Effects of a commercial fermentation byproduct or urea on milk production, rumen metabolism, and omasal flow of nutrients in lactating dairy cattle.

The objective of this study was to evaluate the effects of a fermentation byproduct on rumen fermentation and microbial yield in high producing lactating dairy cattle. Eight ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 60 ± 10 d in milk and 637 ± 38 kg of body weight were assigned to 1 of 2 treatment sequences in a switchback design. Treatment diets contained (dry matter basis) 44% corn silage, 13% alfalfa silage, 12% ground corn, and 31% premix containing either a control mix of urea and wheat middlings (CON) or a commercial fermentation byproduct meal (Fermenten, Arm and Hammer Animal Nutrition, Princeton, NJ) at 3% diet inclusion rate (EXP). Diets were formulated to be isonitrogenous and isocaloric, with similar levels of neutral detergent fiber and starch. The trial consisted of three 28-d experimental periods, where each period consisted of 21 d of diet adaptation and 7 d of data and sample collection. Omasal nutrient flows were determined using a triple-marker technique and double-labeled 15N15N-urea. The EXP diet provided 18 g/d more nonammonia N versus the CON diet, representing 3.0% of total N intake. Energy-corrected milk yield (41.7 and 43.1 kg/d for CON and EXP, respectively), milk fat, and protein yield and content did not differ between treatments. Total dry matter intake was similar between treatments (25.5 and 26.4 kg/d for CON and EXP, respectively). Ammonia N concentration and pool size in the rumen was greater in cows fed the EXP diet. No differences were observed in rumen or total-tract dry matter, organic matter, or neutral detergent fiber digestibility. Ruminal degradation of feed N was 15% lower in cows fed EXP diets, resulting in differences in omasal N flows. Results demonstrated the fermentation byproduct meal had a sparing effect on degradable feed protein, but did not increase microbial N flow from the rumen.

Rumen digestion kinetics, microbial yield, and omasal flows of nonmicrobial, bacterial, and protozoal amino acids in lactating dairy cattle fed fermentation by-products or urea as a soluble nitrogen source.

The objective of this study was to evaluate the effect of a fermentation by-product on rumen function, microbial yield, and composition and flows of nutrients from the rumen in high-producing lactating dairy cattle. Eight ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 60 ± 10 d in milk and 637 ± 38 kg of body weight were randomly assigned to 1 of 2 treatment sequences in a switchback design. Treatment diets contained (dry matter basis) 44% corn silage, 13% alfalfa silage, 12% ground corn, and 31% protein premix, containing either a control mix of urea and wheat middlings (CON) or a commercial fermentation by-product meal (Fermenten, Arm and Hammer Animal Nutrition, Princeton, NJ) at 3% diet inclusion rate (EXP). The trial consisted of three 28-d experimental periods, where each period consisted of 21 d of diet adaptation and 7 d of data and sample collection. A triple-marker technique and double-labeled 15N15N-urea were used to were used to measure protozoal, bacterial, and nonmicrobial omasal flow of AA. Rumen pool sizes and omasal flows were used to determine digestion parameters, including fractional rates of carbohydrate digestion, microbial growth, and yield of microbial biomass per gram of degraded substrate. Fermentation by-product inclusion in EXP diets increased microbial N and amino acid N content in microbes relative to microbes from CON cows fed the urea control. Microbial AA profile did not differ between diets. Daily omasal flows of AA were increased in EXP cows as a result of decreased degradation of feed protein. The inclusion of the fermentation by-product increased nonmicrobial AA flow in cows fed EXP versus CON. Average protozoal contribution to microbial N flow was 16.8%, yet protozoa accounted for 21% of the microbial AA flow, with a range of 8 to 46% for individual AA. Cows in this study maintained an average rumen pool size of 320 g of microbial N, and bacterial and protozoal pools were estimated at 4 different theoretical levels of selective protozoa retention. Fractional growth rate of all microbes was estimated to be 0.069 h-1, with a yield of 0.44 g of microbial biomass per gram of carbohydrate degraded. Results indicated that fermentation by-product can increase omasal flow of AA while maintaining adequate rumen N available for microbial growth and protein synthesis. Simulations from a developmental version of the Cornell Net Carbohydrate and Protein System indicated strong agreement between predicted and observed values, with some areas key for improvement in AA flow and bacterial versus protozoal N partitioning.

Exogenous fibrolytic enzymes and recombinant bacterial expansins synergistically improve hydrolysis and in vitro digestibility of bermudagrass haylage.

Four experiments were conducted to examine the effects of a recombinant bacterial expansin-like protein (BsEXLX1) from Bacillus subtilis and a commercial exogenous fibrolytic enzyme (EFE) preparation for ruminants on hydrolysis of pure substrates (cellulose and xylan) and in vitro digestibility of bermudagrass haylage (BMH). Recombinant Escherichia coli BL21 strain was used to express BsEXLX1; the protein was purified using an affinity column. In experiment 1, carboxymethylcellulose, Whatman #1 filter paper (General Electric, Boston, MA) and oat-spelt xylan substrates were subjected to 4 treatments (1) sodium citrate buffer (control), (2) BsEXLX1 (162 µg/g of substrate), (3) EFE (2.3 mg/g of substrate), and (4) EFE + BsELX1 in 3 independent runs. Samples were incubated at optimal conditions for both additives (pH 5 and 50°C) or at ruminal (pH 6 and 39°C) or ambient (pH 6 and 25°C) conditions for 24 h and sugar release was measured. In experiment 2, digestibility in vitro of BMH was examined after treatment with the following: (1) control (buffer only), (2) BsEXLX1 (162 µg/g of dry matter), (3) EFE (2.2 mg/g of dry matter), and (4) EFE + BsEXLX1 in 3 independent runs at 39°C for 24 h. Experiment 3 examined effects of EFE and BsEXLX1 on simulated preingestive hydrolysis and profile of released sugars from BMH after samples were suspended in deionized water with sodium azide at 25°C for 24 h in 2 independent runs. In experiment 4, the sequence of the BsEXLX1 purified protein was compared with 447 ruminal bacterial genomes to identify similar proteins from the rumen. In experiment 1, compared with EFE alone, EFE and BsEXLX1 synergistically increased sugar release from carboxymethylcellulose and Whatman #1 filter paper under all simulated conditions; however, hydrolysis of xylan was not improved. In experiment 2, compared with EFE alone, treatment with EFE and BsEXLX1 increased neutral detergent fiber and acid detergent fiber digestibility of bermudagrass haylage (by 5.5 and 15%, respectively) and total volatile fatty acid concentrations, and decreased acetate-propionate ratio. In experiment 3, compared with EFE alone. The EFE and BsEXLX1 synergistically reduced concentrations of neutral detergent fiber and acid detergent fiber and increased release of sugars by 9.3%, particularly cellobiose (72.5%). In experiment 4, a similar sequence to that of BsEXLX1 was identified in Bacillus licheniformis, and similar hypothetical protein sequences were identified in Ruminococcus flavefaciens strains along with different protein structures in E. xylanophilum and Lachnospiraceae. This study showed that an expansin-like protein synergistically increased the hydrolysis of pure cellulose substrates and the hydrolysis and digestibility in vitro of BMH.

Effects of the dose and viability of Saccharomyces cerevisiae. 1. Diversity of ruminal microbes as analyzed by Illumina MiSeq sequencing and quantitative PCR.

This study was conducted to examine effects of the dose and viability of supplemental Saccharomyces cerevisiae on the ruminal fermentation and bacteria population and the performance of lactating dairy cows. Four ruminally cannulated lactating cows averaging 284±18d in milk were assigned to 4 treatments arranged in a 4×4 Latin square design with four 21-d periods. Cows were fed a total mixed ration containing 41.7% corn silage, 12.1% brewer's grains, and 46.2% concentrate on a dry matter basis. The diet was supplemented with no yeast (control) or with a low dose of live yeast (5.7×107 cfu/cow per day; LLY), a high dose of live yeast (6.0×108 cfu/cow per day; HLY), or a high dose of killed yeast (6.0×108 cfu/cow per day; HDY). Microbial diversity was examined by high-throughput Illumina MiSeq sequencing (Illumina Inc., San Diego, CA) of the V4 region of the 16S rRNA gene. The relative abundance of select ruminal bacteria was also quantified by quantitative PCR (qPCR). Adding LLY to the diet increased the relative abundance of some ruminal cellulolytic bacteria (Ruminococcus and Fibrobacter succinogenes) and amylolytic bacteria (Ruminobacter, Bifidobacterium, and Selenomonas ruminantium). Adding live instead of killed yeast increased the relative abundance of Ruminococcus and F. succinogenes; adding HDY increased the relative abundance of Ruminobacter, Bifidobacterium, Streptococcus bovis, and Selenomonas ruminantium. The most dominant (≥1% of total sequences) bacteria that responded to LLY addition whose functions are among the least understood in relation to the mode of action of yeast include Paraprevotellaceae, CF231, Treponema, and Lachnospiraceae. Future studies should aim to speciate, culture, and examine the function of these bacteria to better understand their roles in the mode of action of yeast. A relatively precise relationship was detected between the relative abundance of F. succinogenes (R2=0.67) from qPCR and MiSeq sequencing, but weak relationships were detected for Megasphaera elsdenii, Ruminococcus flavefaciens, and S. ruminantium (R2≤0.19).

Ruminal bacteria and protozoa composition, digestibility, and amino acid profile determined by multiple hydrolysis times.

Microbial samples from 4 independent experiments in lactating dairy cattle were obtained and analyzed for nutrient composition, AA digestibility, and AA profile after multiple hydrolysis times ranging from 2 to 168 h. Similar bacterial and protozoal isolation techniques were used for all isolations. Omasal bacteria and protozoa samples were analyzed for AA digestibility using a new in vitro technique. Multiple time point hydrolysis and least squares nonlinear regression were used to determine the AA content of omasal bacteria and protozoa, and equivalency comparisons were made against single time point hydrolysis. Formalin was used in 1 experiment, which negatively affected AA digestibility and likely limited the complete release of AA during acid hydrolysis. The mean AA digestibility was 87.8 and 81.6% for non-formalin-treated bacteria and protozoa, respectively. Preservation of microbe samples in formalin likely decreased recovery of several individual AA. Results from the multiple time point hydrolysis indicated that Ile, Val, and Met hydrolyzed at a slower rate compared with other essential AA. Singe time point hydrolysis was found to be nonequivalent to multiple time point hydrolysis when considering biologically important changes in estimated microbial AA profiles. Several AA, including Met, Ile, and Val, were underpredicted using AA determination after a single 24-h hydrolysis. Models for predicting postruminal supply of AA might need to consider potential bias present in postruminal AA flow literature when AA determinations are performed after single time point hydrolysis and when using formalin as a preservative for microbial samples.

Association of aqueous hydrogen concentration with methane production in continuous cultures modulated to vary pH and solids passage rate.

The objective of this study was to evaluate the effects of altering pH and solids passage rate (kp) on concentration of aqueous H2 [H2(aq)], CH4 production, volatile fatty acids (VFA) production, and fiber digestibility in a continuous culture fermentation system. The present study was conducted as a 2 × 2 factorial treatment arrangement in a Latin square design using continuous culture fermentors (n = 4). Our continuous culture system was converted to a closed system to measure CH4 and H2 emission while measuring H2(aq) concentration and VFA production for complete stoichiometric assessment of fermentation pattern. Treatments were control pH (CpH; ranging from 6.3 to 6.9) or low pH (LpH; 5.8 to 6.4) factorialized with solids kp that was adjusted to be either low (Lkp; 2.5%/h) or high (Hkp; 5.0%/h); liquid dilution was maintained at 7.0%/h. Fermentors were fed once daily (40 g of dry matter; 50:50 concentrate:forage diet). Four periods lasted 10 d each, with 3 d of sample collection. The main effect of LpH increased nonammonia nitrogen flow, and both LpH and Hkp increased nonammonia nonbacterial N flow. We observed a tendency for Hkp to increase bacterial N flow per unit of nonstructural carbohydrates and neutral detergent fiber degraded. The main effect of LpH decreased H2(aq) by 4.33 µM compared with CpH. The main effect of LpH decreased CH4 production rate from 5 to 9 h postfeeding, and Hkp decreased CH4 production rate from 3 to 9 h postfeeding. We found no effect of LpH on daily CH4 production or CH4 produced per gram of neutral detergent fiber degraded, but Hkp decreased daily CH4 production by 33.2%. Both the main effects of LpH and Hkp decreased acetate molar percentage compared with CpH and Lkp, respectively. The main effect of both LpH and Hkp increased propionate molar percentage, decreasing acetate-to-propionate ratio from 2.62 to 2.34. We noted no treatment effects on butyrate molar percentage or total VFA production. The results indicate increasing kp and decreasing pH decreased acetate-to-propionate ratio, but only increasing kp decreased CH4 production; lack of differences for LpH might be a result of compensatory methanogenesis during the second half of the day postfeeding.

Invited review: ruminant ecology and evolution: perspectives useful to ruminant livestock research and production.

The article reviews ruminant ecology and evolution and shows insights they offer into livestock research. The first ruminants evolved about 50 million years ago and were small (<5 kg) forest-dwelling omnivores. Today there are almost 200 living ruminant species in 6 families. Wild ruminants number about 75 million, range from about 2 to more than 800 kg, and generally prefer at least some browse in their diets. Nine species have been domesticated within the last 10,000 yr. Their combined population currently numbers 3.6 billion. In contrast to wild ruminants, domestic species naturally prefer at least some grass in their diets, are of large body weight (BW; roughly from 35 to 800 kg), and, excepting reindeer, belong to one family (Bovidae). Wild ruminants thus have a comparatively rich ecological diversity and long evolutionary history. Studying them gives a broad perspective that can augment and challenge the status quo of ruminant research and production. Allometric equations, often used in ecology, relate BW to physiological measurements from several species (typically both wild and domestic). They are chiefly used to predict or explain values of physiological parameters from BW alone. Results of one such equation suggest that artificial selection has increased peak milk energy yield by 250% over its natural level. Voluntary feed intake is proportional to BW(0.9) across wild and domestic ruminant species. This proportionality suggests that physical and metabolic factors regulate intake simultaneously, not mutually exclusively as often presumed. Studying the omasum in wild species suggests it functions primarily in particle separation and retention and only secondarily in absorption and other roles. Studies on the African Serengeti show that multiple species, when grazed together, feed such that they use grasslands more completely. They support the use of mixed-species grazing systems in production agriculture. When under metabolic stress, wild species will not rebreed, but rather will extend lactation (to nourish their current offspring). This bolsters the suggestion that lactation length be extended in dairy operations. Cooperation between animal scientists and ecologists could generate more valuable insight.

Variability in in situ ruminal degradation parameters causes imprecision in estimated ruminal digestibility.

The ruminal digestibility of dietary ingredients is frequently estimated with degradation parameters (e.g., rate and extent of degradation). Mean degradation parameters (e.g., those in a feed library) are often used, but limited data suggest considerable variation around these means, potentially leading to imprecise digestibility estimates. This experiment quantified degradation parameter variation for forage hays and determined the impact of this variation on the precision of ruminal digestibility estimates. Degradation data were those previously published by our laboratory and included degradation rate, lambda(d) (h(-1)), fraction instantly degraded, a (g x g(-1)), potential extent of degradation, (a + b) (g x g(-1)), and fraction not instantly degraded that is potentially degradable, b (g x g(-1)) of alfalfa, grass, and grass-legume hays. Ruminal digestibilities of chemical fractions (dry matter, neutral detergent fiber, acid detergent fiber, hemicellulose, crude protein) were estimated using these data. Ninety-five percent confidence limits of digestibility were determined using propagation of uncertainty with measured standard deviations of degradation parameters. Values for coefficients of variation of degradation parameters were large; averaged across chemical fractions, they were 24.8, 28.6, 20.7, and 12.6% for lambda(d), a, b, and (a + b). Ninety-five percent confidence limits of digestibility were large (80.5% of digestibility means) and often overlapped each other, even when digestibility means differed greatly numerically. Consequently, digestibility values computed with mean degradation parameters may have little biological and practical significance. When uncertainty in all parameters but lambda(d) was set to zero (lambda(d) alone had uncertainty), 95% confidence limits still encompassed 54.5% of digestibility means. Thus, uncertainty in lambda(d) alone caused considerable imprecision in estimated digestibility. These results caution against using mean degradation parameters to estimate digestibility.

A mechanistic model for predicting intake of forage diets by ruminants.

Accurate voluntary feed intake (VFI) prediction is critical to the productivity and profitability of ruminant livestock production systems. Simple empirical models have been used to predict VFI for decades, but they are inflexible, restrictive, and poorly accommodate many feeding conditions, such as those of developing countries. We have developed a mechanistic model to predict VFI over a range of forage diets (low- and high-quality grasses and legumes) by wild and domestic ruminants of varying physiological states (growth, lactation, gestation, nonproductive). Based on chemical reactor theory, the model represents the reticulorumen, large intestine, and blood plasma as continuous stirred-tank reactors and the small intestine as a plug flow reactor. Predicted VFI is that which 1) fulfills an empirical relationship between chemostatic and distention feedback observed in the literature, and 2) leads to steady-state conditions. Agreement between observed and actual VFI was great (generally R(2) >0.9, root mean square prediction error <1.4 kg/d, CV <25%). Root mean square prediction error for our model was only 67% that of the Beef NRC (2000) model, the leading empirical prediction system for cattle. Together, these results demonstrate that our model can predict ruminant VFI more broadly and accurately than prior methods and, by consequence, serve as a crucial tool to ruminant livestock production systems.

Comparing relative feed value with degradation parameters of grass and legume forages.

Relative feed value (RFV) was evaluated relative to in situ degradation parameters of grass and legume forages. Early-cut alfalfa (n = 20), late-cut alfalfa (n = 26), cool-season grass (n = 11), warm-season grass (n = 4), and grass-legume (n = 20) samples were collected from duplicate hay bales submitted to the 2002 and 2003 Missouri State Fair Hay Contests. Subsamples were incubated in the rumen of 2 lactating Holstein cows for 0, 6 or 8, 12, 24, and 48 h to determine in situ degradation of DM, ADF, NDF, CP, and hemicellulose over time. Degradation data were fit to a variety of candidate models to estimate degradation parameters. Correlation coefficients between degradation parameter estimates [sorted according to forage (early-cut alfalfa, late-cut alfalfa, grass, or grass-legume)] and RFV were determined. For further comparison, correlations between NDF degradation parameter estimates and digestible DMI were determined with data from a previous study. Degradation data were best fit to a single, gamma 2-distributed pool model without a lag phase. Relative feed value was significantly correlated (P < 0.05) with potentially digestible DM and CP for early-cut alfalfa, potentially digestible DM for late-cut alfalfa, and potentially digestible DM, NDF, and hemicellulose for grass-legume. The percentage of significant correlations (10.7%) across the entire data set was low and no correlations were significant for grass. Relative feed value did not account for the variation in degradation parameters, especially for grasses. A further correlation analysis, which compared digestible DMI with degradation parameter estimates reported from another data set, revealed that digestible DMI and degradation parameter estimates were related for grass but not for alfalfa forages. These results suggest that RFV is limited by its failure to include degradation parameters.