Effect of hay type on cecal and fecal microbiome and fermentation parameters in horses.

The effect of hay type on the microbiome of the equine gastrointestinal tract is relatively unexplored. Our objective was to characterize the cecal and fecal microbiome of mature horses consuming alfalfa or Smooth Bromegrass (brome) hay. Six cecally cannulated horses were used in a split-plot design run as a crossover in two periods. The whole plot treatment was ad libitum access to brome or alfalfa hay fed over two 21-d acclimation periods with subplots of sampling location (cecum and rectum) and sampling hour. Each acclimation period was followed by a 24-h collection period where cecal and fecal samples were collected every 3 h for analysis of pH and volatile fatty acids (VFA). Fecal and cecal samples were pooled and sent to a commercial lab (MR DNA, Shallowater, TX) for the amplification of the V4 region of the 16S rRNA gene and sequenced using Illumina HiSeq. The main effects of hay on VFA, pH, and taxonomic abundances were analyzed using the MIXED procedure of SAS 9.4 with fixed effects of hay, hour, location, period, and all possible interactions and random effect of horse. Alpha and beta diversities were analyzed using the R Dame package. Horses fed alfalfa had greater fecal than cecal pH (P ≤ 0.05), whereas horses fed brome had greater cecal than fecal pH (P ≤ 0.05). Regardless of hay type, total VFA concentrations were greater (P ≤ 0.05) in the cecum than in feces, and alfalfa resulted in greater (P ≤ 0.05) VFA concentrations than brome in both sampling locations. Alpha diversity was greater (P ≤ 0.05) in fecal compared with cecal samples. Microbial community structure within each sampling location and hay type differed from one another (P ≤ 0.05). Bacteroidetes were greater (P ≤ 0.05) in the cecum compared with the rectum, regardless of hay type. Firmicutes and Firmicutes:Bacteroidetes were greater (P ≤ 0.05) in the feces compared with cecal samples of alfalfa-fed horses. In all, fermentation parameters and bacterial abundances were impacted by hay type and sampling location in the hindgut.

Caecal fermentation characteristics of commonly used feed ingredients.

BACKGROUND: Commercial horse feeds use cereal grains and by-products; however, their effects on the caecal environment remain poorly characterised. OBJECTIVE: Characterise the effect of commonly used feed ingredients on caecal pH and volatile fatty acid (VFA) concentration. STUDY DESIGN: 6 × 6 Latin square. METHODS: Two days prior to the start of the study, 6 caecally cannulated Quarter horses were moved into individual stalls where Smooth bromegrass hay (brome) was offered at 2.0% BW/d split between 2 feedings (0600 and 1800). On day 0, caecal digesta was collected every 2 h for 12 h relative to the 0600 feeding to establish control values for horses consuming only brome (HAY). On day 1, horses began consuming their respective treatments which consisted of beet pulp (BP), maize (M), dehydrated alfalfa (A), oats (OAT), soybean hulls (SBH), or wheat middlings (WM) at 0.25% BW/d split into 2 feedings. On day 7 of each treatment period, caecal digesta was collected every 2 h for 12 h and analysed for pH and VFA. Data were analysed using mixed ANOVA with repeated measures, fixed effects of treatment and time and random effects of horse and period. RESULTS: There was a main effect of hour (P ≤ .05) indicative of post-prandial shifts in caecal metabolites. There were no main effects of treatment on pH or VFA concentration (P ≥ .3). Effects of hour × treatment (P ≤ .04) were observed for all response variables. Regardless of treatment or hour, caecal pH remained well within normal limits. Three horses exhibited signs of lower oesophageal choke immediately after consumption of BP pellets. LIMITATIONS: Interactions between ingredients when mixed for formulation of a concentrate warrant further research. Furthermore, a short adaptation period was chosen to mimic common management practices. CONCLUSION: Minimal differences in caecal fermentation parameters were detected when ingredients were fed at a common inclusion level.

Effects of Sodium Caseinate and Varying Protein Sources on In Vitro Fermentation of Forages by Mixed Equine Cecal Microorganisms.

To assess the impact of protein on fermentation by equine cecal microorganisms, cecal fluid from 4 cecally cannulated horses was used to inoculate fermentation bottles containing buffer, forage, and supplemental protein. In experiment 1, sodium caseinate (SC) provided 0, 0.5%, 1%, 2%, or 4% additional crude protein (CP) to bottles containing alfalfa or native warm-season prairie grass hay. Bottles were equipped with continuous gas pressure monitors and placed into a shaking incubator for 48 hours at 39°C. Cultures with alfalfa had greater (P < .0001) in vitro dry matter disappearance (IVDMD), neutral detergent fiber disappearance (NDFD), acid detergent fiber disappearance (ADFD), cumulative gas production, and total volatile fatty acid (VFA). Sodium caseinate increased gas production (P ≤ .05) and decreased pH (P < .003) in cultures with grass hay. Sodium caseinate at 1%, 2%, or 4% additional CP increased IVDMD, NDFD, and ADFD (P < .01), while 4% additional CP also increased total VFA (P < .01). For experiment 2, SC, fishmeal, soybean meal (SBM), whey, porcine blood plasma, and L-lysine hydrochloride were added to supply 2% additional CP to cultures with grass hay. All protein sources decreased pH and increased IVDMD, NDFD, and ADFD (P ≤ .01), with the largest effects elicited by SC, L-lysine, and whey (P ≤ .05). Total VFA (P ≤ .04) and gas (P ≤ .05) production increased with L-lysine, whey, SC, SBM, and fishmeal. While protein supplementation had minimal effects on cultures containing alfalfa, it altered fermentation of grass hay, more notably with more soluble protein sources.

Digestibility of diets containing calcium salts of fatty acids or soybean oil in horses.

Calcium salts of fatty acids (CSFAs) frequently are fed to ruminants, but their fate in the equine digestive system is unknown. The purpose of this study was to compare Enertia s/f, a proprietary CSFAs, and soybean (SB) oil with respect to impact on apparent total tract nutrient digestion and cecal fermentation parameters in horses. Eight cecally cannulated Quarter Horses were used in a crossover design in which horses consumed a diet for 32 d consisting of 1.5% body weight (BW) (as-fed) smooth bromegrass hay and 0.5% BW (as-fed) pelleted concentrate containing 4.9% CSFAs or 4.1% SB oil. Fecal samples were collected every 4 h from day 30 to 32 of each period and analyzed for apparent total tract digestibilities of dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), crude fat (CF), and gross energy (GE) using acid detergent insoluble ash as an internal marker. Cecal digesta was obtained at 0, 2, 4, 6, 8, 10, and 12 h following the morning meal on day 29 of each period and analyzed for pH and concentrations of volatile fatty acid (VFA) and long-chain fatty acids (LCFAs). Serum was collected on day 33 of each period following a 16-h fast and analyzed for triglycerides and cholesterol. Apparent total tract digestibilities of DM, NDF, ADF, CP, CF, and GE were unaffected by lipid source (P > 0.10). Serum triglycerides tended to be greater in horses consuming CSFAs compared to SB (P = 0.10); however, serum cholesterol was not different (P = 0.45). In horses consuming SB, cecal pH decreased below baseline (hour 0) at hours 2, 4, and 6 (P < 0.01), whereas cecal pH in horses consuming CSFAs was below baseline at hours 4 and 6 (P < 0.01). There were no treatment or time effects on cecal pH (P > 0.10). Cecal concentrations of total VFAs were greater in horses consuming SB compared to CSFAs at hour 2 (P = 0.01). Cecal concentrations of acetate, propionate, acetate:propionate (A:P), and butyrate were affected by time (P < 0.01). Propionate was less at hour 2 in horses fed CSFAs compared to horses fed SB (P = 0.04). A treatment × time interaction was detected for total cecal LCFAs concentration (P < 0.01); LCFAs concentration was greater at hour 2 for horses consuming CSFAs compared to horses fed SB (P = 0.02). SB oil and CSFAs have similar effects on the digestion of DM, NDF, ADF, GE, CF, and CP.

Effect of Cobalt Chloride on Fermentation of Alfalfa and Smooth Bromegrass Hays by Horse Cecal Microorganisms.

Effects of Co on fiber digestibility in horses are largely unknown. Our objective was to evaluate effects of Co chloride on in vitro gas production, VFA production, and dry matter (DM) disappearance (IVDMD) using cecal fluid from 4 cannulated Quarter Horses. Five grams DM of alfalfa or smooth bromegrass hay were provided as substrate with Co added at 0.0, 0.5, 5.0, 25.0, or 50.0 mg/kg substrate DM. Ten milliliters cecal fluid and 140 mL McDougall's buffer were incubated in duplicate, and pH, VFA concentrations, and IVDMD were measured after 48 hours. Gas production, used as an indicator of fermentative activity, was recorded every 15 minutes. Terminal pH was not affected by Co or forage type (P > .19). There was greater IVDMD in cultures containing alfalfa (29.2%) compared with smooth bromegrass hay (19.4%; P < .01). There was a forage × Co interaction (P < .05) in which gas production was greater in cultures containing alfalfa, and 5 mg Co/kg substrate DM led to greater gas production than 50 mg Co/kg substrate DM in cultures containing brome (P < .05). Gas production was affected by forage, Co, and time (P < .01). Production of individual and total VFA, as well as acetate:propionate (A:P) ratio were increased in cultures containing alfalfa (P < .05); however, there were no effects of Co on VFA concentrations (P > .05). While gas production was influenced by Co in cultures containing brome, no effects of Co were observed for pH, VFA, or IVDMD.

Effects of sodium caseinate on hindgut fermentation and fiber digestion in horses.

Eight cecally cannulated Quarter Horses were used in a replicated 4 × 4 Latin square experiment conducted in four 14-d periods to determine effects of sodium caseinate (casein) on hindgut fermentation and fiber digestion. During each period, horses were assigned to one of four treatments consisting of control (water; CON), 0.125 g casein/kg BW (LOW), 0.25 g casein/kg BW (MED), or 0.5 g casein/kg BW (HI). Casein was solubilized in 800 mL water and dosed directly into the cecum at 0700 and 1900 hours using a metal dosing syringe. Smooth Bromegrass hay (CP 8.50%), water, and salt were provided ad libitum. New hay was fed at 0700 and 1900 hours, and orts were recorded at 1900 daily. During the final 3 d of each period, cecal digesta were collected every 6 h, pH was measured, and samples were frozen for subsequent analyses of VFA and NH3 concentrations. Feed intake during the final 4 d of each period was recorded. Feces were collected during the 3-d sampling period, pooled, subsampled, and frozen. Fecal samples were analyzed for pH and used to determine digestibilities of DM, OM, NDF, and ADF. Statistical analyses were performed via the GLIMMIX procedure of SAS 9.4. Linear and quadratic effects of sodium caseinate on pH, VFA concentrations, and apparent digestibility were assessed by SAS. Digestibilities of DM, OM, ADF, and NDF were unaffected by treatment (P > 0.40). Horses dosed with CON and MED treatments had greater cecal pH than those fed LOW or HI treatments (P < 0.01). Cecal NH3 concentrations increased linearly in response to the amount of casein administered (P < 0.01). Cecal NH3 decreased 6 h after dosing and addition of new hay, regardless of treatment (P < 0.01). Total cecal VFA were unaffected by treatment (P > 0.10), but VFA changed over time with the greatest concentrations observed 6 h after treatments were administered and introduction of new hay (P < 0.01). Treatment did not affect DMI (P ≥ 0.17). In this experiment, cecal infusions of sodium caseinate had minimal to no effect on fermentation parameters or fiber degradation in the horse. A type II error may have occurred due to small population size or the medium quality hay fed to these horses provided sufficient N for microbial fermentation.