| Sheep are seasonal breeding animals,and the unexplained follicular cysts and nonpregnancy can affect the normal reproductive cycle of sheep,and significantly affect the benefits of sheep industry.This experiment was conducted to investigate the fecal microbial diversity and blood metabolome characteristics of non-pregnant ewes and follicular cyst ewes,and to explore the possible causes of non-pregnant sheep and follicular cyst in randomly mating sheep population after weaning.The ewes(Chinese Merino ram x small-tailed Han sheep,2 ～ 3 parities)were fed and managed under the same condition.Ewes are mated by random mating.Thirty days after ram withdrawal,ovary and fetal sac and other organs of sheep were detected by transrectal ultrasonic diagnosis using B mode ultrasound.According to the results of the ultrasonic image sheep were divided into non-pregnant ewes and pregnant ewes.Then,according to the follicle diameter and duration,the sheep in the non-pregnant ewes were divided into follicular cyst group and normal follicle group.Six sheep(weight 65.70±1.90 kg)were randomly selected from each group.Fecal and blood samples were collected and analyzed for fecal microbial diversity and serum differential metabolites by Illumina Miseq sequencing and liquid chromatography tandem mass spectrometry(LC-MS/MS),respectively.Firstly,the intestinal microorganisms and blood metabolism groups of nonpregnant ewes and pregnant ewes in randomly mating population were studied.The results showed that: 1)In terms of alpha diversity,compared with the pregnant ewes,the Shannon index was significantly decreased(P<0.05)in non-pregnant group;The results of PCo A and ANOSIM showed that there were significant differences in the microbial composition between the pregnant group and non-pregnant group at both phylum and genus levels(P<0.05).Linear discriminant analysis(LDA)screened out genus-level microbes with LDA value greater than 2.5.Compared with the pregnant group,Actinobacteriota,Ruminiclostridium and Corynebacterium in the non-pregnant group was significantly down-regulated(P<0.05),Desulfovibrio and Turicibacter were significantly up-regulated in the fecal microbes(P<0.05)in non-pregnant ewes.2)A total of 78 different serum metabolites were found between pregnant group and nonpregnant group(VIP>1,P<0.05),among which Lyso PC(O-18:0),6-deoxohomodolichosterone,3-acetyloxy-2-hydroxypropyl icosanoate and asparaginylalanine were significantly increased in the non-pregnant group(P<0.05).Serum levels of Deoxyinosine,Berteroin and C16-Sphinganine were significantly lower than those in the pregnant group(P<0.05).Differential metabolites were significantly enriched in 11 KEGG pathways(P<0.05).3)Procrustes analysis showed that microbiome and metabolomic expression of the samples in the pregnant group and the non-pregnant group were significantly consistent(P<0.05).The correlation analysis between differential microorganisms and differential metabolites showed that Desulfovibrio was significantly positively correlated with 3a,7a,12b-Trihydroxy-5b-cholanoic acid(P<0.05).Secondly,the gut microbiome and blood metabolome of ewes with normal follicle development and follicular cysts ewes were studied in the non-pregnant ewes.The analysis results showed that: 1)Compared with the normal follicular ewes,there was no significant difference in the alpha diversity index in fecal microorganisms with ewes with follicular cysts.Linear discriminant analysis screened out the genus-level microbes with LDA value greater than 2.The results showed that compared with the normal follicle group,Bacteroides,Anaerosporobacter and Angelakisella were significantly increased in follicular cyst group(P<0.05).2)A total of 44 different metabolites were found between the two groups(VIP>1,P<0.05),6-deoxohomodolichosterone,3-acetyloxy-2-hydroxypropyl icosanoate,3,4-dimethyl-5-pentyl-2-furanpentadecanoic acid,lndoleacetic acid,asparagine-alanine,D-pipecolic acid,linoleic acid,etc.were significantly up-regulated in follicular cysts ewes(P<0.05),while L-proline and lndoleacetic acid were significantly down-regulated(P<0.05).Among them,asparaginealanine,6-deoxyhydroxyprogesterone,and 3-acetyloxy-2-hydroxypropyl icosanoate were three different metabolites that were co-existed in the blood of non-pregnant ewes and follicular cysts ewes.Differential metabolites were significantly enriched in 6KEGG pathways(P<0.05).3)Procrustes analysis showed that the microbiome and metabolomics of the normal follicle and follicular cysts were significantly consistent(P<0.05),and the correlation analysis of microorganisms and metabolites found that Lproline were significantly negative correlated with Bacteroides(P<0.05),organic acids and derivatives(asparagine-alanine)and Lipids and lipid-like molecules(6-deoxohomodolichosterone,3-acetyloxy-2-hydroxypropyl icosanoate)was significantly positively correlated with Anaerosporobacter(P<0.05).In conclusion,the gut microbiota composition and serum metabolomic profile of ewes with non-pregnant or follicular cysts were significantly different(P<0.05).Anaerosporobacter was significantly positively correlated with metabolites such as asparagine-alanine,6-deoxohomodolichosterone and 3-acetyloxy-2-hydroxypropyl icosanoate(P<0.05).The combined action of gut microbes and serum metabolites may contribute to the formation and maintenance of follicular cysts. |
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