Genotype-associated heritable rumen bacteria can be a stable microbiota passed to the offspring

Abstract Recent studies have reported that some rumen microbes are “heritable” (those have significant narrow sense heritability) and can significantly contribute to host phenotype variations. However, it is unknown if these heritable rumen bacteria can be passed to the next generation. In this study, the rumen bacteria from mother cows (sampled in 2016) and their offspring (sampled in 2019) were assessed to determine if vertical transmission occurred between the two generations. The analysis of relationship between host genotypes and heritable bacterial abundances showed that potential of five host genotypes can affect the relative abundances of two unclassified species level heritable bacteria (Pseudoscardovia and p-251-o5). The G allele of BTB-01532239 and A allele of ARS-BFGL-NGS-8960 were associated with a higher relative abundance of p-251-o5. The A allele of BTB-00740910 and BovineHD1300021786 and G allele of BovineHD1900005868 were associated with a higher relative abundance of Pseudoscardovia. The mother–offspring comparison revealed that the heritable rumen bacteria had higher compositional similarity than nonheritable bacteria between two generations, and the predicted heritable microbial functions had higher stability than those from nonheritable bacteria. These findings suggest that a high stability exists in heritable rumen bacteria, which could be passed to the next generation in dairy cows.


INTRODUCTION
Linkage between rumen microbes and production traits of dairy and beef cattle, such as milk production and quality (1,2) and feed efficiency (3,4) has been widely reported recently.A recent study by Wallace et al. (5) identified 39 genera of core rumen bacteria that have significant heritability and showed significantly higher explanatory power for dairy cow's phenotypes compared to other non-heritable core microbes.Similarly, our previous study found 32 heritable bacteria at species level and identified 63 host single nucleotide polymorphisms (SNP) associated with heritable bacteria (6).In beef cattle, 59 rumen microbial taxa (at various classification levels) were identified to be heritable and five SNP were identified that were associated with both rumen heritable microbial taxa and feed efficiency traits (4).Increasing evidence has shown that heritable bacteria were keystone members of rumen microbial interaction networks and are associated with various traits in beef (4) and dairy cattle (5), highlighting their roles in affecting microbial interactions and rumen functionality that related to production traits.Hence, manipulation of rumen heritable bacteria can be one of the effective strategies to optimize productive performance in cattle.Previous studies have primarily focused on the overall rumen microbiota, while the potential for greater phenotypic gains from manipulating heritable bacteria has not been assessed.
In cattle, the rumen microbiota undergoes dynamic changes throughout the growth process, and the development of rumen microbiome can have a direct effect on adult rumen microbiome (7).In recent years, many studies revealed that calf's gut microbiota can be traced back to their mother cows (8,9).For example, Klein-Jöbstl et al. (8) found that the mother cow's vaginal microbiota was similar to those in the calf's feces.Hence, the selection of adult cows and their offspring possessing mature and stable rumen microbiota would be more significant for investigating the composition and functionality of rumen microbiota in dairy cows.This is because the rumen microbiota in adult cows and their offspring exhibit a more stable composition and function compared to newborn or young cows.Our previous study revealed that heritable bacteria had a more significant average contribution to the lactation phenotype compared to non-heritable bacteria (6).
However, it remained unknown whether heritable bacteria can be transmitted from mother to offspring.We hypothesized that the host genotype could be associated with the rumen heritable bacterial relative abundance, and heritable rumen bacteria can be passed from cows to the offspring and have similar functions.Therefore, the objectives of this study were to identify the host loci that can affect heritable bacterial abundance and to investigate the composition and function of the rumen bacterial similarity between mother and their offspring.

Animals, sampling, and phenotypic measurement
The animal care and experimental procedures were approved by the Animal Care Committee of Zhejiang University (Hangzhou, China).Seventeen pairs of mother cows and their respective offspring were selected from a cohort of 361 healthy mid-lactation Holstein dairy cows housed at a commercial dairy farm (6), based on their pedigree records.The animals were reared under the same dairy farm environment and management with a concentrate-to-forage ratio of 57:43 (dry matter basis) (6).Rumen contents were collected using an oral stomach tube before morning feeding and were used to measure volatile fatty acids (VFAs).Blood samples were collected from jugular venous blood and were stored for genotyping analysis.

Genotyping detecting the animals' single nucleotide polymorphisms
The DNA extraction and genotyping analysis have been described previously (6).We performed genotyping data analysis in Genome Studio V2.0 software (https://support.illumina.com/array/array_software/genomestudio/downloads.html) and PLINK V1.07 (10).The genotyping was accomplished through the application of DNA SNP microarray technology, whereby the genotypes were ascertained based on the obtained detection results, facilitating the classification of SNP microarrays according to their genotypic profiles.The genotypes of the SNP were represented by the alleles A, T, C, and G.

16S rRNA gene sequencing, rumen microbiota composition and function analysis
The rumen fluid was collected from two populations of dairy cows (mother population sampled in 2016 and the offspring population sampled in 2019).The 32 species level rumen heritable bacteria and 674 species level non-heritable bacteria were obtained from the previously published study and public database (Accession Number PRJNA597489 and PRJNA741384) (6).For both cohorts from the mother cows (n=254) and the offspring cows (n=107), DNA extraction and 16S rRNA gene amplicon sequencing followed the methodology described previously (2).Briefly, the DNA of the mother group extracted from each rumen fluid sample was amplified using 341F/806R primer set (11) (5'-CCTAYGGGRBGCASCAG-3' / 5'-GGACTACNNGGGTATCTAAT-3'), and genomic DNA of the offspring group was amplified using the 515F/806R primer set (5'GTGY-CAGCMGCCGCGGTAA-3' / 5'-GGACTACNVGGGTWTCTAAT-3').To further identify the long-term effect of host genetics on heritable bacteria, we analyzed the heritable and non-heritable bacteria relative abundance change in 17 pairs of mother cows and their offspring.The presence of batch effect was analyzed and when there was no batch effect, the data were merged into a unified dataset for further downstream analysis.
The quality control of the dataset and sequence demultiplexing was performed using QIIME2 v2.0.6 (12) (https://view.qiime2.org)with q2-demux plug-in to demultiplex the original sequence data and DATA2 for denoising.Potential batch effects were ameliorated in the DADA2 pipeline to resolve singleton amplicon sequence variants (13).SILVA138 database (June 2020 release) was used to map the amplicon sequence variants.PICRUSt2 v2.1.0-b( 14) (https://github.com/picrust/picrust2/wiki)software was used to predict microbial functions.The present study quantified microbial functional enrichment using relative abundance percentages as a metric to assess the degree of enrichment.

Assessment of rumen bacteria stability across generations
Taxa-function stability was used to assess functional stability and defined as the functional shift given a perturbation to the microbiota composition (15).The relationship between taxonomic perturbation magnitude and functional profile shift was defined as follows: where,  is the magnitude of the taxonomic perturbation, and  is the functional shifts.
Two coefficients in the functional shifts, a (attenuation) and  (buffering), were used to describe taxa-function stability.Attenuation (a) describes the expected rate at which increases in the taxonomic perturbation magnitude are expected to increase functional shifts (15).Buffering (b) indicates how large a perturbation must be before a functional profile shift becomes noticeable and approaches the expected shift magnitude defined by attenuation (15).Therefore, the higher the a and b values, the more stable the microbial function.Besides, we used equation (1) to plot the functional response curve and the area under the curve to represent the functional shifts when the taxonomic perturbation happened.

Network analysis
Correlation networks that indicate the relationship among the 32 rumen heritable bacteria were performed in R software with Spearman's method.The list of 32 rumen heritable bacteria was compiled based on previous studies as follows (6) Enterorhabdus_sp.The correlation coefficients with R > 0.4 or R < -0.4 and P < 0.05 were considered significant.The correlation network was generated by Spearman's rank correlation and visualized by Cytoscape v3.9.1.

Statistical analysis
The microbial taxa similarity was calculated based on Bray-Curtis distance method in the R package Vegan (16), and the similarity calculations were based on matrices derived from microbial abundance at both the mother and offspring, as well as the microbial functional abundance profiles.The taxa and function batch effect was removed by R package sva (17).The analyses of the difference between heritable and non-heritable microbiota in taxa similarity, functional similarity, and functional stability were assessed using the t-test.The analyses of two-generation (mother and their offspring) difference in taxa relative abundance and functional stability were assessed using the paired t-test.The taxa's relative abundance and rumen's VFAs among different genotypes of dairy cows were assessed using the nonparametric Kruskal-Wallis test, and we defined the frequency of alleles as the number of different genotypes (18).All differential analyses were performed using GraphPad Prism V 9.4.1; the significant difference was declared at P < 0.05.

Relationship between host genotypes and the relative abundances of heritable bacteria or VFAs concentrations
We analyzed the relationship between host genotypes and heritable bacteria, or VFAs, and found that 5 potential single-nucleotide polymorphisms (SNP associated with the relative abundance of 2 heritable bacteria (Pseudoscardovia, p-251-o5) and concentration of propionate and valerate (Table 1).The frequency of G allele (SNP: BovineHD1900005868) was positively associated with the relative abundance of Pseudoscardovia and concentration of propionate and valerate (Fig. 1A).As for SNP: BTB-00740910, the genotype of GA had higher relative abundance of Pseudoscardovia and propionate and valerate concentration than the GG genotype (Fig. 1B).The A allele (SNP: BovineHD1300021786) had a significant effect on Pseudoscardovia abundance, and on propionate and valerate concentrations (Fig. 1C), Despite this, we found that the A allele (SNP: ARS-BFGL-NGS-8960) was positively associated with the p-251-o5 relative abundance, but was negatively associated with the propionate and valerate concentrations (Fig. 2A).The dairy cows with a higher frequency of G allele (SNP: BTB-01532239) exhibited a higher relative abundance of p-251-o5 and lower concentrations of propionate and valerate (Fig. 2B).p-251-o5 showed significantly (P < 0.05) high correlations with correlations with other heritable bacteria (Fig. 3).

The similarity between the mother and offspring in the composition of heritable rumen bacteria
The similarity between mother and offspring was assessed based on calculated values for heritable (Fig. 4A, ranging from 0.89 to 0.99) and non-heritable bacteria (Fig. 4B, ranging from 0.72 to 0.93), with higher mean similarity for heritable bacteria [0.94 ± 0.01; mean ± SE (standard error of mean)] than for non-heritable bacteria (0.85 ± 0.01; mean ± SE) (Fig. 4C).Sankey diagram depicted the top abundant 20 rumen bacteria identified as heritable (Fig 5A ) and non-heritable (Fig 5B ) in both mother and offspring.According to our findings, the relative abundance of Firmicutes, which was the most abundant phylum among heritable bacteria, was higher in the offspring's rumen than in that of their mothers.However, the relative abundance of the phylum Firmicutes in non-heritable bacteria was lower in the offspring compared to their mothers' rumen non-heritable.In the case of the key heritable bacteria we identified earlier, which had a significant association between their genotype and the relative abundance of heritable bacteria, the relative abundance of p-251-o5 in mothers was higher compared to that of their offspring.

The influence of host genotype on key heritable bacteria in two generations
Across generations, The A allele (SNP: ARS-BFGL-NGS-8960) and G allele (SNP: BTB-01532239) were significantly associated with the relative abundance of p-251-o5 (Fig. 6A).The mother group had more A allele and G allele than the offspring group, and the relative abundance of p-251-o5 showed a significant difference between mother and offspring (Fig, 6A).The SNP (SNP: BovineHD1900005868, SNP: BTB-00740910, SNP: BovineHD1300021786) genotype were not different between mother and offspring, and the relative abundance of Pseudoscardovia was not different between mother and offspring (Fig. 6B).

The effects of the mother's genotypes on the functions of heritable rumen bacteria and their stability in offspring
In total, 191 and 236 predicted biological functions were obtained for heritable and nonheritable bacteria, respectively.The functional similarity and functional stability of heritable and non-heritable bacteria were analyzed between mother cows and their offspring.The functional similarity was calculated between mother and offspring of both heritable (Fig. 7A, ranging from 0.87 to 0.96) and non-heritable (Fig. 7B, ranging from 0.51 to 0.98).There was no significant difference between mother and offspring (Fig. 7C).
For heritable bacteria, the offspring had lower buffering value than mother cows, but no difference was found in attenuation value between two generations (Fig. 8A).However, the offspring had lower attenuation value in non-heritable bacteria than mother cows, with no difference found for buffering index (Fig. 8B).We estimated the functional shift amplitude for heritable and non-heritable bacteria (Fig. 8C, D), and found that heritable bacteria had higher functional stability than non-heritable ones (Fig. 8E).

DISCUSSION
Our previous study has found 32 heritable and 674 non-heritable bacteria in the rumen of Holstein dairy cows (6).Building on this study, our current research provides additional evidence on how host genotype influences the abundance of rumen bacteria.7.05E -07

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-03 1 For each microbial taxonomic feature, P value was adjusted into genome-wide false discovery rates (FDRs) using the Benjamini-Hochberg method.Associations with P adj < 0.01 were considered as significant, and associations with 0.01 < P adj < 0.05 were regarded as suggestively significant.
Our findings uncovered two rumen heritable bacteria (p-251-o5, Pseudoscardovia), which their abundances are associated with host genotypes, and several genotypes (AA, GG, GA, AG) had different potentials to affect the relative abundance of heritable bacteria.Similarly, Yang et al. (18) mapped a quantitative trait locus that were significantly associated with the abundance of Erysipelotrichaceae species in pigs and found the frequency of the A allele (AO gene) increased the Erysipelotrichaceae abundance.Their study focused on monogastric animals, while our current study demonstrates a similar relationship can exist in ruminants, suggesting that the potential to manipulate rumen heritable bacteria via host genotype modulation.The SNP: BTB-01532239 located in leucine rich repeat and fibronectin type III domain containing 2 (LRFN2) gene associated with the relative abundance of the bacterial species belonging to the unclassified genus of p-251-o5.The function of this gene has been found to be involved in the modulation of chemical synaptic transmission and regulation of postsynapse organization (19), indicating that the host has the potential to modulate the abundance of rumen bacteria that exhibit heritability through the detection of signaling molecules present in the rumen.However, research is Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 needed to determine what signal molecules would influence relationship between LRFN2and p-251-o5.The SNP: BovineHD1900005868 (had significant effects on the abundance of the unclassified genus of Pseudoscardovia) is located within the gene SLC13A2 (solute carrier family 13 member 2) on BTA 19.The plasma membrane transporter SLC13A2, a member of the Na+-dependent SLC13 family, exhibits high sensitivity towards succinic acid and is capable of transporting it (20), and has been reported to be involved in intestinal host-microbe interactions in mice (21).Slawinska et al. (22) found that the SLC13A2 takes part in pathways involved in the transport of glucose and other sugars amine compounds.Although the function of the gene SLC13A2 has not been well studied in cattle, it is important to study the genetic variation of this gene and its expression of this gene in the rumen epithelial tissue.This suggests that the host genetics-based microbial regulation strategy (sensing and transporting small molecules) can be a potential mechanism for efficiently regulating host-microbiome interaction in the rumen.We further delineated bacterial composition similarity in mother cows and their offspring.Here, we found that heritable bacteria had greater bacterial composition similarity than the non-heritable bacteria between mother cows and their offspring.Lima et al.(9) showed that bovine calf fecal microbiota clustered closely to the maternal fecal microbiota, and Zhu et al. (24) provided evidence that the fecal microbiome of the calf may be originated from maternal sources.According to Guo et al. (25), the rumen microbiota of calves showed significant similarity to that of cows during the first two weeks of life.Specifically, the majority of the microbiota found in the rumen of calves was derived from that of mother cows.The findings indicate that the maternal influence on the microbial community of the offspring is substantial.When the host genetics effect on the rumen heritable bacteria was compared between two generations, it revealed that significant effect of host genetics on shaping the p-251-o5, Pseudoscardovia relative Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024 Downloaded from https://academic.oup.com/ismecommun/advance-article/doi/10.1093/ismeco/ycad020/7513491 by guest on 24 January 2024

Table and Figure legends:TABLE 1 .
Identified bovine SNPs influenced heritable rumen microbial taxa abundance.