Exploration of mobile genetic elements in the ruminal microbiome of Nellore cattle.

Metagenomics has made it feasible to elucidate the intricacies of the ruminal microbiome and its role in the differentiation of animal production phenotypes of significance. The search for mobile genetic elements (MGEs) has taken on great importance, as they play a critical role in the transfer of genetic material between organisms. Furthermore, these elements serve a dual purpose by controlling populations through lytic bacteriophages, thereby maintaining ecological equilibrium and driving the evolutionary progress of host microorganisms. In this study, we aimed to identify the association between ruminal bacteria and their MGEs in Nellore cattle using physical chromosomal links through the Hi-C method. Shotgun metagenomic sequencing and the proximity ligation method ProxiMeta were used to analyze DNA, getting 1,713,111,307 bp, which gave rise to 107 metagenome-assembled genomes from rumen samples of four Nellore cows maintained on pasture. Taxonomic analysis revealed that most of the bacterial genomes belonged to the families Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, Saccharofermentanaceae, and Treponemataceae and mostly encoded pathways for central carbon and other carbohydrate metabolisms. A total of 31 associations between host bacteria and MGE were identified, including 17 links to viruses and 14 links to plasmids. Additionally, we found 12 antibiotic resistance genes. To our knowledge, this is the first study in Brazilian cattle that connect MGEs with their microbial hosts. It identifies MGEs present in the rumen of pasture-raised Nellore cattle, offering insights that could advance biotechnology for food digestion and improve ruminant performance in production systems.

Feeding amylolytic and proteolytic exogenous enzymes: Effects on nutrient digestibility, ruminal fermentation, and performance in dairy cows.

Exogenous enzymes are added to diets to improve nutrient utilization and feed efficiency. A study was conducted to evaluate the effects of dietary exogenous enzyme products with amylolytic (Amaize, Alltech) and proteolytic (Vegpro, Alltech) activity on performance, excretion of purine derivatives, and ruminal fermentation of dairy cows. A total of 24 Holstein cows, 4 of which were ruminally cannulated (161 ± 88 d in milk, 681 ± 96 body weight, and 35.2 ± 5.2 kg/d of milk yield), were blocked by milk yield, days in milk, and body weight, and then distributed in a replicated 4 × 4 Latin square design. Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation and the last 7 d were used for data collection. Treatments were as follows: (1) control (CON) with no feed additives, (2) amylolytic enzyme product added at 0.5 g/kg diet dry matter (DM; AML), (3) amylolytic enzyme product at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.2 g/kg of diet DM (low level; APL), and (4) amylolytic enzyme products added at 0.5 g/kg diet DM and proteolytic enzyme product at 0.4 g/kg of diet DM (high level; APH). Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). Differences between treatments were analyzed by orthogonal contrasts: CON versus all enzyme groups (ENZ); AML versus APL+APH; and APL versus APH. Dry matter intake was not affected by treatments. Sorting index for feed particles with size <4 mm was lower for ENZ group than for CON. Total-tract apparent digestibility of DM and nutrients (organic matter, starch, neutral detergent fiber, crude protein, and ether extract) were similar between CON and ENZ. Starch digestibility was greater in cows fed APL and APH treatments (86.3%) compared with those in the AML group (83.6%). Neutral detergent fiber digestibility was greater in APH cows compared with those in the APL group (58.1 and 55.2%, respectively). Ruminal pH and NH3-N concentration were not affected by treatments. Molar percentage of propionate tended to be greater in cows fed ENZ treatments than in those fed CON. Molar percentage of propionate was greater in cows fed AML than those fed the blends of amylase and protease (19.2 and 18.5%, respectively). Purine derivative excretions in urine and milk were similar in cows fed ENZ and CON. Uric acid excretion tended to be greater in cows consuming APL and APH than in those in the AML group. Serum urea N concentration tended to be greater in cows fed ENZ than in those fed CON. Milk yield was greater in cows fed ENZ treatments compared with CON (32.0, 33.1, 33.1, and 33.3 kg/d for CON, AML, APL, and APH, respectively). Fat-corrected milk and lactose yields were higher when feeding ENZ. Feed efficiency tended to be greater in cows fed ENZ than in those fed CON. Feeding ENZ benefited cows' performance, whereas the effects on nutrient digestibility were more pronounced when the combination of amylase and protease was fed at the highest dose.

Dietary replacement of soybean meal with heat-treated soybean meal or high-protein corn distillers grains on nutrient digestibility and milk composition in mid-lactation cows.

Lactation diets dependent on rumen undegradable protein (RUP) sources derived from soybean meal (SBM) products are generally high in Lys and poor in Met. We conducted an experiment to evaluate the effects of increasing dietary RUP and altering digestible AA supply by inclusion of heat-treated soybean meal (HTSBM) or high-protein corn dried distillers grains with soluble (DDGS) on performance in mid-lactation dairy cows. Twenty-four Holstein cows (200 ± 40 d in milk and 30.0 ± 3.92 kg/d of milk yield) blocked according to parity, milk yield, and days in milk were used in a 3 × 3 Latin square design experiment with 21-d periods. Treatments were (1) control (CON), a diet with 6.0% RUP containing 15.9% SBM as the main protein source; (2) HTSBM, a diet with 6.7% RUP containing 4.4% HTSBM partially replacing SBM; and (3) high-protein DDGS (FP; FlexyPro, SJC Bioenergia), a diet with 6.9% RUP containing 5.34% FP partially replacing SBM and ground corn. Diets had similar crude protein (16.9%) and net energy of lactation. Data were submitted to ANOVA using the mixed procedure of SAS software (SAS Institute Inc.). Treatment differences were evaluated using orthogonal contrasts: (1) increasing RUP (SBM vs. HTSBM + FP) and (2) altering digestible AA supply (HTSBM vs. FP). Cows fed HTSBM and FP had greater intake (values in parentheses represent treatment means of CON, HTSBM, and FP, respectively) of neutral detergent fiber (7.14, 7.35, and 7.69 kg/d), crude protein (4.27, 4.37, and 4.51 kg/d), and ether extract (0.942, 0.968, and 1.04 kg/d) compared with cows fed CON. Feeding FP resulted in greater intake of neutral detergent fiber and ether extract compared with HTSBM. Cows fed HTSBM and FP had lower sorting index for feed particles <4 mm than cows fed CON (1.029, 1.008, and 1.022). Feeding FP resulted in greater intake of feed particles <4 mm compared with HTSBM. Treatments containing HTSBM or FP tended to decrease organic matter digestibility (72.4, 71.2, and 71.1%), but no other effects were detected in digestibility of neutral detergent fiber, crude protein, or ether extract. No evidence for differences among treatments was detected in excretion of purine derivatives in milk and urine. Milk yield was greater in cows fed HTSBM or FP than in cows fed CON (28.0, 28.9, and 28.8 kg/d, respectively). Cows fed HTSBM or FP tended to have greater energy-corrected milk and protein yield compared with those fed CON. Milk protein concentration was greater in DDGS cows than those in the HTSBM group (3.45 and 3.40%, respectively). No differences were detected in milk fat yield and concentration, milk urea nitrogen, feed efficiency, or serum concentrations of urea and glucose. Overall, increasing dietary RUP by feeding HTSBM or FP improved intake of nutrients and milk yield without affecting feed efficiency. Altering digestible AA supply while maintaining similar dietary RUP had negligible effects on performance of cows.

Feeding encapsulated pepper to dairy cows during the hot season improves performance without affecting core and skin temperature.

Peppers (Capsicum spp.) contain capsaicin, an organic compound with a group of alkaloids that has shown thermoregulation properties in humans and mice, and may influence glucose and lipid metabolism in ruminants. An experiment was conducted to evaluate different doses of a feed additive containing encapsulated pepper on milk yield and composition, dry matter intake, feed sorting index, total-tract apparent digestibility of nutrients, purine derivatives excretion, and serum concentrations of urea-N and glucose, N excretion, respiration rate, rectal temperature, and skin temperature in different regions (forehead, face, and rumen). Thirty-six Holstein cows (150 ± 102.1 d in milk and 29.3 ± 5.81 kg/d milk yield) were used in a 9-wk randomized complete block (n = 12) design experiment. Following a 2-wk covariate period, cows were blocked according to parity, days in milk, and milk yield and were randomly assigned to the following treatments: 0 (CAP0), 0.75 (CAP75), or 1.5 (CAP150) g/d of a feed additive containing encapsulated pepper (1 g/kg, Capcin; NutriQuest) added to the concentrate along with minerals. Treatment differences were evaluated through orthogonal contrasts (CAP0 vs. CAP75 + CAP150 or CAP75 vs. CAP150). The average temperature-humidity index during the experiment was 72.0 ± 2.07. Dry matter intake was greater in cows fed a feed additive containing encapsulated pepper (CAP) treatments (CAP75 and CAP150) compared with CAP0. Cows fed CAP150 tended to have greater dry matter intake than those in CAP75 group. Feeding CAP decreased sorting for feed particles with size between 8 and 4 mm. An interaction effect between treatment and week was observed for crude protein digestibility whereas cows fed CAP150 had the greatest digestibility on the third week of experiment. Orthogonal contrasts did not detect differences in serum concentrations of glucose and urea-N, or purine derivatives excretion. Nitrogen excretion (as % of N intake) in milk, urine, and feces was not altered by treatments. Feeding CAP increased yields of 3.5% fat-corrected milk, fat, protein, and lactose. A tendency toward greater milk protein content was observed for cows fed CAP150 than CAP75. No differences were detected on respiration rate, rectal temperature, and skin temperature of cows. A feed additive containing encapsulated pepper fed at 0.75 or 1.5 g/d can improve yield of fat-corrected milk and milk solids by increasing feed intake without affecting nutrient digestibility and body temperature of lactating cows during the hot season.

Dry malt extract from barley partially replacing ground corn in diets of dairy cows: Nutrient digestibility, ruminal fermentation, and milk composition.

Dry malt extract (DME) has been used in animal nutrition as an alternative source of rapidly fermentable carbohydrate. An experiment was conducted to evaluate the partial replacement of ground corn with DME in diets of dairy cows on apparent digestibility, ruminal fermentation, predicted rumen microbial protein supply, N excretion, serum urea-N concentration, and milk yield and composition. Twenty-eight Holstein cows (35.3 ± 5.88 kg/d milk yield and 148 ± 78 d in milk), 4 of which were rumen cannulated, were blocked according to the presence of rumen cannulas, parity, milk yield, and days in milk and enrolled into a crossover design experiment. Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation and 7 d were used for data collection and sampling. Treatment sequences were composed of control (CON) or DME from barley (Liotécnica Tecnologia em Alimentos) replacing ground corn at 7.62% diet dry matter (~2 kg/d). Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc.) modeling the fixed effects of treatment, period, and their interaction, in addition to the random effect of animal. Ruminal fermentation data were analyzed as repeated measures including time and its interaction with treatment in the previous model as fixed effects. Treatments did not affect nutrient intake or feed sorting. Dry malt extract increased apparent digestibility of CP. Feeding DME decreased ruminal pH and molar percentage of butyrate and increased molar percentage of acetate. No treatment effects were detected for predicted rumen microbial protein supply or N excretion. Cows fed DME had lower serum urea-N concentration than CON cows. Dry malt extract increased yields of actual milk, 3.5% fat-corrected milk, fat, and protein, and improved feed efficiency (fat-corrected milk ÷ dry matter intake). Cows fed DME had lower milk urea nitrogen content in comparison with CON cows. Dry malt extract can partially replace ground corn in the diet while improving milk yield and feed efficiency.

Increasing doses of carbohydrases: Effects on rumen fermentation, nutrient digestibility, and performance of mid-lactation cows.

The objective of this study was to evaluate the effects of exogenous enzymes on nutrient intake and digestibility, rumen fermentation, and productivity of mid-lactating cows. Experiment 1 was designed to test increasing doses [0, 0.5, 1.0, or 1.5 g/kg of dry matter (DM)] of a combination of 2 enzyme products with xylanase and ß-glucanase activities (Ronozyme Wx and Ronozyme VP, respectively; DSM Nutritional Products) on rumen fermentation and total apparent digestibility. Enzyme combinations had a ratio of endo-1,3(4)-ß-glucanase to endo-1,4-ß-xylanase of 8:2 (wt/wt). For experiment 1, 8 rumen cannulated lactating cows were used into a double 4 × 4 Latin square design experiment with 14 d of diet adaptation and 7 d of sampling. Despite no differences in feed intake, carbohydrases linearly increased neutral detergent fiber digestibility. Treatments marginally affected rumen fermentation, where a linear trend for lower rumen pH and a linear trend for greater isobutyrate concentration were observed with increasing enzyme dose. A trend for lower rumen NH3-N concentration was observed for cows receiving carbohydrases in comparison with control group. When comparing all enzyme treatments against control group, cows fed enzymes tended to produce more 3.5% fat-corrected milk (FCM), produced more milk fat, and had greater blood glucose concentration. Experiment 2 evaluated 3 doses (0, 0.5, or 0.75 g/kg of DM) of the same combination of enzyme products on performance of cows (n = 36) in a complete randomized block (n = 12) design. Cows received treatments for 9 wk. No interaction effects between treatments and time were observed for all variables assessed in this study. In agreement with experiment 1, no differences were detected for feed intake, but cows fed the enzyme products tended to produce more 3.5% FCM and milk fat compared with control. In addition, cows fed enzymes exhibited greater efficiency of FCM production (FCM ÷ DM intake) compared with control. No differences were detected for intake and productivity when comparing the 2 doses of carbohydrases. In summary, the enzyme products tested in this study may improve feed efficiency due to greater milk fat concentration.

Effects of a blend of live yeast and organic minerals or monensin on performance of dairy cows during the hot season.

The objective of this study was to evaluate the effects of feed additives on intake and digestibility of nutrients, milk yield and composition, feeding behavior, and physiological parameters of dairy cows during the hot season. Forty Holstein cows were assigned to a randomized block design experiment with a 2 × 2 factorial treatment arrangement to evaluate (1) control diet without inclusion of additives; (2) monensin (MON), 20 mg/kg diet dry matter sodium monensin (Rumensin; Elanco); (3) Milk Sacc+ (MS+), inclusion of 40 g/cow per d of Milk Sacc+ (a blend of live yeast and organic minerals, Alltech); and (4) combination of MON and MS+. The average temperature-humidity index throughout the experimental period was 73 ± 2.84 (standard deviation). The experiment lasted 11 wk, including 2 preliminary weeks for covariate adjustments. Cows fed MS+ increased dry matter intake (% body weight), milk yield, 3.5% fat-corrected milk, and solids yield, and cows fed MON had greater milk urea nitrogen content in comparison with counterparts. Feeding MS+ increased the intake of feed particles with size between 8 and 19 mm and decreased the intake of particles shorter than 4 mm compared with other treatments. Rumination time (min/d) and chewing time (min/kg of neutral detergent fiber) were lower for cows fed MS+. Physiologic parameters (i.e., heart and respiratory rates, and body temperature) were not affected by the treatments. Overall, the use of monensin did not differ from control, and Milk Sacc+ improved performance of cows.

Partial replacement of corn silage with whole-plant soybean and black oat silages for dairy cows.

This study aimed to evaluate the effects of partially replacing corn silage (CS) with whole-plant soybean silage (SS) or black oat silage (OS) on nutrient intake and digestibility, in vitro neutral detergent fiber degradability of silages, feeding behavior, rumen fermentation, and performance of dairy cows. Twenty-four lactating Holstein cows (6 of which were rumen-cannulated) with 32.5 ± 4.92 kg/d milk yield, 150 ± 84.8 days in milk, and 644 ± 79.0 kg of body weight were used in a 3 × 3 Latin square design to evaluate the following treatments: (1) corn silage diet (CSD): using corn silage as the only forage source in the diet [48% dietary dry matter (DM)]; (2) whole-plant soybean silage diet (SSD): SS replacing 16% of corn silage from CSD; and (3) black oat silage diet (OSD): OS replacing 16% of corn silage from CSD. The inclusion of OS and SS decreased intakes of DM, organic matter, and crude protein. Corn silage had the greatest in vivo effective degradability of DM, and SS had the least effective degradability of neutral detergent fiber. The OSD treatment decreased milk and protein yields, whereas SSD increased rumen ammonia nitrogen concentration compared with the other diets. Cows fed OSD exhibited a greater preference for feed with small particles (<4 mm) compared with those fed SSD. Cows fed treatments containing either SS or OS at the expense of CS had increased rumination and chewing activities. Although replacing CS with OS and SS reduced feed intake, SS had no effect on productive performance of dairy cows.

Effects of organic acids in total mixed ration and feeding frequency on productive performance of dairy cows.

This study aimed to evaluate the effects of organic acid (OA; Mold-Zap, Alltech, Nicholasville, KY) inclusion in the total mixed ration (TMR) and feeding frequency of TMR for lactating dairy cows on intake, total-tract apparent digestibility, sorting index, feeding behavior, ruminal fermentation, milk yield and composition, nitrogen balance, and serum metabolites. Twenty-four lactating Holstein cows, 4 with rumen cannulas, with (mean ± standard error) 247 ± 22.2 d in milk, 672 ± 14.6 kg of body weight, and 31.1 ± 1.09 kg of milk yield at the beginning of the experiment were used. The cows were distributed in a balanced and contemporary 4 × 4 Latin square experimental design and randomly assigned in a 2 × 2 factorial arrangement to evaluate OA [0 (OA-) or 0.5 (OA+) L of Mold-Zap/tonne of TMR on a natural matter basis] and feeding frequency of TMR offered once a day (1×) or twice a day (2×). Each experimental period lasted 21 d, with 14 d for acclimation and 7 d for data collection. The treatments were tested for TMR, in which its temperature was recorded every 2 h through a 24-h period in each experimental period. Organic acid-treated TMR showed a lower temperature during the 24-h period compared with nontreated TMR. The OA and feeding frequency had no effect on intake and total-tract apparent digestibility of dry matter and nutrients, aside from a tendency to increase neutral detergent fiber digestibility in cows fed 2×. Also, cows fed 1× tended to select more particles between 19 and 8 mm and refused particles smaller than 4 mm, whereas cows fed OA tended to select more particles smaller than 4 mm. Cows fed OA had greater milk yield and milk protein and lactose yields, but tended to have higher 3.5% fat-corrected milk yield. Neither treatment influenced ruminal and serum variables nor milk fat yield and milk production efficiency. Cows fed OA spent less time idling and tended to have lower rumination time, and tended to have higher time spent drinking water and eating, whereas animals fed 1× spent more time drinking water. Under the conditions of this experiment, we conclude that it was possible to reduce the feeding frequency of TMR, without negative effects on dairy cow performance. However, the use of OA resulted in higher milk yield and mitigated TMR temperature rise regardless of feeding frequency. The effect of external factors such as collective stimulation of intake and stage of lactation on feeding frequency effect must be surveyed in further research.

Increasing dietary levels of docosahexaenoic acid-rich microalgae: Ruminal fermentation, animal performance, and milk fatty acid profile of mid-lactating dairy cows.

This study aimed to evaluate the effects of increasing dietary levels of microalgae (ALG), rich in docosahexaenoic acid (DHA; All-G-Rich, Alltech, Nicholasville, KY), in isolipidic diets, on animal performance, nutrient digestibility, ruminal fermentation, milk fatty acid profile, energy balance, microbial protein synthesis, and blood serum metabolites in mid-lactating dairy cows. Twenty-four Holstein cows [130.3 ± 15.4 d in milk, and 30.8 ± 0.543 kg/d of milk yield (mean ± standard error)] were used in a 4 × 4 Latin square design experiment to evaluate the following treatments: control diet, without addition of ALG; and increasing levels of ALG [2, 4, and 6 g/kg of dry matter (DM)]. The ALG decreased DM intake and increased total-tract DM apparent digestibility. A tendency was observed for a quadratic effect on total-tract NDF digestibility by ALG inclusion, with peak value of the quadratic response at 4.13 g/kg of DM dose. Moreover, ALG increased ruminal pH and decreased acetate and total volatile fatty acid concentrations. Fat-corrected milk and energy-corrected milk were quadratically affected, and a tendency for a milk yield effect was observed when ALG levels increased, whereas maximal yields were observed with intermediate doses. Milk fat, protein, and lactose concentrations were diminished, whereas productive efficiency was improved by the increase of ALG levels. Saturated fatty acid proportions were decreased, whereas polyunsaturated fatty acid proportions were increased when ALG was fed. There was low DHA transfer into milk; however, ALG inclusion decreased C18:0, C18:1 cis-9, C18:2 cis-9,12, and C18:3 cis-9,12,15 proportions, and increased C18:2 cis-9,trans-11, C18:1 trans-9, and C18:1 trans-11 proportions. Gross energy intake was decreased, whereas no effect was observed on digestible, metabolizable, or net energy intake. The ALG inclusion quadratically affected the microbial protein synthesis, with maximal enhancement at 3.24 g/kg of DM dose, and also increased serum cholesterol concentration. Under the conditions of this experiment, the inclusion of ALG in diets for mid-lactating dairy cows decreased feed intake and increased nutrient digestibility, improving productive efficiency and modifying milk fatty acid profile. Estimated intermediate doses (1.22 to 2.90 g/kg of DM) of DHA-rich ALG may be beneficial to milk, fat-corrected milk, and energy-corrected milk yields, and is recommended for dairy cows.

Effects of exogenous fibrolytic and amylolytic enzymes on ruminal fermentation and performance of mid-lactation dairy cows.

Lactation diets are composed mostly of carbohydrates that are not fully fermented by rumen microbes. The aim of this study was to evaluate exogenous fibrolytic (Fibrozyme, Alltech Inc., Nicholasville, KY) and amylolytic (Amaize, Alltech Inc.) enzymes on nutrient intake, sorting index, total-tract apparent digestibility, ruminal fermentation, nitrogen utilization, milk yield, and composition of dairy cows in mid-lactation. Thirty-two multiparous Holstein cows (181 ± 35 d in milk, 571 ± 72.7 kg of body weight, and 29.6 ± 5.24 kg/d of milk yield at the start of experiment) were blocked according to milk yield and randomly allocated to treatments in a 4 × 4 Latin square design. Treatments were (1) control, basal diet without exogenous enzymes; (2) fibrolytic enzyme (FIB), dietary supplementation of Fibrozyme at 12 g/d (51 IU of xylanase activity/kg of diet dry matter); (3) amylolytic enzyme (AMY), dietary supplementation of Amaize at 8 g/d (203 fungal amylase units/kg of diet dry matter); and (4) both fibrolytic and amylolytic enzymes (FIB+AMY) added at the same dose of the individual treatments. Enzyme products were added to the concentrate during its preparation (once a week). The supply of FIB and AMY had no effect on nutrient intake and digestibility. However, an interaction effect was observed on sorting index of feed particle size between 8 and 19 mm. Amylolytic enzyme increased the sorting for feed particles between 8 and 19 mm, only when fed without FIB. In addition, AMY decreased the sorting for feed with particle size greater than 19 mm. An interaction effect was observed between FIB and AMY for ruminal butyrate concentration and N excretion. Amylolytic enzyme increased ruminal butyrate concentration in cows treated with FIB. Further, FIB decreased milk protein production and feed efficiency only in cows not fed AMY. Amylolytic enzyme reduced urinary N excretion. Exogenous enzymes had no effect on milk production and composition of dairy cows. This study lacks evidence that fibrolytic and amylolytic enzymes can affect nutrient digestibility, ruminal fermentation, and performance of mid-lactation cows.