The newborn hindgut microbiome plays important metabolic and nutritional functions (1), with one of its main roles being the development of the intestinal barrier (2) and the maturation of the innate immune system in early life (3). Available studies in humans, lambs, and foals have focused on understanding the hindgut microbiome when microbial colonization starts (4–6). However, few studies have focused on the calf microbiome (7–9).

Whether the fetal gut and the maternal uterus harbor a microbiome prior to delivery has long been controversial (10). For instance, a number of studies have reported that the uterus is sterile and that mammals are exposed to exogenous microorganisms for the first time only at birth (11, 12). However, in recent years, a microbiota has been detected in amniotic fluid, placenta, and umbilical cord of humans during pregnancy and hindgut of newborn calves prior to colostrum feeding (7, 13, 14). Consequently, these data have given rise to the hypothesis of vertical transmission of the microbiome from mothers to offspring (4, 6). Although the precise source of the newborn gut microbiome is not known with certainty (4), some scholars considered that the newborn meconium arises from intrauterine seeding (15–17). In bovine, Klein-Jöbstl et al. (18) and Alipour et al. (19) evaluated the possibility of colonization of the calf fecal microbiota, and concluded that it was maternally related.

SourceTracker script has been used to evaluate quantitatively the contribution of different maternal microbiome constituents in the meconium of the offspring (4). For instance, He et al. (4) working with humans explored maternal transfer characteristics by comparing the microbiome in meconium with that in various maternal sites and concluded that the microbiome in meconium was inoculated from amniotic fluid, feces, vaginal fluid, and saliva, with the amniotic fluid making the greatest contribution. It is unknown to what extent, if any, similar events occur in livestock species such as dairy calves.

We hypothesized that hindgut flora colonization of newborn dairy calves is strongly influenced by different maternal sources including placenta, umbilical cord, amniotic fluid, colostrum and feces. To address this objective, we screened 6 cow-calf pairs to analyze the contribution of the microbiome in the maternal feces, placenta, amniotic fluid, colostrum, and umbilical cord to the seeding of the meconium microbiome using 16S rRNA sequencing technology and SourceTracker software.

Animal care and experimental procedures were approved by the Animal Welfare and Ethics Committee of Heilongjiang Bayi Agriculture University, DaQing, China. Animal care and handling followed the guidelines of the regulations of the Administration of Affairs Concerning Experimental Animals (State Science and Technology Commission of China, 1988).

Several studies have previously investigated the presence of microorganisms in the prenatal fetal gut and compared the offspring hindgut microbiome with that from different maternal sites such as the vagina, colostrum, and maternal feces (5, 6, 18, 30). Studies used healthy mare-foal, ewe-lamb, and cow-calf pairs as animal models and concluded that the prenatal gut harbored active microorganisms and that the fetal gut microbiome was seeded antenatally (7, 31). Regarding the hindgut microflora of calves, the current literature indicates that microbial communities across hindgut segments differ (32, 33). Thus, by examining the microbiome in meconium and different maternal sample types we were able to explore the possible sources of the hindgut microbiome in newborn calves. To obtain repeat and subsequent samples without euthanasia, a non-invasive and practical method (i.e., meconium examination) was chosen. The meconium microbiome represented the gut microbiome at birth without environmental influences such as feeding.

Meconium is the feces present in the hindgut of the calf before birth and can largely reflect the condition of the fetus' intestinal flora in the mother's womb. Both, the placenta after birth. Amniotic fluid is the only environment for the fetus to survive in the mother's womb and the placenta and umbilical cord are important ways for the mother to transfer nutrients to the fetus and for the fetus to metabolize them. This suggests that the similarity of the early fetal gut flora structure to its environment at the phylum level can reveal important information about the origin of microbial colonization in utero.

The bacterial microbiome of the samples from each site was analyzed using the Illumina Nova sequencing platform. At the phylum level, the dominant microbial components of the placenta, umbilical cord, amniotic fluid, colostrum, meconium, and cow feces included Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria.

Proteobacteria are gram-negative bacteria that can produce LPS (lipopolysaccharide) that can enter the blood, reduce the number of hindgut barrier cells, and increase hindgut permeability (34). Bacteroidetes produce butyrate, a product of colonic fermentation with antineoplastic properties, and are beneficial to interactions within the immune system of the host, which can activate T cell-mediated responses (35) and limit the colonization of potentially pathogenic bacteria in the GI tract (35). A previous study found that Proteobacteria, which colonized the intestines of young mice at an early stage, can activate the young mouse's immune system. The high levels of Proteobacteria in meconium in this study may also be related to the construction of the early immune system in calves (36). Thus, the presence of these bacteria in the hindgut of the neonatal calf is advantageous in the context of health (37). Actinobacteria can use carbohydrates to produce lactic acid, which can maintain the acidity of the environment and suppress the growth of pathogenic bacteria in the intestine (38). In addition, microbiota-depleted mice inoculated with Bacteroidetes and Firmicutes reflected that the two phyla both have distinct effects on hindgut immunity by differentially inducing primary and secondary response genes (39).

Similar to previous studies (18, 19, 30), the results in the present study indicated that the core community in meconium was enriched in Halomonadaceae, Pseudomonadaceae, Dietziaceae, Ruminococcaceae, Moraxellaceae, and Enterobacteriaceae. These responses were also similar to those in human studies (40). Accordingly, we speculate that the microbiome in the abovementioned core community is the “pioneer flora” during early life of the offspring. As such, it plays a central role in shaping the community of anaerobic organisms. Results from other studies (19, 30) can also be corroborated by differences in the composition of the microbiome between meconium and cow feces in the present study: compared with those in newborn calves, Proteobacteria levels were reduced in young and adult calves, and Firmicutes and Bacteroides dominated the fecal microbiome (33). These results indicated that the increasing diversity and richness in hindgut microbiome communities as the animal ages are indicative of progressive establishment of a complex microbiome during early life stages (33).

The presence of Pseudomonas, Limnobacter, and Brevundimonas as the most abundant genera in colostrum underscored the key role of colostrum in helping colonize the neonatal gut with microflora with obvious beneficial effects (e.g., probiotic effect) (41). Pseudomonas has been consistently reported to be the dominant microbe in colostrum (42) and raw milk (43). Li et al. (44) concluded that Pseudomonas, Lactococcus, and Acinetobacter were the most common genera, and Hang et al. (45) found that Streptococcus, Acinetobacter, Enterobacter, and Corynebacterium were the dominant microbiota. Although those studies differed from our results, it is possible that differences in experimental approaches and even environment account for most of the discrepancies. For example, Hang et al. (45) squeezed colostrum samples into a non-sterile bucket and collected them directly from the bucket after mixing. Clearly, use of a non-sterile container likely would have contaminated the samples.

Transmission of microbiota from milk to the developing offspring may exert many short- or long- term influences on the physiology of the offspring (46). For example, Lactobacilli in milk include species associated with the hindgut microbiome (47). These microorganisms can produce a large quantity of lactic acid, which can inhibit the growth of pathogenic bacteria (38). Lactic acid can also be converted to butyrate, which maintains the acidity of the environment and suppresses growth of pathogens in the intestine (48). In addition, it has been documented that flora in the maternal gut can reach the mammary gland via intestinal mononuclear cells during late gestation and lactation, also suggesting the existence of bacterial transmission via intestinal-lacteal routes (46). The offspring's intestinal microbiota and its immune evolution are related to milk microbiota, which are derived from the maternal entero-mammary pathway (49). Interestingly, although calves did not have access to the udder at the time of sampling in this experiment, at the phylum level the colostrum flora matched the meconium flora by 14.4%, which was very close to the match between the placental flora and the meconium flora (15.5%). This suggested that colostrum microorganisms have some influence on meconium. DiGiulio et al. (50) suggested that the perinatal transfer of beneficial microorganisms from the maternal gut to the mammary gland via the bloodstream, i.e., the adjustment of the oligo-oligosaccharides, immune factors and microbial communities in milk before delivery helped prepared, so that these prepared “beneficial bacteria” for transmission to the offspring through milk after birth. This may be a specific evolutionary phenomenon.

Quercia et al. (6) concluded that amniotic fluid and intestinal ecosystems can also contribute uniquely to the meconium microbiome community in foals. He et al. (4) studied the association of the microbiome in infant meconium with that in maternal vagina, saliva, amniotic fluid, and feces samples. Their data indicated that the meconium microbiome was seeded from multiple maternal body sites, with amniotic fluid microbiome contributing the most. Thus, vertical transmission of the microbiome from the mother to the offspring may exist. A hypothetical “enteromammary” pathway was proposed in which the selected bacteria in the maternal intestine can access the mammary glands, and dendritic cells and CD18+ cells can take up non-pathogenic bacteria from gut epithelial cells and carry them to other locations (51). The placental microbiome in mice is colonized by invasion and crossing of the endothelial lining (52), a process thought to occur during early vascularization and placentation (15). Dendritic cells from the mare penetrate the host epithelia including hindgut epithelium carrying luminal bacteria or bacterial antigens that are then released into the placenta via the bloodstream (6). Once the amniotic fluid is reached, these microbial factors may have access to the fetal gut and become a part of the meconium ecosystem (53). Various bacteria can also be released into the breast through the blood. Thus, we speculate that the main biological function of microbial factor transfer from the intrauterine region to the fetus may be beneficial to the development of digestive function and the construction of the immune system of newborn calves after delivery.

SourceTracker analysis showed that in the maternal transmission process, cow feces mainly transmitted acid-producing bacteria such as Saccharofermentans, Acetitomaculum, and Pseudoclavibacter. Saccharofermentans are fibrolytic (54) and produce short-chain fatty acids and low-density lipoprotein cholesterol (6) both of which help maintain health and provide energy for the developing intestinal wall (55). Acetitomaculum and Pseudoclavibacter produce mainly acetic acid and butyric acid, respectively (55, 56). Butyrate, as an energy source for host epithelial cells, can regulate growth and the differentiation-related activator protein 1 (AP-1) signaling pathway (57) leading to an increase in the number of immunoregulatory T regulatory (T-reg) cells. These functions may reduce the likelihood of maternal rejection of the fetal allograft (58).

Other groups of bacteria that appear mainly transmitted from cow feces are common in the digestive tract and are associated with nutrition such as the aerobic denitrification bacteria Thauera, Lysinibacillus, and Peptostreptococcus and Novosphingobium, which are core members of the gut flora in the cow (18). Cellulosilyticum, Saccharofermentans, and Ruminobacter, the main cellulose-degrading bacteria (59, 60), and Bradyrhizobium, Mogibacterium, Alcanivorax, Fastidiosipila, Saccharofermentans, and Ruminobacter, are all common bacteria in alimentary canals involved in fiber degradation (59, 61, 62). These are all key microbiome communities transmitted by amniotic fluid. In fact, these bacteria were the main transmitted bacteria that were not only in cow feces and amniotic fluid, but also in placenta and umbilical cord and dominated in the succession that occurred in early life (58). Algoriphagus, Pseudoxanthomonas, Bradyrhizobium, and Novosphingobium were the main cellulose-degrading microbiome constituents (63–65) transmitted via colostrum. In addition, it was reported that the special genus Truepera, which was mainly transmitted by colostrum, was the core flora constituent in bedding used to house cows (66).

Overall, microbial communities of the cow-calf pair encompassed a complex and shared microbiome that likely interacted to maintain health in both cows and calves (40, 67). Although there were differences in microbial community structure among different sample types of dams and offspring, the microbiome involved in cellulose degradation, fermentation, and the common flora in alimentary canals were seeded into the calf via the maternal transmission process and affected the calf's nutrition and the microbial communities existing in the calf intestine.

Some limitations in the present study must be mentioned. First, the number of cow-calf pairs could be considered small for a robust evaluation of maternal transmission. Second, a deviation in PCR results may have occurred in the analysis of low-microbial-biomass samples, and the possibility of contamination of samples cannot be completely excluded. Third, the inherent limits of molecular analyses did not allow for studying whether live or dead bacteria, even microbial debris, were present in the samples collected. Lastly, we identified numerous flora in the meconium, but the origins and timing of the colonization were not investigated, and knowledge about the influence of these microflora on the metabolism and immune function remains limited.

Data provide evidence that the fetal hindgut microbiome of the calf may arise from different maternal parts. The composition of the meconium microflora originated from multiple maternal sites including umbilical cord, placenta, colostrum, and amniotic fluid. Characteristics of the microorganisms in the placenta, umbilical cord, colostrum, and meconium were more obvious than those in amniotic fluid, and differences in the microbial characteristics between meconium and cow feces were the largest. Microflora with digestive functions such as cellulose decomposition and rumen fermentation were highly matched during the maternal transmission process. Overall, the present findings advanced our understanding of the calf gut microbiome and lays a foundation for improving the growth and development of offspring, hindgut health, and lactation potential of calves by intervening in the gut microecology of pregnant cows. Further studies are required to gain an in-depth understanding of the origin, composition, function, dynamics, and colonization time of the calf gut microbiome. Elucidating the effects of the fetal gut microbiome on development, immunity, and health throughout early life will be an important undertaking.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The data presented in the study are deposited in the NCBI BioProject dataset repository, and the BioProject ID is PRJNA768139.

The animal study was reviewed and approved by Animal Welfare and Ethics Committee of Heilongjiang Bayi Agriculture University.

HZ, CX, and YQ: conceptualization, methodology, and supervision. MY and SY: experiments and data curation. HZ and LL: writing-original draft preparation and software. JL, AE, and WW: reviewing and editing. All authors read and approved the final version of the manuscript.

This research was funded by the National Natural Science Foundation of China (Grant Nos. 32072758, U20A2062, and 31902186), the Natural Science Foundation of Heilongjiang Province of China (LH2021C069), and the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars (Grant No. ZRCLG201903).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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