Perturbed maternal nutrition during critical developmental windows in gestation can have long-term impacts on postnatal calf growth and production efficiency via negatively influencing in utero fetal programming (Caton et al., 2019; Crouse et al., 2019; Reynolds et al., 2019). Recent developments derived from rodent animals and humans suggest that the maternal gut microbiota is involved in metabolic (Kimura et al., 2020) and neurological (Vuong et al., 2020; Sun et al., 2023) programming of offspring beginning at the embryonic stage. These developments further support the notion of the microbiome involvement in the developmental origins of health and disease (DOHaD) (Stiemsma and Michels, 2018; Stinson, 2020; Amat et al., 2022). Despite the known intimate relationship between the diet and gut microbiome and host health (Singh et al., 2017), the impact of perturbed nutrition on the gut microbiota and gut microbiota-mediated fetal programming in cattle remains elusive. In this study, we evaluated the effects of maternal nutrition (restricted dietary intake) and OCM supplementation from breeding to d 63 of gestation on maternal ruminal, vaginal and blood microbiota in beef cattle.
Our 16S rRNA gene sequencing results revealed that the ruminal microbiota underwent changes during the first 63 days of pregnancy due to time and restricted gain. While the impact of pregnancy on the ruminal microbiota in cattle has less been characterized, there is an ample evidence from human research showing that the maternal gut microbiota undergoes profound changes over the course of pregnancy (Collado et al., 2008; Koren et al., 2012; Nuriel-Ohayon et al., 2016; Smid et al., 2018). As pregnancy progresses from the 1st to the 3rd trimester, the maternal gut microbiota of women becomes less diverse (Koren et al., 2012), but increases in microbial cell density (Collado et al., 2008). In the present study, ruminal microbiota diversity remained stable while overall community structure and species richness changed from pre-breeding to day 63 of pregnancy. Overall species richness decreased as pregnancy progressed during the course of this study. Such changes in the ruminal microbiota following pregnancy might partially be driven by the increased metabolic demands from the developing fetus (Smid et al., 2018; Codagnone et al., 2019).
Diet is the most important factor shaping the ruminal microbiota (Henderson et al., 2015). Diet composition and eating patterns have profound impact on the composition and function of the gut microbiota (Klingbeil and de La Serre, 2018) including ruminal microbiota (Wang et al., 2020). The impact of caloric restriction on the gut microbiota in humans has been well documented (Sbierski-Kind et al., 2022; Kern et al., 2023). Thus, the significant impact of restricted diet on the ruminal microbiota of RES heifers was expected. The effects associated with restricted diet on the ruminal microbiota structure and composition were the most evident on d 63 of gestation. On d 63, beta diversity of the ruminal microbiota diverged between RES and CON heifers, and microbial richness was increased in RES heifers. Likewise, species richness of the ruminal microbiota in lambs fed a diet with low (9.2 MJ/kg) metabolizable energy was higher than the lambs fed a diet containing a higher metabolizable energy (10.4 MJ/kg; Wang et al., 2020). In the present study, composition of the diet given to RES and CON heifers was the same, but the daily energy intake resulting from the diet restriction may have promoted an increase in richness of the ruminal microbiota in RES heifers in order to extract more energy from the diet to meet the energy demand by both ruminal microbes and the host. Another factor that could attributed to the difference in species richness of the ruminal microbiota observed between RES and CON heifers might be due to the difference in the ruminal fluid volume resulting from the different amounts of feed ingested into the rumen between the two groups. Caloric restricted diet (Sowah et al., 2022) and time-restricted eating (TRE; (Pieczyńska-Zając et al., 2023) have been reported to be associated with increased alpha diversity in the human gut microbiota. A meta-analysis done by Pieczyńska-Zając also observed that TRE and fasting did not influence the alpha diversity of the gut microbiota in rodent animals, but enhanced microbial fluctuation (Pieczyńska-Zając et al., 2023). We did not observe the impact of restricted diet on Shannon diversity indices of the ruminal microbiota.
The alterations in the relative abundance of phylum and genera observed in RES heifers on d 63 highlights significant modulation of the taxonomic composition of the ruminal microbiota due to restricted intake. Firmicutes, Actinobacteriota and Chloroflexi were enriched following 63 days of restricted dietary intake. Firmicutes is one of the most dominant phyla found in the rumen (Holman and Gzyl, 2019) and human gut (Hou et al., 2022), and it is the phylum most frequently reported to be affected by diet restriction and eating patterns (Kern et al., 2023). In the mouse gut, the abundance of Firmicutes increased following intermittent fasting (Beli et al., 2018) while it was reduced in both the rodent and human gut when the hosts subjected to caloric intake reduced ad libitum intake by 10 to 30% (Kern et al., 2023). Increased abundance of Firmicutes in the rumen has been associated with increased average daily gains in beef steers (Myer et al., 2015) and milk-fat yield in dairy cattle (Jami et al., 2014), suggesting its positive correlation with feed efficiency (Myer et al., 2015). The change in Firmicutes abundance results in change to the Firmicutes-to-Bacteroidetes ratio, which has previously been correlated with enhanced feed efficiency in sheep (Zhang et al., 2021) and cattle (Jami et al., 2014). There is little evidence showing either positive or negative correlations of phyla Actinobacteriota or Chloroflexi in cattle or other ruminant species. Nevertheless, Firmicutes enrichment induced by restricted gain may reflect taxonomic changes in the ruminal microbiota that might indicate re-assembly towards a more efficient energy extraction state to maximize limited feed ingestion. This notion is further supported by the alterations of the abundance of 34 bacterial genera, in which 25 of them become more abundant in RES heifers on d 63. Many of these bacterial genera enriched in RES heifers have been reported to have positive associations with animal health and feed efficiency. Among the enriched genera were SCFA producers including Butyrivibrio [the main butyrate-producing genus in the rumen (Palevich et al., 2017)], Christensenellaceae R-7 group [acetate and butyrate producers in the rumen (Andrade et al., 2022) that are also associated with increased feed efficiency (Perea et al., 2017; Andrade et al., 2022; Fonseca et al., 2023)], and the acetate producing genus Acetitomacum (Greening and Leedle, 1989). The SCFAs produced from the gut microbiota have many important roles such as serving as an energy source to the host, and acting as signaling molecules between the gut and extraintestinal organs (Canfora et al., 2015) and regulating the central appetite (Frost et al., 2014). Potentially, RES heifers harbored greater abundance of SCFA producers in their rumen as compared to CON heifers as to modulate their appetite to adapt to the restricted caloric intake and/or to reduce fat accumulation. Acetate can reduce appetite via a central homeostatic mechanism (Frost et al., 2014), and butyrate can suppress insulin-mediated fat accumulation by SCFA receptor GPR43 (G protein coupled receptor) (Kimura et al., 2013). Another important factor driving the enrichment of SCFA producing bacteria in the rumen of RES pregnant heifers might be due to increased demand for SCFAs by the growing fetus. In rodent animal models, it has been demonstrated that the SCFAs produced from the maternal gut microbiota are provided to embryos via maternal circulation where they involve in regulation of fetal glucose homeostasis via the SCFA-GPR41/ 43 axis and imparting resistance to obesity in the offspring (Kimura et al., 2020). The maternal gut microbiota derived SCFAs are also involved in the regulation of fetal neurodevelopment (Vuong et al., 2020). In the present study we did not measure the SCFA production in the rumen of these pregnant heifers. However, our results point out that the ruminal bacteria associated with SCFAs might be key members affected by restricted dietary intake and maternal nutrition perturbations in early gestation. The implications of SCFA production in early gestation on fetal programming and maternal health warrants further investigation.
Some of the genera whose relative abundance was altered by restricted dietary intake on d 63 of gestation including Lachnospiraceae, Prevotella, and Ruminococcus are often positively or negatively associated with feed efficiency in cattle (Lopes et al., 2021; Liu et al., 2022; Fonseca et al., 2023). In addition to the changes observed in the relative abundance of 34 genera, overall genera-genera interaction network structure was also influenced by 63 days of restricted dietary intake (Fig. 4), resulting in more intense interactions that are centered around fewer hubs as compared to CON heifers. It is challenging to make inferences on the biological implications of the altered interaction network structure observed in RES heifers; however, active interactions between different microbial species are important for maintaining the stability and functional features of the microbiota associated with the gastrointestinal (Foster et al., 2017; Coyte and Rakoff-Nahoum, 2019) and respiratory tract (Amat et al., 2023). Intensive interactions with balanced positive (cooperation) and negative (competition) proportions are positively associated with the functional activities and stability of the gut microbiota (Foster and Bell, 2012; Fiegna et al., 2015; Venturelli et al., 2018). Accordingly, the intensified interactions between the ruminal genera of RES heifers may be an indication of a positive shift in the ruminal ecology in response to dietary intake restriction. Taken all together, restricted diet intake from breeding to 63 days of gestation resulted in significant alterations in maternal ruminal microbiota, which are characterized by the community structure, species richness, and composition at the phyla and genera level, and overall interaction network structure. How such microbial compositional and interaction network structural alterations that accompanied restricted dietary intake during the first trimester, which as a reminder, is a critical window of developmental programming events relating to skeletal muscle formation, organogenesis, and metabolic and neurodevelopment (Caton et al., 2019; Costa et al., 2021; B Menezes et al., 2022; McCarthy et al., 2022; Reynolds et al., 2022), should be the focus of feature research.
Supplementation of OCM from breeding to d 63 of gestation had minimal effect on the ruminal microbiota. Supplementation of OCM in this study was designed to test its ability to mitigate the undesired impact of the restricted dietary intake on fetal programming events. Immediately following fertilization, major epigenetic modifications including demethylation of paternal and maternal DNA, and embryonic genome re-methylation takes place (Li, 2002; Morgan et al., 2005; Messerschmidt et al., 2014). These epigenetic events require adequate amount of OCM as they are essential for synthesis of the methyl donor S-adenosylmethionine (SAM), used for DNA and histone methylation (Abuawad et al., 2021; Krautkramer et al., 2021). One-carbon metabolites such as folate, butyrate, and vitamin B12 can be produced by microbial fermentation in the gut (Jiménez-Chillarón et al., 2012; Nicholson et al., 2012; Krautkramer et al., 2021); however, three essential B vitamins [folate (B9), B12, and B6) utilized in the folate cycle are not supplied in sufficient amounts in the diet and must be supplied through de novo synthesis by the gut microbiota (Krautkramer et al., 2021). To the best of the author’s knowledge, this is the first study to evaluate the impact of OCM supplementation on maternal ruminal microbiota in cattle. While the impact on the dam’s ruminal microbiota was not evaluated, one study reported that rumen-protected methionine supplementation during the last 28 days of gestation resulted in alterations of the fecal microbiota of Holstein dairy calves, which were characterized by the enrichment of butyrate-producing bacteria, and microbial functional genes associated with antibiotic biosynthesis pathways (Elolimy et al., 2019). There are several factors that could contribute to the resistance of ruminal microbiota modulation by OCM supplementation in the present study. One of which might be due to the rumen-protective coating of the choline and methionine, which limits microbial degradation of these OCMs in the rumen; thereby leading to negligible dietary influence. Another factor may be due to the robustness and resilient nature of the mature ruminal microbiota in these pregnant heifers (> 14 months old; (Weimer, 2015; Costa-Roura et al., 2022). The dose of OCM supplementation may not have been high enough to induce changes in the ruminal microbiota, or noticeable alterations of the ruminal microbiota composition induced by OCM may take longer and be evident in the mid to late gestation periods.
Although the extent of dietary restriction from breeding to 63 days of gestation on vaginal microbiota is not as extensive as what was observed in the ruminal microbiota, it is interesting to detect a distinct community structure, altered phylum abundance, and different interaction network structure in the vaginal microbiota of RES heifers as compared to CON heifers on d 63. The effects of diet and eating patterns on vaginal microbiota had largely been underexamined in both humans (Rosen et al., 2022) and livestock animals. However, considering the increased appreciation of the role of vaginal microbiota in protecting the pregnant uterus from pathogen invasion (Adnane and Chapwanya, 2022), from spontaneous preterm birth (Freitas et al., 2018), as well as its role as a microbial seeding source of offspring perinatally (Guzman et al., 2020; Amat et al., 2021a; Messman and Lemley, 2023), the dietary impact on the vaginal microbiota particularly during early gestation in cattle deserves full scale investigation. Diet could indirectly influence the vaginal microbiota through modulation of the immune system and the availability of micronutrients such as vitamins and minerals involved in overall host health (Barrientos-Durán et al., 2020; Adnane and Chapwanya, 2022). Additionally, changes in the gut microbiome due to dietary changes could alter the vaginal microbiome through the transfer of fecal microbiota to the vagina given the proximity of the anus to the vulva in cattle (Laguardia-Nascimento et al., 2015). Our group recently observed significant alterations of the vaginal microbiota composition and diversity following 112 days of feeding two different high concentrate diets in beef heifers (Winders et al., 2023). While we are unable to provide clear insights into the mechanisms of modulation of the vaginal microbiota by restricted dietary intake and OCM supplementation, our results show that maternal nutrition and caloric restriction during early pregnancy can influence vaginal microbiota of cattle.
Future research is warranted to investigate the effects of vaginal microbiota alterations on fetal programming due to restricted diet intake, and on feto-maternal crosstalk and offspring microbiome development. Focus should be given to the impact of altered relative abundance of the main bacterial phyla implicated in reproductive health, pregnancy maintenance, and offspring microbial seeding. In the present study, we observed changes in the vaginal microbiota characterized by the increase in the phyla Actinobacteriota and decrease of Proteobacteria and Fusobacteria of RES heifers. These three phyla are important members of the microbial communities in the vagina and uterus of cattle, and their presence has been reported in fetal samples (Luecke et al., 2022; Messman and Lemley, 2023). These phyla are also dominant phyla correlated with gut, reproductive, and respiratory tract-associated microbiota of newborn calves (Luecke et al., 2023).
Our results also revealed that vaginal microbiota of pregnant heifers underwent significant changes immediately after fertilization which can be seen by the sharp increase in species richness, and diversity (Shannon and Inverse Simpson diversity) from pre-breeding to 35 days post-breeding, followed by further increase from d 35 to d 63. Vaginal microbiota in women has been reported to undergo significant changes over the course of pregnancy (Aagaard et al., 2012; MacIntyre et al., 2015; Rasmussen et al., 2020). In contrast to our findings, other studies reported that species richness and diversity of vaginal microbiota reduced as pregnancy progressed in women (Aagaard et al., 2012; MacIntyre et al., 2015). One of the explanations for such change is to protect both mother and the fetus from pathogen invasion by reducing the pH, which can be initiated by increased lactic acid production and immune modulation (Di Simone et al., 2020; Rasmussen et al., 2020). A healthy vaginal microbiota in women is typically characterized by a low-diversity microbial community mainly dominated by lactic acid-producing Lactobacillus (Baud et al., 2023). The increased richness and diversity of the vaginal microbiota have been associated with spontaneous preterm birth (Freitas et al., 2018). Our results suggest that vaginal microbiota in cattle increases in richness and diversity following impregnation and throughout the first 63 days of gestation. Whether the increased richness and diversity of the maternal vaginal microbiota remains throughout the 2nd and 3rd trimester is a question for future studies.
Increasing evidence derived from humans and sheep revealed the presence of microbial DNA signatures in blood samples, suggesting the presence of blood-associated microbiota (Schierwagen et al., 2019; D'Aquila et al., 2021; Peña-Cearra et al., 2021; Cheng et al., 2023). Although the hypothetical presence of a unique microbiome specific to the blood is not supported by the results of recent large-scale study conducted to evaluate the blood microbiota of healthy individuals (n = 9,770; (Tan et al., 2023), it was identified that the bloodstream of healthy individuals contains DNA from more than 100 different microbial species, and the bloodstream allows these microbes to move between different body sites including the gut, mouth, and urogenital tract. These identified microbial species were distinct from pathogens detected in hospital blood cultures. Replication rate analyses revealed that some of these microbes might be live and can replicate actively in the blood stream (Tan et al., 2023). Yet, it is still debated if there is a self-sustaining and unique microbial community in the bloodstream of healthy animals. However, the presence of peripheral blood mononuclear cell-associated microbiota in goats has recently been reported (Peña-Cearra et al., 2021). Given that the blood may serve as a microbial transfer medium from the gut to extra-gastrointestinal microbial niches including the uterus, we were interested in characterization of the microbial DNA from whole blood of both RES and CON pregnant heifers at d 63. We identified microbial DNA signatures of bacterial species within 23 different phyla, and 358 different genera. All top 7 phyla and the majority of top 20 genera (Fig. 7C and D) are commonly present in the rumen (e.g. Rikenellaceae RC9 gut group), reproductive (e.g. Cutibacterium) and respiratory (e.g. Mycoplasma) tracts of cattle. While the phyla and genera level taxonomic composition of the blood microbiota supports the notion by Tan and colleagues (Tan et al., 2023) that the blood may not harbor blood specific microbiota, but instead harbors transient microbes using the bloodstream to translocate between the gut and extra-gastrointestinal microbial niches, our results presented in the Venn diagram (Fig. 8A), heatmap (Fig. 8B) and potential core ASVs table (Table 1) suggest otherwise. We identified over 1900 ASVs unique to the blood and not found in the ruminal and vaginal samples, and only 2 ASVs (ASV62 and 31) were shared by more than 65% of all ruminal, vaginal and blood samples. As shown in the heatmap, the ASVs found in blood are distinctively different in terms of frequency and relative abundance from the ASVs found in the ruminal fluid. This suggests that the ruminal microbiota may not be the only seeding source for the microbes present in the blood stream. Other microbial sources such as the hindgut (Rakow et al., 2019), oral (Kitamoto et al., 2020), and urogenital tract (Flores-Mireles et al., 2015) associated microbial communities may contribute microbes. No effects of restricted dietary intake were observed on blood microbial community composition and diversity.