Initial diet shapes resistance-gene composition and fecal microbiome dynamics in young ruminants during nursing.

This study was conducted to examine how colostrum pasteurization affects resistance genes and microbial communities in calf feces. Forty female Holstein calves were randomly assigned to either the control (CON) group, which received unheated colostrum, or the pasteurized colostrum (PAT) group. The calves body weight was measured weekly before morning feeding. Calf starter intake were measured and recorded daily before morning feeding. Samples of colostrum were collected before feeding. Blood was collected on d 1 and 70 before morning feeding. Ten calves were randomly selected from each group (n = 20 calves total) for fecal sampling on d 3, 28, 56 and 70 for subsequent DNA extraction and metagenomic sequencing. Total bacterial counts in the colostrum were markedly higher in the CON group than in the PAT group. Pasteurized colostrum administration substantially reduced the ARO diversity and diminishes the abundance of Enterobacteriaceae, thereby decreasing their contribution to resistance genes. Pasteurization also reduced glucoside hydrolase-66 activity in 3-day-old calves which led to an increase in the activity of aminoglycoside antibiotics, resulting in 52.63 % of PAT-enriched bacteria acquiring aminoglycoside resistance genes. However, from the perspective of overall microbial community, the proportion of aminoglycoside, beta-lactam and tetracycline resistance genes carried by microbial community in PAT group was lower than CON group (P < 0.05). Fecal samples from the PAT group contained greater abundances of Subdoligranulum (P < 0.05) and Lachnospiraceae_NK4A136_group (P < 0.05) on days 28 and 70 compared to CON. Network analysis and abundance variations of the different bacteria obtained by linear discriminant analysis effect size analysis showed that pasteurized colostrum feeding reduced the interactions among related bacteria and maintained stability of the hind-gut microbiome. In conclusion, these findings underscore the intricate interactions between early diet, calf resistance-gene transmission and microbial dynamics, which should be carefully considered in calf-rearing practices.

Metagenomics reveals the temporal dynamics of the rumen resistome and microbiome in goat kids.

BACKGROUND: The gut microbiome of domestic animals carries antibiotic resistance genes (ARGs) which can be transmitted to the environment and humans, resulting in challenges of antibiotic resistance. Although it has been reported that the rumen microbiome of ruminants may be a reservoir of ARGs, the factors affecting the temporal dynamics of the rumen resistome are still unclear. Here, we collected rumen content samples of goats at 1, 7, 14, 28, 42, 56, 70, and 84 days of age, analyzed their microbiome and resistome profiles using metagenomics, and assessed the temporal dynamics of the rumen resistome in goats at the early stage of life under a conventional feeding system. RESULTS: In our results, the rumen resistome of goat kids contained ARGs to 41 classes, and the richness of ARGs decreased with age. Four antibiotic compound types of ARGs, including drugs, biocides, metals, and multi-compounds, were found during milk feeding, while only drug types of ARGs were observed after supplementation with starter feed. The specific ARGs for each age and their temporal dynamics were characterized, and the network inference model revealed that the interactions among ARGs were related to age. A strong correlation between the profiles of rumen resistome and microbiome was found using Procrustes analysis. Ruminal Escherichia coli within Proteobacteria phylum was the main carrier of ARGs in goats consuming colostrum, while Prevotella ruminicola and Fibrobacter succinogenes associated with cellulose degradation were the carriers of ARGs after starter supplementation. Milk consumption was likely a source of rumen ARGs, and the changes in the rumen resistome with age were correlated with the microbiome modulation by starter supplementation. CONCLUSIONS: Our data revealed that the temporal dynamics of the rumen resistome are associated with the microbiome, and the reservoir of ARGs in the rumen during early life is likely related to age and diet. It may be a feasible strategy to reduce the rumen and its downstream dissemination of ARGs in ruminants through early-life dietary intervention. Video Abstract.

Altered microbiota, antimicrobial resistance genes, and functional enzyme profiles in the rumen of yak calves fed with milk replacer.

IMPORTANCE: Yaks, as ruminants inhabiting high-altitude environments, possess a distinct rumen microbiome and are resistant to extreme living conditions. This study investigated the microbiota, resistome, and functional gene profiles in the rumen of yaks fed milk or milk replacer (MR), providing insights into the regulation of the rumen microbiome and the intervention of antimicrobial resistance in yaks through dietary methods. The abundance of Prevotella members increased significantly in response to MR. Tetracycline resistance was the most predominant. The rumen of yaks contained multiple antimicrobial resistance genes (ARGs) originating from different bacteria, which could be driven by MR, and these ARGs displayed intricate and complex interactions. MR also induced changes in functional genes. The enzymes associated with fiber degradation and butyrate metabolism were activated and showed close correlations with Prevotella members and butyrate concentration. This study allows us to deeply understand the ruminal microbiome and ARGs of yaks and their relationship with rumen bacteria in response to different milk sources.

Transcriptomic and metabolomic insights into the roles of exogenous ß-hydroxybutyrate acid for the development of rumen epithelium in young goats.

Beta-hydroxybutyric acid (BHBA), as one of the main metabolic ketones in the rumen epithelium, plays critical roles in cellular growth and metabolism. The ketogenic capacity is associated with the maturation of rumen in young ruminants, and the exogenous BHBA in diet may promote the rumen development. However, the effects of exogenous BHBA on rumen remain unknown. This is the first study to investigate the mechanisms of BHBA on gene expression and metabolism of rumen epithelium using young goats as a model through multi-omics techniques. Thirty-two young goats were divided into control, low dose, middle dose, and high dose groups by supplementation of BHBA in starter (0, 3, 6, and 9 g/day, respectively). Results demonstrated the dietary of BHBA promoted the growth performance of young goats and increased width and length of the rumen papilla (P < 0.05). Hub genes in host transcriptome that were positively related to rumen characteristics and BHBA concentration were identified. Several upregulated hub genes including NDUFC1, NDUFB4, NDUFB10, NDUFA11 and NDUFA1 were enriched in the gene ontology (GO) pathway of nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) activity, while ATP5ME, ATP5PO and ATP5PF were associated with ATP synthesis. RT-PCR revealed the expression of genes (HMGCS2, BDH1, SLC16A3, etc.) associated with lipolysis increased significantly by BHBA supplementation (P < 0.05). Metabolomics indicated that some metabolites such as glucose, palmitic acid, cortisol and capric acid were also increased (P < 0.05). This study revealed that BHBA promoted rumen development through altering NADH balance and accelerating lipid metabolism, which provides a theoretical guidance for the strategies of gastrointestinal health and development of young ruminants.

Early Solid Diet Supplementation Influences the Proteomics of Rumen Epithelium in Goat Kids.

It is well known that solid diet supplementation in early life can significantly promote rumen development and metabolic function in young ruminants. However, the changes in the expressed proteome and related metabolism in rumen epithelium in response to a supplemented solid diet remain unclear. In this study, rumen epithelial tissue from goats in three diet regimes including milk replacer only (MRO), milk replacer supplemented concentrate (MRC), and milk replacer supplemented concentrate plus alfalfa pellets (MCA) were collected for measurement of the expression of epithelial proteins using proteomic technology (six per group). The results showed that solid diet significantly improved the growth performance of goats, enhanced the ability of rumen fermentation, and promoted the development of epithelial papilla (p < 0.05). Proteome analysis revealed the distinct difference in the expressed protein in the MRC and MCA group compared with the MRO group (42 upregulated proteins and 79 downregulated proteins in MRC; 38 upregulated proteins and 73 downregulated proteins in MCA). Functional analysis showed that solid diet supplementation activated a variety of molecular functions in the epithelium, including protein binding, ATP binding, structural constituent of muscle, etc., in the MRC and MCA groups. Meanwhile, the expression of proteins related to fatty acid metabolism, the PPAR signaling pathway, valine, leucine, and isoleucine degradation, and butanoate metabolism were upregulated, being stimulated by solid feed. In contrast, the proteins associated with carbohydrate digestion and absorption and glycosaminoglycan degradation were downregulated. In addition, the protein expression of enzymes involved in ketone body synthesis in the rumen was generally activated, which was caused by solid feed. In summary, solid feed promoted the development of rumen epithelium by changing the expression of proteins related to fatty acid metabolism, energy synthesis, and signal transduction. The ketone body synthesis pathway might be the most important activated pathway, and provides energy for rumen development.

Obstructive sleep apnea-related knowledge, attitude, experience, and behaviors among orthodontic professionals: a survey.

PURPOSE: To assess obstructive sleep apnea (OSA)-related experience, knowledge, attitude, and behaviors among orthodontic professionals in China and identify factors associated with their knowledge levels, attitude toward referring, and self-confidence in the management of patients with OSA. METHODS: An online cross-sectional survey was conducted using a 31-item questionnaire developed with a professional online survey tool ( www.wjx.cn ) and distributed via WeChat (Tencent, Shenzhen, China). Data were collected between January 16 and 23, 2022 and analyzed using the chi-square test, Fisher's exact test, and multivariate generalized estimation equations. RESULTS: A total of 1760 professionals responded to the survey, and responses to 1611 questionnaires were valid. The average score of correct answers to the 15 OSA knowledge questions was 12.1 ± 2.0. Most of the professionals agreed that it was necessary to identify patients who might have OSA in practice. The top three sources for gaining knowledge of OSA according to the survey were classrooms and textbooks (76.3%), medical lectures (75.7%), and academic conferences (73.2%). The level of knowledge was significantly correlated with self-confidence in treatment (P < 0.001) and willingness to refer patients to otolaryngologists or clinicians of related disciplines (P < 0.001). CONCLUSIONS: Most orthodontic professionals agreed that there was a need to identify patients with OSA and learn further about related problems. Treatment confidence and willingness of professionals to refer patients were related to the level of OSA knowledge. These findings suggest that promotion of OSA-related education may help improve the care of patients with OSA.

Dietary medium-chain fatty acid and Bacillus in combination alleviate weaning stress of piglets by regulating intestinal microbiota and barrier function.

The present study evaluated the effects of dietary medium-chain fatty acid (MCFA) and Bacillus on growth performance, nutrient digestibility, antioxidant capacity, colonic fermentation, and microbiota of weaning piglets. A total of 400 weaned piglets were randomly divided into 4 treatments, with 10 replicates per treatment and 10 pigs per replicate. The treatment included: basal diet (control, CON), basal diet with 0.588 g/kg MCFA (MCF), basal diet with 1.3 × 109 CFU/kg Bacillus (BAC), and basal diet with 0.588 g/kg MCFA and 1.3 × 109 CFU/kg Bacillus (SYN). Compared with CON group, the average daily gain of MCF and SYN in the early (1 to 9 d) and whole stage (1 to 36 d) of trail were improved (P < 0.05), the feed to gain ratio of MCF in later (10 to 36 d) and whole stage of trial were decreased (P < 0.05), and the diarrhea rate of SYN in the early stage (1 to 9 d) of trial decreased (P < 0.05). The digestibility of dry matter, ether extract, acid detergent fiber digestibility of MCF were decreased (P < 0.05) compared with CON. The serum d-lactic acid in MCF, BAC, and SYN were lower (P < 0.05) compared with CON group. Compared with CON group, the contents of total antioxidant capacity, superoxide dismutase, and glutathione peroxidase were greater (P < 0.05), whereas the content of malondialdehyde and the contents of colonic isobutyrate and isovalerate were lower (P < 0.05) in MCF. The microbial Shannon and Simpson diversity was lower in MCF (P < 0.05) than that in BAC and SYN. The relative abundance of Prevotella was greater (P < 0.05), whereas the Treponema and Oscillibacter were lower (P < 0.05) in MCF than that in BAC and SYN. In addition, the metabolic pathways of bacteria such as pentose phosphate pathway, adenosine nucleotides degradation II were enhanced (P < 0.05), whereas the pathways such as incomplete reductive TCA cycle, and TCA cycle IV (2-oxoglutarate decarboxylase) were decreased (P < 0.05) in MCF compared with BAC. The results indicated that dietary MCFA and Bacillus in combination improved the intestinal barrier function of piglets by changing the intestinal microbiota and its metabolic function, and finally alleviated the diarrhea rate in early weaning stage and improved growth performance in whole trial period. In addition, MCFA was effective in improving feed efficiency and antioxidant capacity of piglets.

Weaning is the most stressful stage in the growth of piglets. Weaning stress can reduce the feed intake of piglets, cause diarrhea and even death of piglets, and finally result in economic losses to livestock production. To alleviate the weaning stress of piglets after the prohibition of antibiotics in feed, this study evaluated the effect and mechanism of medium-chain fatty acid (MCFA) and Bacillus in combination on regulating the intestinal microbiota balance and health status of weaned piglets. It was found that dietary MCFA and Bacillus in combination improved the intestinal barrier function of piglets by changing the intestinal microbial community and metabolic pathway encoded by bacteria, and finally alleviated the diarrhea rate in the early weaning stage and improved the growth performance in whole trial period. In addition, MCFA was effective in improving feed efficiency and antioxidant capacity of piglets.

Dietary ß-hydroxybutyric acid improves the growth performance of young ruminants based on rumen microbiota and volatile fatty acid biosynthesis.

Introduction: The ketone body ß-hydroxybutyric acid (BHB) plays critical roles in cellular proliferation and metabolic fuel utilization; however, its effects on the rumen microbiota remain unknown. Methods: Here, three doses of BHB (low, medium, and high) were supplemented to early-weaned goat kids. Results: Compared with controls, the beneficial effects of BHB on growth and rumen development were observed in goats at 90 days of age (d). The low dose of dietary BHB increased the concentration of rumen acetate, propionate, and butyrate on d90. The sequencing results of the rumen microbiota revealed marked shifts in rumen microbial community structure after early-weaned goat kids consumed BHB for 2 months. The signature bacterial ASVs for each treatment were identified and were the main drivers contributing to microbial interactions in the rumen. The bacteria associated with rumen weight were also correlated with body weight. Some classified bacterial signatures, including Prevotella, Olsenella umbonate, and Roseburia faecis, were related to rumen volatile fatty acids and host development. Conclusion: Overall, dietary BHB altered rumen microbiota and environments in young goats, which contributed to rumen development and growth.

Impact of dietary supplementation of ß-hydroxybutyric acid on performance, nutrient digestibility, organ development and serum stress indicators in early-weaned goat kids.

The objective of this study was to determine the effect of dietary supplementation of ß-hydroxybutyric acid (BHBA) on performance, nutrient digestibility, organ development, and serum composition in early-weaned goat kids. Sixty-four goat kids at 30 d of age were assigned to 4 treatments in a completely randomized design: 1) control (basal diet); 2) low (basal diet with 3 g/d per animal BHBA); 3) medium (basal diet with 6 g/d per animal BHBA; and 4) high (basal diet with 9 g/d per animal BHBA). Subsequently, 48 (6 kids per treatment) goat kids were randomly selected and slaughtered at 60 and 90 d of age. Compared with the control group, BHBA at low and high doses increased body weight (P < 0.05), average daily gain (P < 0.01), and average daily starter intake (P < 0.01). The BHBA improved organ development, especially at the lowest dose (P < 0.01). The digestibility of dry matter and crude protein increased with age (P < 0.05). However, BHBA did not affect nutrient digestibility. Compared with the control group, serum ceruloplasmin increased (P < 0.05) with high BHBA level at 90 d of age. However, the serum creatinine (P < 0.05) increased over time but was not affected by BHBA. The serum total antioxidant capacity and superoxide dismutase decreased with the high dose of BHBA at 90 d of age (P < 0.01). In contrast, the serum glutathione peroxidase and malondialdehyde increased with the high doses of BHBA (P < 0.01). Overall, low doses of BHBA were positive for growth performance, organ development, and health status against weaning stress. Whereas high doses of BHBA in the long term could negatively affect antioxidant status.

Solid diet manipulates rumen epithelial microbiota and its interactions with host transcriptomic in young ruminants.

Solid diet supplementation in the early life stages of ruminants could improve rumen microbiota and tissue development. However, most studies focus on bacteria in the rumen content community. The microbiota attached on rumen epithelium are rarely investigated, and their correlations with rumen content bacteria and host transcripts are unknown. In this study, rumen digesta attached in the epithelium from goats in three diet regimes (milk replacer only, milk replacer supplemented concentrate and milk replacer supplemented concentrate plus alfalfa pellets) were collected for measurement of the epithelial microbiota using next generation sequencing. Correspondingly, the rumen tissues of the same animals were measured with the host transcriptome. The distinct microbial structures and compositions between rumen content and epithelial communities were associated with solid diet supplementation. Regarding rumen development in pre-weaning ruminants, a solid diet, especially its accompanying neutral detergent fibre nutrients, was the most significant driver that influenced the rumen microbiota and epithelium gene expression. Compared with content bacteria, rumen epithelial microbiota had a stronger association with the host transcriptome. The host transcriptome correlated with host phenotypes were associated with rumen epithelial microbiota and solid diet. This study reveals that the epithelial microbiota is crucial for proper rumen development, and solid diet could improve rumen development through both the rumen content and epithelial microbiota.

Predicting the Digestive Tract Development and Growth Performance of Goat Kids Using Sigmoidal Models.

The transition from monogastric to rumination stage is crucial in ruminants' growth to avoid stressors-weaning and neonatal mortalities. Poor growth of the digestive tract could adversely affect the performance of the animal. Modeling informative growth curves is of great importance for a better understanding of the effective development pattern, in order to optimize feeding management system, and to achieve more production efficiency. However, little is known about the digestive tract growth curves. For this reason, one big goat farm of Laiwu black breed was chosen as a basis of this study. Forty-eight kids belonging to eight-time points (1, 7, 14, 28, 42, 56, 70, and 84 d; 6 kids for each) were selected and slaughtered. The body weight, body size indices, rumen pH, and stomach parts were determined and fitted to the polynomial and sigmoidal models. In terms of goodness of fit criteria, the Gompertz model was the best model for body weight, body oblique length, tube, and rumen weight. Moreover, the Logistic model was the best model for carcass weight, body height, and chest circumference. In addition, the Quadratic model showed the best fit for dressing percentage, omasum weight, abomasum weight, and rumen volume. Moreover, the cubic model best fitted the ruminal pH and reticulum percentage. The Weibull model was the best model for the reticulum weight and omasum percentage, while the MMF model was the best model describing the growth of chest depth, rumen percentage, and abomasum percentage. The model parameters, R squared, inflection points, area under curve varied among the different dependent variables. The Pearson correlation showed that the digestive tract development was more correlated with age than body weight, but the other variables were more correlated with body weight than age. The study demonstrated the use of empirical sigmoidal and polynomial models to predict growth rates of the digestive tract at relevant age efficiently.

Effects of Age and Dietary Factors on the Blood Beta-Hydroxybutyric Acid, Metabolites, Immunoglobulins, and Hormones of Goats.

The study was aimed to examine the effects of age and dietary beta-hydroxybutyric acid (BHBA) on blood BHBA and blood health indicators in goat kids. Thirty male goats of five ages (1, 2, 3, 6, and 12 months old) were selected for blood sampling to determine the influence of age. Another 64 goat kids (half males and half females) were weaned at 1 month old and fed with starter diets with control, low, medium, and high BHBA doses (0, 3, 6, and 9 g/animal/day, respectively). Six goats per treatment were selected for blood analysis at 2 and 3 months of age. There were significant effects (p < 0.01) of ages on the blood parameters of goat kids. The 6- and 12-month-old goats showed significantly lower blood total protein, globulin, BHBA, IgA, and IgM concentrations than did young goats, while they had a higher albumin-to-globulin ratio than young goats. The blood glucose decreased (p < 0.01) and IgG increased over time (p < 0.01). In blood, growth hormone (GH) and insulin-like growth factor I (IGF-I) were lower (p < 0.01) at 1- and 3-month-old goats than 12-month-old goats. The high dietary BHBA improved (p < 0.05) the ratio of albumin to globulin of 2-month-old kids compared with control. The blood GH and IGF-I were lower (p < 0.01) in the medium BHBA dose at 2 months of age than control. These results suggested that age greatly impacted blood composition, especially around weaning, and dietary BHBA showed beneficial regulating effects on blood total protein level in young goats.

Longitudinal Investigation of the Gut Microbiota in Goat Kids from Birth to Postweaning.

Early microbial colonization in the gut impacts animal performance and lifelong health. However, research on gut microbial colonization and development in young ruminants, especially after weaning, is currently limited. In this study, next-generation sequencing technology was performed to investigate the temporal dynamic changes of the microbial community in the jejunum and colon of goats at 1, 7, 14, 28, 42, 56, 70, and 84 days (d) of age. As age increased, significant increases in microbial diversity, including the number of Observed OTUs and the Shannon Index, were observed in both the jejunum and colon. Regarding beta diversity, significant shifts in community membership and structure from d1 to d84 were observed based on both Bray-Curtis and Jaccard distances. With increasing age, dominant genera in the jejunum shifted from Lactobacillus to unclassified Ruminococcaceae, unclassified Lachnospiraceae and unclassified Clostridiales through starter supplementation, whereas colonic dominant genera changed from Lactobacillus and Butyricicoccus, within d1-d28, to unclassified Ruminococcaceae, unclassified Clostridiales and Campylobacter after solid diet supplementation. The linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed bacterial features that are stage-specific in the jejunum and colon, respectively. In the jejunum and colon, a significantly distinct structure and membership of the microbiota was observed across all ages. The growth stage-associated microbiota in each gut compartment was also identified as a marker for biogeography. Our data indicate the temporal and spatial differences of the gut microbiota in goats are important for their performance and health. Early microbial colonization can influence microbial composition in later life (e.g., post-weaning phase). This study provides insights that the temporal dynamics of gut microbiota development from newborn to post-weaning can aid in developing feeding strategies to improve goat health and production.

The Signature Microbiota Drive Rumen Function Shifts in Goat Kids Introduced to Solid Diet Regimes.

The feeding regime of early, supplementary solid diet improved rumen development and production in goat kids. However, the signature microbiota responsible for linking dietary regimes to rumen function shifts are still unclear. This work analyzed the rumen microbiome and functions affected by an early solid diet regime using a combination of machine learning algorithms. Volatile fatty acids (i.e., acetate, propionate and butyrate) fermented by microbes were found to increase significantly in the supplementary solid diet groups. Predominant genera were found to alter significantly from unclassified Sphingobacteriaceae (non-supplementary group) to Prevotella (supplementary solid diet groups). Random Forest classification model revealed signature microbiota for solid diet that positively correlated with macronutrient intake, and linearly increased with volatile fatty acid production. Bacteria associated with carbohydrate and protein metabolism were also identified. Utilization of a Fish Taco analysis portrayed a set of intersecting core species contributed to rumen function shifts by the solid diet regime. The core community structures consisted of the specific, signature microbiota and the manipulation of their symbiotic partners are manipulated by extra nutrients from concentrate and/or forage, and then produce more volatile fatty acids to promote rumen development and functions eventually host development. Our study provides mechanisms of the microbiome governed by a solid diet regime early in life, and highlights the signature microbiota involved in animal health and production.