Effects Of Dietary Fiber Level On Follicle Development Of Gilts And The Underlying Mechanisms

Dietary fiber,known as the"The seventh nutrient",is widely used in the diets of sows,which can promote gastrointestinal development,reduce oxidative stress and improve immunity.However,there are few reports on the application of dietary fiber in gilts due to the limitation of food intake in gilts and the negative effect of dietary fiber on nutrient utilization.Studies have reported that dietary fiber can affect follicular development,improve oocyte quality,and increase early embryo survival,but the mechanism remains unclear.The ovaries of gilts not only continuously supply high-quality oocytes for fertilization,but also have sufficient large follicles before mating.Previous research by our team found that the increased energy intake of gilts would affect the number of large follicles before mating.Dietary fiber interacts with intestinal microorganisms to produce a large number of metabolites,especially short-chain fatty acids,which can promote the synthesis of serotonin and further affect the expression of mTOR,AMPK,Caspase3 and other signaling molecules.This may be an important factor affecting follicular development.Therefore,this experiment mainly takes the gilts as the research object.Firstly,the optimum dietary fiber level for follicular development was determined.The effects of dietary fiber and energy interaction on follicular development of gilts were further studied.To explore the possible mechanism of dietary fiber on follicular development of gilts from the perspective of intestinal microorganism-serotonin-mTORC1 and AMPK signaling protein.The main research contents and results are as follows:Experiment 1,Effects of dietary fiber level on follicular development and intestinal microbial composition of giltsSeventy-six gilts（LY,average weight 33.2 kg,average age 92.6 days）were randomly divided into four groups with different dietary fiber intake levels:basal dietary fiber intake group（1-fold dietary fiber intake group,1.0DF）,1.5-fold dietary fiber intake group（1.5DF）,1.75-fold dietary fiber intake group（1.75 DF）and 2.0-fold dietary fiber intake group（2.0DF）,with 19 gilts in each treatment group and 1 gilt in each replicate.The gilts from basal dietary fiber intake group（1-fold dietary fiber group,1.0DF）were fed with basal corn-soybean meal diet,while the other groups were fed with different levels of dietary fiber in addition to basal dietary fiber intake.Because the primordial follicles of gilts ovaries were mainly activated at the stage of 50kg body weight,6 sows were randomly selected from each group to collect bilateral ovaries on the 30^th day（about 123days old）.The remaining gilts in each group were further investigated for follicular development,age at first estrus and oocyte quality at the third estrus.The results are as follows:（1）The average dietary fiber intakes of the gilts from 1.0DF,1.5DF,1.75DF and2.0DF groups during the experiment were about 300g/d,450g/d,525g/d and 600g/d,respectively.The additional intake of 50%to 100%dietary fiber per day did not affect the onset of estrus.（2）Dietary fiber levels had no significant effect on the relative weight of ovaries.However,the relative weight of uterus and the length of uterine horn on both sides were increased linearly with the increase of dietary fiber intake of gilts（Linear,P<0.05）.（3）With the increase of dietary fiber intake of gilts,the proportion of primordial follicles tended to be increased linearly（Linear,P=0.059）,while the proportion of atresia follicles decreased linearly（Linear,P<0.05）on day 30 of experiment;the proportion of primordial follicles of ovaries in the third estrus still tended to be increased linearly（Linear,P=0.065）,while the proportion of antral follicles and atresia follicles decreased linearly（Linear,P=0.065）.With the increase of dietary fiber intake,polar body excretion rate of the COCs in vitro was increased linearly（Linear,P<0.05）.（4）High throughput sequencing of fecal microorganisms showed that the microbial community structure was changed significantly when dietary fiber intake increased by 75%or 100%compared with the control group.With the increase of dietary fiber intake,the relative abundances of intestinal microorganisms such as Lactobacillus,Prevotella＿9,Alloprevotella,Sphaerochaeta,Fibrobacter,Faecalibacterium at genus level were increased linearly or quadarticly（Linear or Quadarticly,P<0.05）,while that of Clostridium＿sensu＿stricto＿1,Succinivibrio,Escherichia-Shigella at genus level were decreased linearly（Linear,P<0.05）.（5）With the increase of dietary fiber intake,the concentrations of short-chain fatty acids,especially propionic acid and butyric acid in feces and colonic content of gilts were increased linearly on the 30^th day of experiment（Linear,P<0.05）.（6）With the increase of dietary fiber intake,the serotonin concentration in the serum was increased linearly on the 30^th day of experiment and the 19^th day of third estrus in gilts（Linear,P<0.05）.Meanwhile,serotonin concentrations in colon and ovary tissues of gilts were increased linearly with the increase of dietary fiber intake（Linear,P<0.05）.（7）The results of gene and protein analysis showed that the apoptotic genes Bax and Caspase 3 of granulosa cells in pre-puberty and post-puberty gilts decreased linearly with the increase of dietary fiber intake（Linear,P<0.05）.While the anti-apoptotic genes Bcl-2,and 5-HTR₇,5-HTR_2C were increased linearly（Linear,P<0.05）.The ratios of p-mTOR/mTOR,p-S6/S6,p-ERK_1/2/ERK_1/2 and Caspase-3/actin in ovarian tissues of pre-puberty gilts were significantly decreased（Linear,P<0.05）.On the contrary,the ratio of p-AMPK/t-AMPK was increased linearly（Linear,P<0.05）.Quantitative analysis of granulosa cell genes in gilts before mating showed that the expression of apoptotic genes Bax and Caspase 3 was decreased linearly with the increase of dietary fiber intake（Linear,P<0.05）.The anti-apoptotic genes Bcl-2,and 5-HTR₇,5-HTR_2B and 5-HTR_2C were increased linearly（Linear,P<0.05）.Meanwhile,the relative expression levels of GDF-9and AMH genes were increased linearly with the increase of dietary fiber intake（Linear,P<0.05）.The ratios of p-mTOR/mTOR and p-S6/S6 in ovary tissue of gilts before mating were decreased linearly with the increase of dietary fiber intake（Linear,P<0.05）,while the ratios of p-ERK_1/2/ERK_1/2（Linear,P=0.053）and Caspase-3/actin（Linear,P=0.064）trended to decrease linearly.On the contrary,the ratio of p-AMPK/t-AMPK trended to increase linearly（Linear,P=0.086）.In conclusion,（1）Under the conditions of this experiment,dietary fiber intake of 600g/d（SF:ISF=1:6）on average changed the composition of intestinal microbiota to the greatest extent,increased the concentration of propionic acid and butyric acid in colon,and increased the concentration of serotonin.（2）Under the conditions of this experiment,dietary fiber intake of 600g/d（SF:ISF=1:6）on average reduced the activation of primordial follicle to the greatest extent,and reduced the atresia of antral follicles,and improved the maturation of oocytes.（3）Under the conditions of this experiment,dietary fiber intake of600g/d（SF:ISF=1:6）on average maximized the down-regulation of ovarian mTORC1protein expression,while serotonin may be the key metabolic signal of dietary fiber regulating the ovarian mTORC1 pathway and the activation of primordial follicle.Experiment 2,Effects of energy and dietary fiber levels on follicular development in giltsThirty-two Landrace×Yorkshire crossbred gilts of similar body weight（91.7±3.7 kg;Mean±SD）and age（161.5±0.5 d;Mean±SD）were selected and allocated to one of four nutritional groups:low energy（7.9 Mcal/d DE）and low dietary fiber（300 g/d DF）（LNLF）,low energy and high dietary fiber（600 g/d DF）（LNHF）,high energy（10.0 Mcal/d DE）and low dietary fiber（HNLF）,high energy and high dietary fiber（HNHF）.The LNLF gilts were fed 2.4kg basal diets The LNHF group gilts were fed 2.4kg basal diets and an additional 300 g/d dietary fiber per day,which consisted of inulin and cellulose（1:4）.The HNLF group gilts were fed 2.4kg basal diets and an additional 240 g/d soybean oil,and the HNHF group gilts were fed 2.4kg basal diets and an additional 240 g/d soybean oil and300 g/d dietary fiber per day.There were 8 replicates in each treatment group and 1 sow in each replicate.The results are as follows:（1）High energy intake significantly increased BF of the gilts at puberty and led to early estrus（P<0.05）.However,energy and dietary fiber intake had an interactive effect on the backfat gain（P=0.075）,and high dietary fiber intake significantly reduced backfat gain under high energy intake conditions.（2）High energy intake significantly increased triglycerides,free fatty acids,and total cholesterol in the blood of the gilts（P<0.05）.High dietary fiber intake showed a tendency to reduce concentrations of free fatty acids（P=0.061）and total cholesterol（P=0.088）.However,high energy and high dietary fiber intake had significant interaction effects on plasma free fatty acid content（P=0.039）.High dietary fiber intake significantly reduced the serum glucose concentration and the AUC of the glucose curve in gilts（P<0.05）,while significantly decreased insulin concentration and the AUC of the insulin（P<0.05）.The concentration of glucose and insulin changed periodically,both were increased rapidly after feeding and decreased gradually with time.（3）High dietary fiber intake significantly increased the relative weight of stomach,large intestine and length of large intestine（P<0.05）.High energy intake significantly increased the length and weight of small intestine（P<0.05）.High dietary fiber intake significantly increased the length of bilateral uterine horns and the relative weight of uterus（P<0.05）.（4）The high energy intake significantly increased the number of large follicles before mating（P<0.05）.High dietary fiber intake significantly had a tendency to increase the number of primordial follicles（P=0.082）.At the same time,high dietary fiber intake significantly increased the number of antral follicles（P<0.05）and decreased the number of atretic follicles（P<0.05）.High energy intake had a tendency to increase the number of atretic follicles（P=0.086）,while high dietary fiber intake significantly decreased the number of atretic follicles（P<0.05）.（5）High-throughput sequencing of intestinal microorganisms revealed that high energy intake significantly reduced the relative abundance of Treponema＿2,Prevotella＿9,Prevotella＿7,Sphaerochaeta,Anaerovibrio（P<0.05）.High energy intake significantly increased the relative abundance of Eubacterium＿coprostanoligenes＿group（P<0.05）.High dietary fiber intake significantly increased the relative abundance of Rikenellaceae＿RC9＿gut＿group,Alloprevotella,Sphaerochaeta,Bifidobacterium（P<0.05）,and significantly decreased the relative abundance of Streptococcus,Clostridium＿sensu＿stricto＿1（P<0.05）.The effects of energy and dietary fiber on Streptococcus,Treponema＿2,Rikenellaceae＿RC9＿gut＿group showed significant interaction（P<0.05）.High dietary fiber intake significantly increased the relative abundance of Aloprevotella,Sphaerochaeta,Fibrobacter,Erysipelotrichaceae＿UCG-001,Bifidobacterium,Faecalibacterium,Akkermansia and related specie Lactobacillus＿gasseri and Lactobacillus＿gasseri related species（P<0.05）.However,high energy intake significantly reduced the relative abundance of Prevotella＿9,Sphaerochaeta,Fibrobacter and Lactobacillus＿gasseri species（P<0.05）.Energy and dietary fiber levels had significant interaction with Erysipelotrichaceae＿UCG-001 and Faecalibacterium（P<0.05）.（6）High dietary fiber intake significantly increased the colon content of propionic acid and butyric acid in colon content of gilts（P<0.05）.High energy intake significantly reduced the propionic acid concentration in the colon content of gilts（P<0.05）.（7）High energy intake significantly increased the serum estrogen concentration in the gilts（P<0.05）.High dietary fiber intake significantly increased serum serotonin concentration（P<0.05）.Moreover,the serum concentration of sows showed periodic changes.After feeding,the serum concentration of sows gradually increased and reached a peak about 2 hours after feeding,and then gradually decreased to the level before feeding.High dietary fiber intake significantly increased the serotonin concentration in the colonic tissue,follicular fluid and ovarian tissue of gilts（P<0.05）.High-energy intake had a tendency to reduce serotonin concentration in ovarian tissue（P=0.059）.（8）The pro-apoptosis-related genes Caspase3 and Bax were significantly（P<0.05）up-regulated in ovarian tissue of gilts fed with a high energy diet.However,the anti-apoptosis-related gene BCL-2 was significantly（P<0.05）up-regulated while Caspase3 and Bax were significantly（P<0.05）down-regulated in the ovarian tissue of gilts fed high dietary fiber diets.In addition,the serotonin receptor genes 5-HTR_1D,5-HTR_2B and 5-HTR₇ were significantly（P<0.05）up-regulated in ovarian tissue of gilts fed with a diet high in fiber.However,the serotonin receptor genes 5-HTR_1D（P=0.092）and 5-HTR_2B（P=0.068）tended to be down-regulated in ovarian tissue of gilts fed with the high energy diet.（9）The level of the apoptosis-related protein Bax and Caspase3 was significantly（P<0.05）up-regulated,while anti-apoptosis-related pretein BCL-2 was significantly（P<0.05）up-regulated in the ovarian tissue of gilts fed with a high energy diet.High dietary fiber intake significantly down-regulated the relative expression of pro-apoptosis related protein Bax,and significantly up-regulated the relative expression of anti-apoptosis related protein bcl-2（P<0.05）,and showed a trend of decreasing the relative expression of caspase-3（P=0.098）.In summary,（1）Under the conditions of this experiment,intake of 600g dietary fiber per day promoted the development of gastrointestinal tract and uterus of gilts,increased the concentration of propionic acid and butyric acid in colon,and increased the concentration of serotonin.（2）Under the conditions of this study,daily intake of 10.0Mcal DE increased the number of large ovarian follicles,but resulted in excessive apoptosis of granulosa cells and increased antral follicular atresia.Intake of 600g dietary fiber per day alleviated excessive granulosa cell apoptosis and the excessive antral follicular atresia caused by high energy.（3）Under the conditions of this study,dietary fiber intake of 600g per day may regulate apoptosis-related protein expression through serotonin receptor,which is involved in the regulation of ovarian granulosa cell apoptosis and excessive atresia of antral follicles.Experiment 3,Effect of serotonin precursor 5-hydroxytryptophan on follicular development in miceOne hundred and twenty 4-week-old C57BL/6J female mice were randomly divided into three diet groups with 40 mice in each group:dietary fiber-free basic diet（control group,Con,0%dietary fiber）,high dietary fiber diet（dietary fiber group,HDF,10%dietary fiber）and 5-hydroxytryptophan group（5-HTP,0.2%dietary 5-HTP）.The experiment diets were fed from 4 weeks to 12 weeks of age.Twenty mice were slaughtered in each treatment.The results are as follows:（1）Compared with the control group,high fiber significantly increased the feed intake of mice（P<0.05）.The average total feed intake per mouse increased by 4.0%（128.8g VS133.9g）in total 8 weeks,so the total energy intake per mouse in high fiber group decreased by about 1.0%.The average weight of mice in the high fibre group decreased significantly about 3.7%（20.58 g VS 21.38 g）at the 8^th week（P<0.05）compared with the control group.There was no significant difference in feed intake and body weight between 5-HTP group and control group.（2）Compared with control group and 5-HTP group,high dietary fiber intake significantly increased the concentration of propionic acid and butyric acid in intestinal contents（P<0.05）,and trended to increase the concentration of acetic acid（P=0.075）.（3）Compared with the control group,the blood glucose concentration of mice in the high dietary fiber group decreased significantly（P<0.05）.Serotonin levels in serum and ovarian tissues of mice in 5-HTP group were significantly higher than that in control group and high dietary fiber group（P<0.05）.Serotonin levels in serum and ovarian tissues of mice in high dietary fiber group were significantly higher than that in control group（P<0.05）.There was no significant difference in serotonin concentration in colon tissue between 5-HTP group and high dietary fiber group.But the serotonin concentration in colon tissue in 5-HTP group was significantly higher than that in control group（P<0.05）.（4）Compared with the control group,5-HTP and high dietary fiber significantly increased the proportion of primordial follicles（P<0.05）,and significantly decreased the proportion of primary and secondary growth follicles（P<0.05）.（5）Compared with the control group,serotonin receptors 5-HTR_1D,5-HTR_2C,5-HTR₇and GDF-9,BMP15 and Bcl-2 in ovarian tissues were significantly increased（P<0.05）,while the expression of Caspase-3 and Bax in ovaries was significantly down-regulated（P<0.05）by 5-HTP and high dietary fiber.（6）5-HTP and high dietary fiber significantly decreased p-mTOR/mTOR,p-ERK1/2/ERK1/2 and Caspase-3/actin ratio in ovarian tissue（P<0.05）.On the contrary,the ratio of p-AMPK/t-AMPK increased significantly（P<0.05）.In conclusion,（1）Under the conditions of this experiment,0.2%5-HTP and 10%dietary fiber could increase serotonin concentration in blood and ovarian tissue of mice.（2）Under the conditions of this experiment,0.2%5-HTP and 10%dietary fiber inhibited the activation of primordial follicles in mice,and reduced the expression of apoptosis-related genes and proteins in granulosa cells.（3）This experiment further proved that dietary fiber may affect ovarian follicle development through serotonin.Collectively,the results of three experiment demonstrated that:（1）The average intake of 600g of dietary fiber（SF:ISF=1:6）per day of gilts changed the composition of intestinal microbiota,increased the concentration of propionic acid and butyric acid in colon,increased the concentration of serotonin and decreased primordial follicular activation and increased oocyte maturation.（2）High energy intake increased the number of large follicles and the activation of primordial follicles and atresia of antral follicles.The appropriate dietary fiber intake decreased the activation of primordial follicles and atresia of antral follicles.（3）Dietary fiber down-regulated the protein expression of ovarian mTORC1 and Caspase3 pathway.Serotonin may be a key fator in the regulation of follicular development by dietary fiber.