| There are two limitations for a sustainable swine production,including the shortage of feed resources with high protein content,as well as the excretions of nitrogen in feces and urine to the environment.The efficient use of protein feed resources is becoming the focus and frontier in the field of animal nutrition and feed science.Nitrogen excretions from pregnant sows account for up to 20% of the pig industry.Although low-protein diet technology is a traditional technology to reduce nitrogen excretion from growing and finishing pigs,it is not clear whether it can be also effective for pregnant gilts.The previous findings both from our research team and other research groups have confirmed the importance of microbial metabolism of dietary fiber for pregnant sows,yet both dietary fiber and intestinal microbiota significantly affect the digestion and metabolism of dietary protein or nitrogen,and it remains unclear whether the efficiency of low-protein diet could be affected by dietary fiber for pregnant gilts.To test this,the present doctorial paper was conducted to investigate the effects and the underlying mechanisms of dietary fiber on the nitrogen metabolism for pregnant sows,using the technologies of animal feeding trial,nitrogen balance trial,in vitro culture of microbiota,RT-PCR,16 Sr RNA and metabolomics.Four trials were included as follows:Experiment 1,Effects of dietary fiber and low protein diets on nitrogen metabolism and reproductive performance of pregnant gilts.According to a 2×2factorial design,28 LY first-parity gestating gilts with similar body condition and genetic background were randomly divided into 4 dietary treatment with 7duplicates of 1 pig.These four diets were formulated to contain similar level of digestive energy(3.20Mcal/kg),total lysine(0.68%),mineral elements and vitamins by adjusting corn starch,oil and other dosage.Gestating gilts were provided with 2.4kg feed per day.Nitrogen balance were conducted at 35-40 days of pregnancy and95-100 days of pregnancy,and the litter performance of sows was recorded,with the following results:(1)Growth performance of gilts during gestation: The effects of crude protein levels,fiber levels,and interaction between the two were not significant for the daily weight gain of sows 0-30 days,31-90 days,0-110 days,and bodyweight 30 days,90 days and 110 days of pregnancy.The interaction between fiber and protein levels on the daily weight gain of 90-110 days of pregnancy were observed,when feeding normal protein levels there was no effects of dietary fiber,but under low protein levels,dietary fiber significantly increased the daily weight gain of sows by62 g/d(P < 0.05).(2)Diet treatment during pregnancy significantly affected the nitrogen utilization: High dietary fiber increased fecal nitrogen excretion(P = 0.094)and reduced urine nitrogen excretion(15.91 g/d vs 13.12 g/d,P = 0.002),reduced the apparent nitrogen digestibility(P = 0.071),increased the nitrogen retention(56.15%vs 63.13%,P = 0.008),improved the net nitrogen utilization rate(47.83% vs53.05%,P = 0.027);Reducing dietary crude protein decreased fecal nitrogen and urine nitrogen excretion(P<0.01),reduced nitrogen apparent digestibility(P<0.01),but had no significant difference in nitrogen retention rate and net nitrogen efficiency(P>0.05);Pregnancy stage significantly affected the nitrogen balance.Urine nitrogen in the late pregnancy(95-100 days)was significantly reduced,and nitrogen retention,nitrogen retention rate,and net nitrogen utilization were significantly greater than those in the early pregnancy(35-40 days)(P <0.01).(3)The influence of dietary treatments during pregnancy on the litter performance of gestating gilts: Crude protein level,dietary fiber level,interaction between crude protein and dietary fiber level during pregnancy on the total litter size and number of live births,showed no significant differences.No effects of mummification,stillbirth,IUGR number,birth weight,and average birth weight were observed.Collectively,the results of experiment 1 revealed that: feeding pregnant gilts with cellulose and inulin as fiber source,increased fecal nitrogen excretion(-0.48g/d),and reduced urinary nitrogen excretion(-2.79g/d),nitrogen deposition efficiency and net utilization efficiency were improved.Experiment 2,Effects of low-protein diet supplemented with dietary fiber on the nitrogen metabolism and reproductive performance of gestating gilts.77 LY primiparous gilts,after confirmation of pregnancy after artificial insemination,were randomly assigned to 4 treatment groups in a 2×2 factorial design,and the four dietary treatment included a normal crude protein(NP: 12.82%CP,total lysine0.61%)or a low-protein diet(LP: 10.53% CP,total lysine 0.61%),in daily intake of feed at 2.15 kg(From day 1 to 90 of pregnancy)or 2.68kg(From day 91 pregnancy to parturition),and additional intake of 193.5g/d(1-90 d of pregnancy),241.2g/d(91days of pregnancy-birth)dietary fiber mixture(DF,cellulose: inulin: pectin prepared at a ratio of 34:10:1)to form four diet treatment groups(NPLF: normal protein and low fiber group,n=19;NPHF: normal protein and high fiber group,n=19;LPLF:X low protein and low fiber group,N=20;LPHF: low protein and high fiber group,n=19).The nitrogen balance trial was conducted at 35-38 days of gestation,65-68 days of gestation,and 95-98 days of gestation.The results were listed as follows:(1)Growth performance of gilts during gestation: No difference in the daily weight gain of pregnant gilts were affected by dietary treatments from days 1-30 of gestation,days of 31-60 gestation,and days of 61-90 gestation(P > 0.05).Feeding a low-protein diet during gestation significantly reduced daily weight gain from 91 to110 days of pregnancy(-162.5 g/d,P = 0.004)and daily weight gain from 1 to 110 days of pregnancy(-36.2 g/d,P = 0.024).The CP level,the interactive effect between DF and CP levels had no significant effects on daily weight gain and backfat thickness during pregnancy(P > 0.05).(2)Nitrogen balance trial: The addition of dietary fiber to the gestation diet of gilts increased the excretion of fecal nitrogen at days 65-68(+24.1%,P = 0.019)and95-98(+13.8%,P = 0.019)of pregnancy;reduced the urine nitrogen excretions at days 35-38(-12.1%,P = 0.015),65-68(-19.5%,P = 0.026),95-98 of pregnancy(-17.8%,P = 0.029);DF addition increased the nitrogen retention ratio(deposited nitrogen/ Nitrogen absorption × 100%)at days 35-38(+12.6%,P = 0.046),65-68(+21.1%,P = 0.049),and 95-98 of pregnancy(+10.4%,P = 0.041).The net utilization efficiency of nitrogen(deposited nitrogen/total intake of nitrogen ×100%),were improved by DF addition at days 35-38(+12.5%,P = 0.028),65-68(+17.4%,P = 0.099),and days 95-98 of pregnancy(+7.6%,P = 0.087).Feeding a low-protein diet reduced the urinary nitrogen excretion of gilts at different stages of pregnancy and increased nitrogen deposition efficiency,and the net utilization efficiency of nitrogen at different stages of pregnancy was not significantly affected by CP levels(P > 0.05).The interactive effects of dietary fiber and crude protein levels have no significant effects on the fecal nitrogen,urine nitrogen,nitrogen deposition efficiency and net nitrogen utilization efficiency of gilts at different gestation stages.At the same time,adding dietary fiber or reducing crude protein level in gestation diets significantly reduced the concentration of urea nitrogen and ammonia in the blood of gilts at different stages of pregnancy(P < 0.05).(3)Microbial protein content in feces: Addition of dietary fiber increased the percentage of dry matter in the feces of sows at different stages of pregnancy(P<0.05).Dietary fiber addition increased the ratio of microbial protein content in feces of gilts at days 35-38(+26.4%),65-68(+20.2%),95-98(+17.9%)of pregnancy(P < 0.01),and dietary fiber significantly increased the amount of microbial protein excretions in feces at days 35-38(+30.0%),65-68(+48.1%),and95-98 of pregnancy(+37.4%)(P < 0.01 or P < 0.05).(4)The litter performance: Decreasing the crude protein level of gilts during pregnancy increased the total litter size(number of live births + number of stillbirths)by 1.6 per litter(P = 0.022),and increased the number of stillbirths(+0.83 per litter,P = 0.046),reduced the birth weight of piglets(1.52 kg vs 1.37 kg,P = 0.006),but did not affect the number of effective piglets at birth,the number of IUGR piglets,the coefficient of variation within litter,the placental weight and the efficiency of the placenta,and the parturition length(P > 0.05).On effects of dietary fiber,and interaction between DF and CP levels on the total litter size,the number of live litters,the number of stillbirths,the effective number of live litters,the number of IUGR piglets,the coefficient of variation within the litter,the placental weight and the efficiency of the placenta were observed(P > 0.05).(5)Hormone secretion and blood biochemical indicators: The blood biochemical indicators such as creatinine,aspartate transaminase activity,alanine transaminase,transglutaminase,total protein and albumin in the blood of sows at different gestational stages were not affected by dietary treatment(P > 0.05).There was no significant difference of the reproductive hormone 17β-estradiol and progesterone concentration in the blood and the ratio of the two hormones on days30 and 90 of pregnancy(P > 0.05).(6)Fecal microbiota and their metabolites SCFAs: Through 16 S r RNA seque ncing,the pregnancy stages significantly affected the intestinal microbial abundance o f the 7 bacterial phyla,while the dietary fiber,crude protein level,and the interaction between the two had no significant effects on the microbial abundance at the phylum l evel.It was found that the crude protein level significantly affected the abundance of only one microbiota unidentified＿Prevotellaceae(P <0.05)at the genus level;The ad dition of dietary fiber had a more significant effect on the relative abundance of micro organisms at the genus level.Dietary fiber addition increased the relative abundance o f Faecalibacterium,Phascolarctobacterium,Oscillibacter and Alloprevotella(P < 0.01),and reduced the relative abundance of Bifidobacterium(P < 0.01)and unidentified＿Christensenellaceae(P < 0.05);From the perspective of pregnancy stage,the relativ e abundance of 23 of the 27 genera is affected by the pregnancy stage,among which u nidentified＿Clostridiales,Bacteroides,Lactobacillus,Terrisporobacte,Succinivibrio,Sarcina,Campylobacter,Romboutsia,unidentified＿Christensenellaceae,Enterocevio ccus(Methanobroviboccus),and the relative abundance of Streptococcus,unidentified XII＿Prevotellaceae,Oscillospira,Parabacteroides,Phoscolarctobacterium,unidentified＿Bacteroidales,Alloprevotella(P < 0.05)reached the peak at farrowing,while the rel ative abundances of Streptococcus,unidentified＿Prevotellaceae,Oscillospira,Paraba cteroides,Phoscolarctobacterium,unidentified＿Bacteroidales,Alloprevotella(P <0.05)reached the bottom at farrowing,while Turicibacter,unidentified＿unidentified＿uni dentified＿Spirocha reaches their maximum levels at day 60 of pregnancy(P < 0.05).Analysis of the concentration of short-chain fatty acids in feces found that dietary treatment had no significant effects on the acetic acid,propionic acid,butyric acid,isobutyric acid,isovaleric acid,and valeric acid concentrations in feces of gilts at day 30 of gestation.On day 90 of pregnancy,dietary fiber addition reduced the content of isovaleric acid in feces,and the interaction effect of dietary fiber and crude protein on the content of acetic acid,propionic acid,isobutyric acid,and total shortchain fatty acids were not affected by dietary treatment(P > 0.05).(7)The addition of dietary fiber to the low-protein diet of pregnant sows significantly affects the synthesis of microbial protein: Dietary fiber added to the diet of sows significantly increases the ability of microbial protein synthesis during the in vitro fermentation of microorganisms at 30 and 90 days of gestation(P<0.05),the interaction effects of dietary crude protein levels,crude protein and dietary fiber treatments in sows during pregnancy had no significant difference in microbial protein synthesis during the in vitro fermentation of fecal microorganisms at 30 and90 days of pregnancy(P> 0.05);At the same time,dietary fiber significantly affected the production of short-chain fatty acids(butyric acid,P= 0.033),(isovaleric acid,P <0.001),and(valeric acid,P= 0.047)during in vitro fermentation of intestinal microorganisms.Collectively,results of trial 1 revealed that,decreasing the level of dietary crude protein or adding dietary fiber reduces the urine nitrogen emissions,but dietary fiber shifts of nitrogen excretion from urine nitrogen to fecal nitrogen,mainly due to the increase of microbial protein synthesis of microbiota by dietary fiber.Additionally,feed a low protein diet to gestating gilts increases the total litter size of sows while increasing the number of stillborns,and reduces the birth weight of piglets.Experiment 3,Effects of adding dietary fiber in semi-purified diets on nitrogen metabolism and reproductive performance of pregnant sows.The above studies have confirmed that dietary fiber changes the nitrogen metabolism of pregnant sows,but also changes the fecal microbial diversity and microbial protein synthesis.The purpose of this experiment is to use fiber-free semi-purified diets to establish a model of gut microbial disorders in pregnant sows,and to reveal the role of gut microbiota in the control of nitrogen metabolism and reproductive performance of pregnant sows by dietary fiber.In this experiment,10 LY sows of similar body weight and age were selected,and they were randomly divided into 2treatment groups after mating,namely fiber-free or fiber group,with 5 replicates for each treatment.The diet formula of the fiber-free group is composed of flour,corn starch,soy protein isolate,fish meal and other raw materials that do not contain dietary fiber.Nitrogen balance experiments were carried out at 35-38 days of gestation and 95-98 days of gestation.Sows were slaughtered at 105 days of gestation to collect tissue samples such as the sow’s intestines and number and weight of fetus was recorded.The results were as follows:(1)The addition of dietary fiber to semi-purified diets significantly affected the nitrogen metabolism of pregnant sows: Pregnant sows of semi-purified basal diets had obvious constipation;At 35-38 days of pregnancy,adding dietary fiber to semipurified diets increased sow fecal nitrogen excretion(P=0.076),decreased urine nitrogen excretion(37.81g/d vs 28.81 g/d,P=0.002),increased nitrogen deposition(13.71g/d vs 19.05g/d,P=0.059),improved the nitrogen retention efficiency(26.55% vs 39.70%,P = 0.019)and the net nitrogen utilization rate(25.51% vs35.44%,P = 0.059).From 95 to 98 days of gestation,the effects of dietary fiber supplementation in semi-purified diets on sows’ fecal nitrogen and urinary nitrogen excretion,nitrogen deposition efficiency,and net nitrogen utilization rate showed similar effects to 35-38 days of pregnancy.(2)The addition of dietary fiber to the semi-purified diet significantly affected the reproductive performance of pregnant sows: The sows were slaughtered at 105 days gestation,and it was found that the addition of dietary fiber to the semihomogenous diet had an effect on the total number of fetuses and the number of live fetuses(14.6 vs 17.25/litter)was not significantly different(P = 0.454),reduced the number of mummies(4.6 vs 0.0 per litter,P = 0.038),and increased the total live fetal weight(11.34 kg vs 17.06 kg,P = 0.009).The addition of dietary fiber increased placental weight(P = 0.070)and pregnancy weight(P = 0.003),but did not affect the average fetal weight.(3)The addition of dietary fiber to the semi-purified diet significantly affected the blood indicators of pregnant sows: The addition of dietary fiber to the semipurified diet significantly reduced the concentration of urea nitrogen in the portal vein blood(P <0.05).Analysis of amino acid showed that dietary fiber significantly XIV increased arginine(0.036 vs 0.136 mmol/L,P <0.001),glutamic acid(1.477 vs2.671 mmol/L,P = 0.009),methylhistidine(P = 0.021),carnosine in portal vein blood(P = 0.0075);measuring the concentration of urea in the anterior vena cava blood of sows at 30,60,and 90 days of gestation showed that dietary fiber significantly reduced the blood urea concentration of sows at all stages of pregnancy(P <0.01 or P <0.1);Measurement of blood ammonia concentration showed that dietary fiber reduced the blood ammonia concentration in sow hepatic portal vein(P= 0.048)and fetal cord blood(P = 0.075).(4)The addition of dietary fiber to the semi-purified diet significantly affected the intestinal development and intestinal microbial abundance of pregnant sows: The intestinal morphology of sows was analyzed,and dietary fiber affected the duodenum,jejunum and ileum of pregnant sows.The effects of villus height and crypt depth were not changed significantly,but addition dietary fibre significantly increased the mucosal thickness of the sow colon(147.03 μm vs 187.47 μm,P =0.018).Using 16 S r RNA sequencing technology to analyze the microbes in the colon and cecal contents,it was found that the addition of dietary fiber to the semipurified diet significantly affected the ratio of Firmicutes,Bacteroidetes,Firmicutes and Bacteroidetes in the colon content(P <0.05);The relative abundance of 29 bacterial genera in the colon was analyzed,and dietary fiber significantly affected the relative abundance of Muribaculaceae＿norank,Ruminococcaceae UCG-002,Alloprevotella,Ruminococcaceae＿uncultured,Family XIII AD3011 group and other bacterial genera.The relative abundance of genus showed no significant difference between diets(P> 0.05).(5)The addition of dietary fiber to semi-purified diets significantly affected the colon contents and metabolism of pregnant sows: Using non-targeted metabonomic analysis,there were 15 different metabolites in the hepatic portal vein blood of the two groups of sows,including amino acids which there were 10 kinds of metabolism related,1 kind of carbohydrate metabolism,2 kinds of lipid metabolism,and 1 kind of nucleotide metabolism.In the identification of different metabolites in cord blood,it was found that there were 20 different metabolites between the two groups of sows,including amino acids which there were 4 kinds of metabolism related,9kinds of carbohydrate metabolism,and 2 kinds of nucleotide metabolism.In the identification of different metabolites in colon contents,it was found that there were19 different metabolites in the two groups of sows.Among them,2 were related to amino acid metabolism,3 were related to bile acid metabolism,3 were related to lipid metabolism,and 2 were related to vitamin metabolism.It showed that dietary fiber significantly affected the amino acid metabolism of the intestine of sows.(6)Analysis of genes related to intestinal and amino acid metabolism found that the addition of dietary fiber to semi-purified diets significantly down-regulated the expression of key genes BCAT2 and BCK related to amino acid metabolism(P<0.05),and up-regulated the genes related to fatty acid metabolism AMPKα,CPT1,LCAD expression(P<0.05).(7)Effects of dietary fiber addition in semi-purified diets on microbial protein synthesis during in vitro fermentation of fecal microbes in pregnant sows: Addition dietary fiber to semi-purified diets significantly increased microbial protein synthesis after the 24 h in vitro fermentation of fecal microbes in sows at 90 days of pregnancy(P = 0.017);the addition of fiber to the sow’s diet significantly increased the levels of propionic acid(P= 0.005),butyric acid(P<0.001),and valeric acid(P =0.054)during in vitro fermentation of fecal microorganisms at 90 days of pregnancy.Collectively,results of trial 3 revealed that: addition of dietary fiber significantly affected the gut microbes and nitrogen metabolism in pregnant sows,and the mechanism might be related to the effects of dietary fiber on the amino acid metabolism of the gut tissue.Overall conclusions:(1)Adding cellulose,inulin,and pectin as fiber source to corn-soybean meal basal diets can increase fecal nitrogen excretion and decrease urine nitrogen excretion in first-part gestating gilts.The greater reduction in urine nitrogen leads to higher nitrogen efficiency;Low protein diets reduce urine nitrogen emissions from pregnant gilts.Although it increases the total litter size,the number of stillbirths also increases.The applicability of low protein diets to pregnant sows still needs further research.(2)The mechanism by which dietary fiber affects nitrogen utilization efficiency is related to changes of the abundance and diversity of intestinal microbes(such as Bacteroides).Intestinal microbiota may improve the nitrogen utilization by the use of non-protein nitrogen and the production of short-chain fatty acids. |
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