.01）and the protein abundance of BAX（P（27）0.05）increased due to choline deficiency.The immunofluorescence analysis showed that the protein expression levels of PCNA and KI67were reduced significantly by choline deficiency.（2）Flow cytometry cycle and apoptosis analysis showed that the number of G0/G1phase cells decreased（P（27）0.01）and the number of G2/M phase cells increased（P（27）0.01）when choline was deficient,whereas the number of S phase cells was not altered by choline deficiency（P（29）0.05）.Increase in the percentage of early apoptosis（P（27）0.05）and total apoptosis（P（27）0.05）in p Tr cells were detected in the 0μM versus 50μM.The percentage of late apoptosis in p Tr cells was not different between the two groups（P（29）0.05）).（3）The m RNA abundances of PEMT,CHDH,IL6,NFKB1 and RELA were up-regulated（P（27）0.05）,whereas the m RNA abundance of GLUT1 decreased（P（27）0.05）due to choline deficiency.A higher transcript abundance of PRKCD,PRKCE and PRKCZ（P（27）0.01）were detected by choline deficiengy.The results showed that choline deficiency decreased the expression of PCNA and KI67 proteins,suggesting the proliferation of p Tr cells was inhibited.Choline deficiency up-regulated BAX protein expression and down-regulated Bcl-2 protein expression,suggesting the apoptosis of p Tr cells was promoted.Meanwhile,choline deficiency disrupted the choline metabolism of p Tr cells,promoted inflammatory response,and reduced glucose transport capacity of p Tr cells.The results suggest that choline deficiency inhibits p Tr cell growth,impels placental development,and affects placental function.Experiment 3:Effects of different choline levels in gestation diet on reproductive performance of sowsThe results of experiments 1 and 2 showed that dietary choline deficiency during pregnancy reduced embryo survival and increased the proportion of mummified fetuses,and choline deficiency impaired placental development and function.Pregnant sows are at risk of choline deficiency based on NRC（2012）recommendation due to improved reproductive performance.Therefore,it is not clear whether increasing dietary choline levels above NRC（2012）recommendation could reduce the risk of choline deficiency and thus improve reproductive performance of sows.A completely randomized block design（RCBD）was used in the study.A total of 260,parities 2 to 8（mean parities 4.29±0.28）,Large White sows,with a weaning-to-estrus interval within 7 days were divided into 5treatment groups on the day of breeding and fed the experimental diets with 1050,1450,1850,2250 and 2650 mg/kg choline concentration respectively from day 0 of gestation until parturition.The choline intake of sows in the 1050mg/kg group was designed to meet the daily choline requirements of gestating sows recommended by NRC（2012）.The batch of sows which were bred in the same week was considered a block.Each treatment group had 52 replicates with 1 sow per replicate.The distribution of parities was similar in all 5treatment groups.The study lasted from day 0 of gestation（first artificial insemination）to weaning at day 18 of lactation,with the day of parturition defined as day 0 of lactation.The reproductive and litter performance were recorded.The plasma samples of precaval vein were collected from the 1050,1850 and 2650 mg/kg groups at days 30,90 and 110 of gestation.The plasma on days 30 and 110 of gestation was used to measure the concentrations of reproductive hormone,related metabolites and the blood biochemical parameters,and the plasma on day 90 was used for a non-target metabolomics analysis.Rectal fecal samples in the three choline groups were collected at days 30 and 110 of gestation for fecal microbiota analysis.The results showed that:（1）No effect of dietary choline level during gestation was found on total number of piglets born,mummified fetuses,sum of born alive and stillborn piglets（P>0.05）.（2）The individual piglet birth weight for total piglets born and piglets born alive increased linearly（P（27）0.05）with the increase in dietary choline level,whereas the within-litter birth weight CV of piglets（P（27）0.05）decreased and the litter weight of weaned piglets improved linearly（P（27）0.05）as dietary choline concentration increased.（3）Compared with 1050 mg/kg choline group,increasing dietary choline level reduced plasma H₂O₂ concentration（P（27）0.05）at day 30 of gestation and plasma HCY concentration（P（27）0.05）at day 110 of gestation.（4）A total of 46 differential metabolites were identified by plasma metabolomics analysis.Increasing dietary choline level significantly elevated the plasma concentrations of L-proline,L-phenylalanine,L-tyrosine,tyramine,dopamine,TMAO and diethanolamine,and significantly decreased the 1-methylhistidine concentrations.KEGG metabolic pathway analysis showed that the differential metabolites were involved in 19 metabolic pathways and five of them were mainly affected which including protein digestion and absorption,amino acid metabolism,mineral absorption,aminoyl-Tr NA biosynthesis and central carbon metabolism.（5）Sows fecal microbial analysis results showed that the alpha diversity（observed＿Species,Chao 1 and ACE）of gut microbiota of sows at day 110 of gestation significantly decreased（P（27）0.01）when dietary choline level reached 2650 mg/kg,compared to the 1050 mg/kg choline group.Compared with 1050 mg/kg choline group,the relative abundance of genus Kocuria at day 110 of gestation was significantly increased（P（27）0.01）in 1805 mg/kg and 2650 mg/kg choline groups.In addition,the relative abundance of genus Bifidobacterium of 2650 mg/kg choline group at day 110 of gestation was significantly lower than that of 1050 mg/kg choline group（P（27）0.01）.The results of this experiment showed that increasing dietary choline level of pregnant sows above NRC（2012）recommendation did not improve litter size of sows.Increasing dietary choline level improved the birth weight of piglets,reduced within-litter birth weight CV of piglets,and increase litter weight of piglets at weaning.Based on the results of this study and the cost of choline addition,the appropriate dietary choline level for pregnant sows is 1850 mg/kg.Based on the above three experiments,the conclusions of this paper are as follows:（1）Under the conditions of this study,dietary choline deficiency decrease energy,nucleotide and amino acid metabolism and subsequent nutrients supply capacity of pregnant sows,and impaired placental development and fetal growth by down-regulating placental IGF2 and VEGFA gene expression,and up-regulating placental H19 gene expression,thereby reducing embryo survival and increasing the proportion of mummified fetus.（2）Possible mechanisms by which choline deficiency influences placental development and function:on one hand,choline deficiency down-regulates PCNA and KI67 protein expression,which suggesting the p Tr cell proliferation is inhibited;The expression of BAX protein was up-regulated and the expression of Bcl-2 protein was down-regulated,which suggesting the apoptosis of p Tr cells was promoted.Thus,the growth of p Tr cells would decrease and the placental development would be impaired by choline deficiency.On the other hand,choline deficiency disturbs the choline metabolism of p Tr cells,promotes cellular inflammation,reduces their glucose transport capacity,thus impairing placental function.（3）The dietary choline level recommended by the NRC（2012）can meet the needs for pregnant sows to produce 12-14 piglets when the litter size was taken as evaluation criterion.Increasing dietary choline level improves birth weight and litter integrity of newborn piglets and increase litter weight of piglets at weaning.Based on the results of the study and the cost of choline addition,the appropriate dietary choline level for pregnant sows is 1850 mg/kg.