A comparison of post-ruminal provision of Ca-gluconate and Ca-butyrate on growth performance, gastrointestinal barrier function, short-chain fatty acid absorption, intestinal histology, and brush-border enzyme activity in beef heifers.

The objective of this study was to compare the effects of post-ruminal provision of Ca-butyrate (CaB) when delivered via abomasal dosing, and Ca-gluconate (CaG) when provided ruminally using a rumen protected form or using an unprotected form via abomasal dosing on short-chain fatty acid (SCFA) concentration throughout the GIT, nutrient digestibility, GIT barrier function, ruminal SCFA absorption, ruminal morphometrics, intestinal brush border enzyme activity, and blood parameters for beef heifers. Thirty-two beef heifers fitted with ruminal cannulas were used in a randomized complete block design and assigned to one of four treatments: 1) negative control (ruminal infusion of double-distilled water; CON); 2) abomasal infusion of CaB (AB; 0.0029% of BW); 3) abomasal infusion of CaG (AG; 0.0077% of BW); and 4) ruminal infusion of a hydrogenated fat-embedded CaG (RG; 0.0192% of BW) to provide ruminal protection. Excluding CON, treatments were designed to deliver the same amount of butyrate in the small intestine. Heifers were housed in individual pens and DMI was limited to 95% of voluntary intake to minimize a potential confounding effect of DMI on treatment responses. Total GIT barrier function was assessed on day 17 and SCFA disappearance was evaluated on day 21 using the temporarily isolated and washed reticulo-rumen technique. On day 28, heifers were slaughtered, and ruminal and colonic digesta were collected to assess SCFA concentration. Additionally, ruminal, jejunal, and colonic tissues were collected to assess SCFA fluxes and regional barrier function ex vivo using the Ussing chamber technique. For colonic digesta, both AB and CaG treatments reduced the proportion of acetate (P < 0.05) and increased the proportion on propionate (P < 0.05) compared to CON. Relative to CON, AB but not CaG treatments increased in vivo ruminal disappearance of total SCFA (P = 0.01), acetate (P = 0.03), propionate (P = 0.01), and butyrate (P > 0.01). Treatments did not affect (P ≥ 0.10) acetate and butyrate fluxes in the ruminal and colonic tissues when measured ex vivo; however, when compared with CON, AB tended to decrease (P = 0.09) mannitol flux across ruminal tissue. In addition, mannitol flux was affected (P < 0.01) by region, with greater mannitol flux across the jejunum than rumen and colon. We conclude that while both abomasal infusion of CaB and CaG affect the molar proportion of acetate and propionate in the colon, only abomasal CaB stimulated ruminal SCFA absorption for growing beef heifers.

Butyrate, a short-chain fatty acid (SCFA), has received attention due to its ability to promote gastrointestinal (GIT) health and development. However, butyrate in its free form presents a strong odor, limiting its use in diet formulation. Supplementation of butyrate precursors, such as gluconate, have been studied to enhance butyrate production in the GIT. This study evaluated the effects of post-ruminal infusion of Ca-butyrate (AB; 0.0029% of BW) and Ca-gluconate (AG; 0.0077% of BW) and ruminal infusion of a hydrogenated fat-embedded Ca-gluconate (RG; 0.0192% of BW) relative to control (CON; ruminal infusion of double-distilled water). Thirty-two beef heifers fitted with ruminal cannulas were fed for 28 d and GIT barrier function and ruminal SCFA absorption were assessed. At slaughter, the rumen, jejunum, and colon tissues were collected and barrier function and SCFA fluxes were assessed ex vivo. Relative to CON, AB but not AG and RG increased in vivo ruminal SCFA absorption and tended to increase ex vivo barrier function. Thus, the data presented in this study shows that butyrate and gluconate do not function through the same mode of action in the GIT of beef heifers.

Effect of feeding calcium gluconate embedded in a hydrogenated fat matrix on feed intake, gastrointestinal fermentation and morphology, intestinal brush border enzyme activity and blood metabolites in growing lambs.

Gluconate salts have been identified as a butyrate precursor when fed to non-ruminant species and may increase the butyrate concentration in the large intestine supporting gastrointestinal health and development. The objective of this study was to evaluate the dose response of hydrogenated fat-embedded calcium gluconate (HFCG) on performance and gastrointestinal tract (GIT) development in growing lambs. Thirty-two wether lambs were used in a randomized complete block design and assigned to 1 of 4 treatments differing in the inclusion of HFCG: 0.0% (CON), 0.075% (LOW), 0.30% (MED), and 0.60% of the diet (HIGH). Lambs were allocated into individual pens and fed ad libitum with feed delivered twice daily. Feed intake was recorded daily, and body weight (BW) was assessed at the beginning and the end of the 29-d period. Blood was sampled on day 21, prior to feeding and 6 h post-feeding to evaluate changes in ß-hydroxybutyrate, glucose, and insulin concentrations. Total fecal collection was conducted during days 25 to 28 to assess apparent total tract digestibility. On day 29, lambs were slaughtered, and the entire GIT was separated by region to enable sampling of tissue and digesta. Data were analyzed to assess linear, quadratic, and cubic effects of HFCG dose. Final BW, average daily gain, and dry matter intake decreased linearly (P ≤ 0.02) with increasing HFCG. Increasing inclusion of HFCG linearly decreased (P = 0.01) the thickness of the stratum corneum in ruminal papillae but did not affect other strata (P ≥ 0.34). Omasal digesta weight linearly decreased (P = 0.01) as the concentration of HFCG increased and abomasal digesta weight was cubically affected (P = 0.03) the increasing dose of HFCG. Short-chain fatty acid concentration in the cecum was cubically affected (P < 0.01) with increasing dose of HFCG where low dose had the greatest concentration. Moreover, increasing the dietary supply of HFCG linearly increased the proportion of acetate (P = 0.04) in the cecum and linearly decreased the proportion of propionate in the digesta of both the cecum (P < 0.01) and colon (P = 0.01). Colon crypt depth was quadratically (P = 0.03) affected with the increasing dose of HFCG, where lambs fed MED had greatest crypt depth. We conclude that feeding HFCG to growing lambs did not increase butyrate concentration in the large intestine and consequently does not increase the absorptive surface area of the whole tract, the size of the GIT, or the functionality of the intestine.

Gluconate salts have been reported to be metabolized by microbes in the gastrointestinal tract to yield butyrate. Butyrate has shown potential to enhance functionality of the gastrointestinal tract by increasing the absorptive surface area, enzyme activity, and the barrier function. This study evaluated the inclusion of four levels of hydrogenated fat-embedded Ca-gluconate (HFCG; 0.0%, 0.075%, 0.30%, and 0.60% of the diet) designed to increase the production of butyrate in the large intestine. Thirty-two wether lambs were fed for 28 d, slaughtered, and eviscerated to allow complete evaluation of the gastrointestinal tract and its contents. Growth and dry matter intake decreased linearly with increasing dose of HFCG. Dose of HFCG cubically affected short-chain fatty acid concentration in the cecum with increased concentrations at the 0.075% dose. Moreover, increasing dose of HFCG linearly increased the proportion of acetate and linearly decreased the proportion of propionate in the cecum without altering the proportion of butyrate. Thus, the supplementation of HFCG did not increase butyrate concentration in the large intestine and did not enhance gastrointestinal tract function.

Persistence of Cellulolytic Bacteria Fibrobacter and Treponema After Short-Term Corn Stover-Based Dietary Intervention Reveals the Potential to Improve Rumen Fibrolytic Function.

Limited lignocellulose degradation is the primary obstacle to feed digestion efficiency in ruminant animals. Low-quality forage with high levels of fibrous components can favor the proliferation of fibrolytic bacteria, but whether this can result a profound microbial shift after dietary intervention remains unclear. In this study, we monitored the microbial communities in the rumens of five ruminally cannulated Hu sheep through dietary transition from alfalfa hay (AH, pre-CS) to corn stover (CS, post-CS) and then back to AH (post-AH), with each treatment lasting for 14 days. The CS intervention significantly increased the relative abundance of microorganisms involved in lignocellulose degradation, including Fibrobacter and Treponema. When the diet was switched back to AH, the microbial community did not completely return to a pre-CS treatment state. In the post-AH microbial community, the relative abundances of Fibrobacter and Treponema were persistently high, and were similar to those in the post-CS community. Meanwhile, the diversity of the microbial community increased after dietary transition from AH to CS and remained significantly higher after transition from CS to AH compared to those under the original AH diet. Enzyme activity measurement verified significant increase of carboxymethyl cellulase (CMCase) and xylanase catalytic activities in the rumen. Microbial functional predictions using Tax4Fun revealed that this microbial persistence may enhance the carbohydrate metabolism pathway in the rumen. In summary, persistence of Fibrobacter and Treponema can be enhanced through a low-quality forage intervention at least for 2 weeks, which may enlighten the reprogram of microbial population in the rumen in the future.

Alfalfa Intervention Alters Rumen Microbial Community Development in Hu Lambs During Early Life.

The pre-weaning period is crucial for rumen developmental plasticity, which can have a long-term impact on animal performance. Understanding the rumen microbiota during early life is important to elucidate its potential role in rumen development. In this study, the rumen microbiota of 10-day-old Hu lambs fed either milk replacer (B-10), milk replacer and starter (STA) or milk replacer and starter supplemented with alfalfa (S-ALF) in the pre- (d17, 24, and 38) and post-weaning periods (d45 and 66) were assessed to characterize rumen microbial colonization during early life and its response to fiber intervention. In the rumens of B-10 lambs, 498 operational taxonomic units belonging to 33 predominant genera were observed, and the top six predicted functions included "Membrane transport," "carbohydrate metabolism," "amino acid metabolism," "replication and repair," "translation," and "energy metabolism." Prevotella, Succinivibrio, Bifidobacterium, and Butyrivibrio abundances were increased at d38 for both STA and S-ALF groups compared to the B-10 group, whereas fibrolytic bacteria of the taxa Lachnospiraceae and Treponema were only increased in the S-ALF group at d38. A number of saccharolytic bacteria (Bacteroidaceae), organic acid-producing bacteria (Coprococcus and Actinomyces), proteolytic and amino acid fermenters (Fusobacterium) and fibrolytic bacteria (unclassified Ruminococcaceae) were significantly decreased in the STA lambs but not in the S-ALF lambs at d38. After weaning and exposed to alfalfa, the rumen microbial composition in the STA group started to appear similar to that of the S-ALF lambs. The relative abundance of unclassified Clostridiales was higher in S-ALF lambs than STA lambs after weaning. Spearman's correlation analysis showed positive relationships between unclassified Lachnospiraceae, unclassified Clostridiales, Treponema, unclassified Bacteroidales, Coprococcus and crude protein intake, neutral detergent fiber intake, and plasma ß-hydroxybutyrate. The unclassified Lachnospiraceae and Treponema were also positively correlated with average daily gain. Our results revealed that alfalfa stimulated changes in rumen microbiota during the pre- and post-weaning periods and was consistent with rumen development for better feed intake and animal performance before and after weaning. The findings of this study provide clues for strategies to improve rumen function through manipulation of the rumen microbiota during early life.

Enhancing the Resolution of Rumen Microbial Classification from Metatranscriptomic Data Using Kraken and Mothur.

The advent of next generation sequencing and bioinformatics tools have greatly advanced our knowledge about the phylogenetic diversity and ecological role of microbes inhabiting the mammalian gut. However, there is a lack of information on the evaluation of these computational tools in the context of the rumen microbiome as these programs have mostly been benchmarked on real or simulated datasets generated from human studies. In this study, we compared the outcomes of two methods, Kraken (mRNA based) and a pipeline developed in-house based on Mothur (16S rRNA based), to assess the taxonomic profiles (bacteria and archaea) of rumen microbial communities using total RNA sequencing of rumen fluid collected from 12 cattle with differing feed conversion ratios (FCR). Both approaches revealed a similar phyla distribution of the most abundant taxa, with Bacteroidetes, Firmicutes, and Proteobacteria accounting for approximately 80% of total bacterial abundance. For bacterial taxa, although 69 genera were commonly detected by both methods, an additional 159 genera were exclusively identified by Kraken. Kraken detected 423 species, while Mothur was not able to assign bacterial sequences to the species level. For archaea, both methods generated similar results only for the abundance of Methanomassiliicoccaceae (previously referred as RCC), which comprised more than 65% of the total archaeal families. Taxon R4-41B was exclusively identified by Mothur in the rumen of feed efficient bulls, whereas Kraken uniquely identified Methanococcaceae in inefficient bulls. Although Kraken enhanced the microbial classification at the species level, identification of bacteria or archaea in the rumen is limited due to a lack of reference genomes for the rumen microbiome. The findings from this study suggest that the development of the combined pipelines using Mothur and Kraken is needed for a more inclusive and representative classification of microbiomes.

Use of Genomic Tools to Improve Cattle Health in the Context of Infectious Diseases.

Although infectious diseases impose a heavy economic burden on the cattle industry, the etiology of many disorders that affect livestock is not fully elucidated, and effective countermeasures are often lacking. The main tools available until now have been vaccines, antibiotics and antiparasitic drugs. Although these have been very successful in some cases, the appearance of parasite and microbial resistance to these treatments is a cause of concern. Next-generation sequencing provides important opportunities to tackle problems associated with pathogenic illnesses. This review describes the rapid gains achieved to track disease progression, identify the pathogens involved, and map pathogen interactions with the host. Use of novel genomic tools subsequently aids in treatment development, as well as successful creation of breeding programs aimed toward less susceptible livestock. These may be important tools for mitigating the long term effects of combating infection and helping reduce the reliance on antibiotic treatment.