繁殖周期内大白猪母猪肠道菌群特征分析

Analysis of the gut microbiota characteristics of sows during reproductive cycle

为了探究繁殖周期内母猪肠道菌群的演替规律和结构特征,试验从山东省莱西市某猪场选取12头大白猪母猪为研究对象,采集其空怀期(配种前2天)、妊娠早期(妊娠第28,42,56天)、妊娠末期(妊娠第70,84,98天)、分娩期(分娩当天)及哺乳期(泌乳第7,14,21天)繁殖周期5个阶段11个时间点的新鲜粪便样本,提取粪便样本基因组DNA,采用16S rRNA高通量测序技术进行测序,分析大白猪母猪繁殖周期不同阶段肠道微生物的多样性及在门、科、属水平上的组成结构特征。结果表明:大白猪母猪繁殖周期肠道微生物在空怀期时的α多样性最低,随着妊娠的进行,α多样性逐渐上升,在妊娠末期达到最高,进入分娩期后急剧下降,但分娩期与哺乳期差异不显著(P>0.05)。空怀期、妊娠早期、妊娠末期、分娩期和哺乳期5个阶段的平均物种数量在400~800种之间,妊娠末期时的物种数量最多,且除空怀期和妊娠早期物种数量差异不显著(P>0.05)外,其余繁殖阶段间均差异显著或极显著(P<0.05或P<0.001)。母猪繁殖周期不同阶段的β多样性存在差异(P=0.001),空怀期与妊娠早期β多样性相似性最高,分娩期与哺乳期相似性较高,空怀期与妊娠末期差异性最大。母猪繁殖周期内肠道菌群共注释到32门283科633属,相对丰度较高的前10个菌门分别为厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、放线菌门(Actinobacteriota)、变形菌门(Proteobacteria)、螺旋菌门(Spirochaetota)、蓝藻菌门(Cyanobacteria)、互养菌门(Synergistota)、疣微菌门(Verrucomicrobiota)、弯曲杆菌门(Campilobacterota)、酸杆菌门(Acidobacteriota),其中厚壁菌门和拟杆菌门是优势菌门,且二者比值在分娩期最高,在空怀期最低;相对丰度大于1%的菌科共注释到29个,其中优势菌科包括梭菌科(Clostridiaceae)、消化链球菌科(Peptostreptococcaceae)和颤螺旋菌科(Oscillospiraceae),妊娠后期和分娩期的优势菌科还包括Muribaculaceae;共存在47个特征性菌属[线性判别分析(linear discriminant analysis,LDA)>2.0],其中空怀期存在9个特征菌属,相对丰度最高的为梭状芽孢杆菌属(Clostridium_sensu_stricto_1);妊娠早期存在8个特异性菌属,相对丰度最高的为克里斯滕森菌属(Christensenellaceae_R_7_group);妊娠末期存在13个特征菌属,相对丰度较高的为梭状芽孢杆菌属UGG_014(Clostridia_UCG_014)和普雷沃氏菌属NK3B31_group(Prevotellaceae_NK3B31_group);分娩期存在7个特征菌属,相对丰度最高的为Muribaculaceae;哺乳期存在11个特征菌属,相对丰度最高的为拟杆菌属(Bacteroides)。说明大白猪母猪繁殖周期不同阶段肠道菌群的多样性存在差异,各有其特征菌属。

To explore the succession and structural characteristics of intestinal flora of sows during the reproductive cycle,twelve Large White sows were selected as the study objects from a pig farm in Laixi,Shandong Province.Fresh fecal samples were collected from 1l time points in 5 stages of the reproductive cycle,including the non-pregnancy period(2 days before mating),the pre-gestation period(28,42 and 56 days afer mating),the late gestation period(70,84 and 98 days afer mating),the farrowing period(the day of delivery)and the lactation period(7,14 and 21 days afer parturition).The genomic DNA was extracted from the samples.Subsequently,high-throughput sequencing technology targeting the 16S rRNA gene was employed to examine the gut microbial diversity and the microbial composition and structure characteristics at the phylum,family,and genus levels at different stages of Large White sow reproductive cycles.The results showed as follows:theα-diversity of gut microorganisms was the lowest at the non-pregnancy period during the reproductive cycle of Large White sows;it gradually increased with the progress of pregnancy;recorded the highest at the late gestation period,and sharply decreased after the farrowing period,and there was no significant difference between the lactation period and the farrowing period(P>0.05).The average number of species at the five stages ranged from 400 to 800,and the number of species in the late gestation period was the largest,and the difference between the number of species at the other stages was significant or extremely significant(P<0.05 or P<0.001),except that there was no significant difference in the number of species between the non-pregnancy stage and the pre-pregnancy stage(P>0.05).There were variations inβ-diversity during different stages of the reproductive cycle(P=0.001).The similarity ofβ-diversity was the highest between non-pregnancy and pre-pregnancy,which was higher between parturition and lactation,and that was the largest between non-pregnancy and late gestation periods.During the reproductive cycle of sows,their intestinal flora was composed of 32 phyla,283 families,and 633 genera.The top ten phyla with the highest relative abundance were Firmicutes,Bacteroidota,Actinobacteriota,Proteobacteria,Spirochaetota,Cyanobacteria,Synergistota,Verrucomicrobiota,Campilobacterota,and Acidobacteriota.Among them,Firmicutes and Bacteroides were the dominant phyla,and the ratio of the two was the highest during the farrowing period and lowest during the non-pregnancy period.A total of 29 families with a relative abundance greater than 1%were annotated,among which the dominant families were Clostridiaceae,Peptostreptococcaceae,and Oscllospiraceae.The dominant bacterial families in the late pregnancy period and the farrowing period also include Muribaculaceae.A total of 47 notable genera were detected(LDA>2.0),among which 9 characteristic bacteria genera existed in the non-pregnancy period,the relative abundance of which was Clostridium_sensu_stricto_1.In the pre-pregnancy period,8 notable genera were observed,the relative abundance of which was Christensenellaceae_R_7_group;13 characteristic genera were present during the late gestation period,the relative abundance of which was Clostridia_UCG_014 and Prevotellaceae_NK3B31_group;7 characteristic genera were present during the farrowing period,the relative abundance of which was Muribaculaceae;and 1l characteristic genera were present during lactation,the relative abundance of which was Bacteroides.These findings suggested that there were differences in intestinal flora diversity at different stages of the reproductive cycle of Large White sows,and each stage had its own characteristic genera.