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Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.

Transient changes in the dry matter (DM) concentration of silages often occur, which will cause transient changes in the ration. To determine the effects of a transient change in silage DM, 24 Holstein cows (116 d in milk) were used in an 8 replicated 3×3 Latin square design with 21-d periods. Treatments were (1) control, (2) unbalanced (UNBAL), and (3) balanced (BAL). The control diet was designed to have a consistent day-to-day forage:concentrate ratio of 55:45 on a DM basis. The UNBAL and BAL diets were the same as the control diet for most of the period except during two 3-d bouts when water was added to the silage (simulating a rain event) to cause a 10-percentage unit decrease in silage DM concentration. During the bouts, the UNBAL diet was the same as that of the control on an as-fed basis, but on a DM basis, the forage:concentrate ratio decreased to 49:51, which reduced dietary concentrations of DM (63.9 vs. 66.2%) and forage NDF (21.0 vs. 23.6%), and increased starch (30.4 vs. 28.4%). The BAL treatment corrected for the change in silage DM by an increase in the inclusion of wet silage and had the same composition as the control diet on a DM basis, except for ration DM (66.2 vs. 63.9%). Over the 21-d period, treatment did not affect DM intake (DMI; 24.0 kg/d); however, DMI of cows on the UNBAL and BAL treatments tended to decrease during the wet bouts, especially during the second bout. The day following both bouts, DMI of cows fed BAL and UNBAL diets were greater than that of cows fed the control diet, which contributed to the lack of a treatment effect on DMI over the entire period. Milk production was greater for the UNBAL than control cows (39.8 vs. 39.3 kg/d) over the 21-d period. That difference was largely caused by increased milk yield during the first bout by cows on the UNBAL diet. Over the 21-d period, milk yield did not differ between control and BAL cows. Some small differences in milk fat and protein concentrations (≤ 0.1 percentage units) were observed between treatments. Total-tract digestibility of most nutrients was not affected by treatment. Overall, a 10-percentage unit decrease in silage DM over short-term bouts (with or without total mixed ration adjustment) had only minor effects on DMI, milk yield, and composition.

A method that accurately quantifies changes in the supply of metabolizable Met following a dietary change will allow accurate economic comparisons of various Met sources. This paper describes a novel method of estimating the relative supply of metabolizable Met based on changes in the concentration of Se in milk. Selenized yeast (Se-yeast) contains selenomethionine (Se-met) and because Se-met and Met are indistinguishable by cells, Se-met can be used as a tracer of Met. We hypothesized that if the size of the Met pool was increased but intake of Se-met was constant, the concentration of Se in milk relative to milk Met would decrease. To test this hypothesis, 6 Holstein cows were fed a diet that contained 0.3 mg of Se from Se-yeast/kg of diet DM and then in a 2-period crossover experiment, were abomasally infused with water (control) or an aqueous solution that provided 9 g of Met/d. Milk was sampled during the infusion and the specific activity (SA) of milk (Se concentration divided by Met concentration) was calculated for each treatment. The SA in milk from Met-infused cows was divided by SA in milk from control cows to calculate the change in supply of metabolizable Met. As hypothesized, infusing Met reduced the SA of milk (84.7 vs. 72.5 microg of Se/mg of Met). The calculated flow of metabolizable Met was 17% greater when cows were infused with 9 g of Met/d compared with cows infused with water (essentially the same difference was measured using SA calculated with N concentrations of milk). Assuming the infused Met was 100% absorbed, the flow of metabolizable Met for control cows was 9/0.17 = 53 g of Met/d, which agreed well with literature data and estimates derived from common nutritional models.

Development of antimicrobial resistance in food animals receiving antimicrobials has been well documented among bacterial isolates, especially pathogens, but information on development of antimicrobial resistance at the microbial community level during long-term feeding of antimicrobials is lacking. The objective of this study was to examine the association between inclusion of tylosin in feed and occurrence of resistance to macrolide-lincosamide-streptogramin B (MLSB) in the entire fecal microbial communities of beef cattle over a feeding study of 168 d. A completely randomized design included 6 pens housed together in 1 barn, with each pen housing 10 to 11 steers. The control and tylosin groups each had 3 pens, with the former receiving no antimicrobial whereas the latter received both tylosin and monensin (11 and 29.9 mg/ kg of feed, respectively, DM) in feed. The abundance of genes conferring resistance to MLSB (erm genes) and tetracyclines (tet genes) were quantified using class-specific, real-time PCR assays. The abundances of erm and tet genes were analyzed with pens as experimental units using the MIXED procedure of SAS. Correlations between abundance of different resistance genes were calculated using the CORR procedure of SAS. We identified 4 classes (B, F, T, and X) of erm genes in fresh fecal samples collected at wk 2, 17, and 21 of feeding. From wk 2 to 17, the abundance of erm(T) and erm(X) increased (P < 0.05), whereas that of erm(B) and erm(F) did not. The abundance of the erm genes did not further change from wk 17 to 21. The tet(A/C), tet(G), and tet gene variants encoding ribosomal protection proteins (including classes M, O, P, Q, S, T, and W) appeared to be co-selected by tylosin feeding. Such co-selection of multiresistance at community level by one antimicrobial drug used in animals has the important implication that future studies should examine resistance to not only the antimicrobials used in animals, but also other antimicrobials, especially those used in human medicine, to fully assess the potential risk associated with antimicrobial use in animals. Both the erm and tet genes appeared to be disseminated among the microbial populations in all steers housed together.

Biohydrogenation (BH) of fatty acids (FA) from fresh alfalfa and alfalfa hay with varying levels of supplemental sucrose and media pH was evaluated in vitro. A multicompartmental model was then developed to estimate pool size and flux of vaccenic acid (VA) during BH of FA in fresh alfalfa. To vary incubation pH, alfalfa samples were inoculated with rumen fluid in 2 media differing in molarity of the bicarbonate buffer. Samples were incubated for 0, 1, 2, 3, 4, 6, 9, and 12 h; pH was measured and tubes were put in ice and stored until analysis. The BH rates of linoleic acid (18:2) and linolenic acid (18:3) were estimated by PROC NLIN of SAS (single pool, first-order kinetic model) and SAAM II (multiple pools, first-order kinetic model). Both methods gave similar estimates for the BH rates of 18:2 and 18:3 as well as the temporal pool size of VA. The BH rates (%/h) in the strong (SB) and weak buffers (WB) were 27.4 (+/-0.7) and 23.5 (+/-0.9) for 18:2, and 43.8 (+/-0.2) and 30.3 (+/-0.6) for 18:3, respectively. The WB decreased the BH rates of 18:2 and 18:3 for both forage sources. However, BH rates of 18:3 were higher from fresh alfalfa than alfalfa hay. There was no effect of sucrose addition on the BH rates of 18:2 and 18:3. Moreover, there was no effect of buffer on the BH of VA estimated by the multiple pools model between the SB and WB (12.5 +/- 2.1 and 14.1 +/- 3.7%/h, respectively). The BH rates of the conjugated linoleic acid isomers were not different between the SB and WB treatments (36.7 +/- 19.8 and 25.9 +/- 27.2, respectively). Because we could estimate fluxes as well as mass of the VA pools, more information was generated from the data when a multiple pools model was used compared with a single pool, first-order kinetic model.

Methionine supplemented as 2-hydroxy-4-(methylthio)-butanoic acid (HMB) has been suggested to alter bacterial or protozoal populations in the rumen. Our objective was to determine if source of Met would change microbial populations in the rumen and to compare those results to samples from the omasum. The ruminal and omasal samples were collected from cows fed control (no Met), dl-Met, HMB, or the isopropyl ester of HMB (HMBi; estimated 50% rumen protection) in a replicated 4×4 Latin square design. In one square, changes in protozoal populations were determined using microscopic counts and denaturing gradient gel electrophoresis (DGGE), whereas changes in bacterial populations were determined using DGGE and ribosomal intergenic spacer length polymorphism (RIS-LP). Neither the protozoal counts nor the DGGE banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by both DGGE and RIS-LP, bacterial populations clustered by treatments in ruminal and especially in omasal samples. Using cows from both Latin squares, the flow of protozoal cells from the rumen was quantified by multiplying protozoal cell count in omasal fluid by the omasal fluid flow (using CoEDTA as a liquid flow marker) or was estimated by rumen pool size of cells multiplied by either the ruminal dilution rate of CoEDTA (after termination of CoEDTA dosing) or the passage rate of Yb-marked particles. Compared with the omasal fluid flow measurement (16.4h), protozoal generation time was approximated much more closely using the particulate than the fluid passage rate from the rumen (generation times of 15.7 and 7.5h, respectively). There seems to be minimal selective retention of protozoal genera in the rumen in dairy cattle fed every 2h. Data support the validity of the omasal sampling technique under our conditions.

The objective of this analysis was to examine the intestinal digestibility of individual long-chain fatty acids (FA) in lactating dairy cows. Available data were collated from 15 publications containing 61 treatments, which reported total and individual FA duodenal flows and calculations of intestinal digestibility. All studies involved lactating dairy cows, and estimates of digestibility were based on measurements either between the duodenum and ileum (18 treatments) or between the duodenum and feces (43 treatments). Fatty acid digestibility was calculated for C16:0, C18:0, C18:1 (cis and trans isomers), C18:2, and C18:3. Digestibility of C18:0 was lower than for C18:1 and C18:3, with no difference in digestibility between saturated FA (C16:0 and C18:0). We weighted the studies by the reciprocal of the variance to generate best-fit equations to predict individual FA digestibility based on duodenal flow of FA and dietary independent variables. The flow of C18:0 negatively affected the digestibility of C18:0 and was also included in the best-fit equations for all other 18-carbon FA using duodenal flow characteristics. The type of fat supplemented had an effect on digestibility of individual FA, with whole seeds having reduced digestibility. Our meta-analysis results showed minimal differences in the digestibility of individual FA. However, C18:0 flow through the duodenum had a negative effect on the digestibility of several individual FA, with the largest negative effect on C18:0 digestibility. The mechanisms that reduce C18:0 absorption at high concentrations are unknown and warrant further investigation.

An important objective is to identify nutrients or dietary factors that are most critical for advancing our knowledge of, and improving our ability to predict, milk protein production. The Dairy NRC (2001) model is sensitive to prediction of microbial protein synthesis, which is among the most important component of models integrating requirement and corresponding supply of metabolizable protein or amino acids. There are a variety of important considerations when assessing appropriate use of microbial marker methodology. Statistical formulas and examples are included to document and explain limitations in using a calibration equation from a source publication to predict duodenal flow of purine bases from measured urinary purine derivatives in a future study, and an improved approach was derived. Sources of specific carbohydrate rumen-degraded protein components probably explain microbial interactions and differences among studies. Changes in microbial populations might explain the variation in ruminal outflow of biohydrogenation intermediates that modify milk fat secretion. Finally, microbial protein synthesis can be better integrated with the production of volatile fatty acids, which do not necessarily reflect volatile fatty acid molar proportions in the rumen. The gut and splanchnic tissues metabolize varying amounts of volatile fatty acids, and propionate has important hormonal responses influencing milk protein percentage. Integration of ruminal metabolism with that in the mammary and peripheral tissues can be improved to increase the efficiency of conversion of dietary nutrients into milk components for more efficient milk production with decreased environmental impact.

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation (R(2)=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner-Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.

2-Hydroxy-4-(methylthio) butanoic acid (HMB) positively affects milk composition and yield, potentially through ruminal actions. Four continuous culture fermenters were used to determine the optimal concentration of HMB for digestibility of organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose and synthesis of microbial N. A highly degradable mix of hay and grain was used as a basal diet to simulate a typical lactation diet. Three concentrations of HMB (0, 0.055, and 0.110%) and one concentration of dl-Met (0.097%) were infused into the fermenters according to a 4 x 4 Latin square design. Digesta samples were collected during the last 3 d of each of the four 10-d experimental periods. Digestibility of OM, hemicellulose, and NDF was largely insensitive to treatment. Digestibility of ADF showed a quadratic effect to supplementation of HMB, with 0.055% having lower digestibility than 0 or 0.110%. Total production of VFA was not influenced by HMB supplementation, but differences in concentration and production of individual VFA were seen. Isobutyrate increased linearly with increasing HMB supplementation. Propionate concentration decreased linearly with increased HMB supplementation, but propionate production showed a quadratic trend (P = 0.13). A higher concentration of acetate was detected for dl-Met compared with the highest HMB concentration. There were trends (P < 0.15) for dl-Met to decrease the production of isobutyrate and to lower the concentration of butyrate when compared with HMB. Microbial efficiency was not different among treatments. The proportion of bacterial N produced from NH3-N decreased linearly with increasing HMB, and bacteria receiving dl-Met synthesized more N from NH3-N than those receiving HMB. These data suggest that supplementation of HMB may have a sparing effect on branched chain volatile fatty acids because the fatty acids are not needed to provide carbon for synthesis of valine, isoleucine and leucine with ammonia. Comparisons of bacterial community structure in the fermenter effluent samples using PCR amplicons containing the ribosomal intergenic spacer region and its flanking partial 16S ribosomal RNA gene showed no distinct banding patterns, though treatments tended to group together. Both Met and HMB affect the rumen microbial population, but Met supplied as dl-Met does not act identically to that supplied as HMB.

Metabolizable protein (MP) supply and amino acid balance were manipulated through selection of highly digestible rumen-undegradable protein (RUP) sources and methionine (Met) supplementation. Effects on production efficiency and N utilization of lactating dairy cows were determined. Thirty-two multiparous (647 kg) and 28 primiparous (550 kg) Holstein cows were assigned during the fourth week of lactation to one of four dietary treatments. Treatments were 1) 18.3% crude protein (CP) with low estimated intestinal digestibility of RUP (HiCP-LoDRUP), 2) 18.3% CP with high digestibility RUP (HiCP-HiDRUP), 3) 16.9% CP with high digestibility RUP (LoCP-HiDRUP), and 4) 17.0% CP with high digestibility RUP and supplemental Met (LoCP-HiDRUP + Met). Diets were balanced to have equal concentrations of net energy for lactation (NE(L)), acid detergent fiber (ADF), neutral detergent fiber (NDF), and ash. Milk yields (40.8, 46.2, 42.9, 46.6 kg/d), protein percentages (2.95, 2.98, 2.99, 3.09%), and fat percentages (3.42, 3.64, 3.66, 3.73%) are reported here for HiCP-LoDRUP, HiCP-HiDRUP, LoCP-HiDRUP, and LoCP-HiDRUP + Met, respectively. Milk urea N and BUN decreased when feeding a lower CP diet. Efficiency of use of N for milk protein production was higher when feeding higher digestibility RUP, especially with the LoCP-HiDRUP + Met diet. A digestibility study followed the production trial, with six cows per treatment group continuing on the same treatment for an additional week. The experimental periods were 5 d long, with 1 d of adjustment and 4 d of total collection of urine and feces. Dry matter intake, milk production, milk protein production, and N digestibility were not significantly different among treatments during the collection trial, whereas N intake and N absorbed increased with the higher CP diets. The quantity of N in feces did not change with diet, but quantity of N in urine decreased in the low CP diets. Milk N as a percentage of intake N and milk N as a percentage of N absorbed showed a trend toward increasing as CP concentration in the diet decreased. The supplementation of Met did not improve the efficiency of N utilization during the digestibility study, in contrast to what was estimated during the production trial. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows during the production trial, indicating that postruminal digestibility of RUP and amino acid balance can be more important than total RUP supplementation.

Six ruminally and duodenally cannulated Holstein cows were used in a 6 × 6 Latin square design to 1) evaluate the potential interaction in effectiveness of neutral detergent fiber (NDF) from whole cottonseed (WCS) when it was substituted for forage NDF (FNDF) and fed with ground (G) or steam-flaked (SF) corn and 2) to determine whether the kinetic properties of NDF digestion further clarify the effectiveness of WCS. The six dietary treatments were: forage control with G corn (21% FNDF), 5% WCS with G or SF corn (18% FNDF), 10% WCS with G or SF corn (15% FNDF), and 15% WCS with G corn (12% FNDF). Based on chewing activity, the NDF from WCS was estimated to be 84% (SE = 36%) as effective as alfalfa silage NDF. Decreasing passage and digestion rates of potentially digestible NDF with increasing WCS increased the evacuated pool size of ruminal DM, apparently explaining the similar ruminal mat consistency among treatments. Measures of effectiveness of WCS treatments did not interact with corn source. Fluid dilution rate was estimated based on a two-compartment model describing Co dilution, but no treatment differences were detected. There was a strong linear bias for estimates of ruminal NDF digestibility based on a single compartment model using the digestion rate of potentially digestible NDF and the passage rate of either indigestible NDF or digestible NDF when compared with NDF digestibility calculated using duodenal flows. Although further verification is needed, these digestion and passage kinetics help explain why WCS are effective at stimulating chewing during eating and rumination.

The nutrient composition of diets can vary from batch to batch because of variation in the nutrient composition of ingredients. The concentration of fat in distillers grains can be highly variable and, coupled with a high dietary inclusion rate, can result in substantial variation in the fat concentration of the total diet. Our hypothesis was that variation in dietary fat concentrations over short periods would have negative effects on production measures of dairy cattle. Twenty-four Holstein cows were used in 8 truncated Latin squares (3 cows × 2 periods). Periods were 16 d long, with a 12-d washout period separating the 2 periods. The 3 treatments were 1) control, 2) moderate variation, and 3) high variation. The control treatment was a consistent diet over the 16 d and contained 5.8% (dry basis) total long-chain fatty acids (LCFA), 23.7% distillers grains, and 1.1% of dry matter as corn oil. The average concentration of LCFA over the 16-d period for the moderate variation and high variation treatments was also 5.9%, but concentrations of LCFA varied over time by the addition or removal of corn oil. The moderate variation treatment had a 4-d phase of 5.4% LCFA, followed by a 4-d phase with 6.4% LCFA to complete the cycle, and then the cycle was repeated. The high variation treatment followed the same cycle pattern, but concentration of LCFA was either 4.8 or 7.0%. Over the 16 d, dry matter intake and milk yield were significantly decreased by the high variation treatment (intake = 21.7, 21.8, and 20.7 kg/d; milk = 36.4, 37.6, and 35.4 kg/d for the control, moderate variation, and high variation treatments). The effect of variation appeared to be cumulative; differences were not significant in the first 8-d cycle but were in the second 8-d cycle. Milk fat concentration was low (2.4%) and was not affected by treatment or cycle × treatment. The concentration of trans-10 C18:1 in milk fat was very high (4.2% of total milk fatty acids) but was not affected by treatment. However, cows on the high variation treatment had higher concentrations of trans-10, cis-12 C18:2 in milk fat (0.036, 0.042, and 0.047% of milk fatty acids for the control, moderate variation, and high variation treatments). Overall, an extreme short-term fluctuation in dietary fat concentration was needed before negative effects were observed.

The effect of additional dietary potassium in early lactation dairy cows was evaluated with the addition of potassium carbonate sesquihydrate, which increased dietary K from 1.3 to 2.1% of dry matter (DM) from wk 3 to 12 of lactation. Cows fed potassium carbonate sesquihydrate in the form of DCAD Plus (Church & Dwight Co. Inc., Princeton, NJ) had increased DM intake, milk fat percentage and yield, energy-corrected milk, and efficiency of milk production per unit of DM intake. Milk fat of cows fed higher dietary K had a lower concentration of trans fatty acids, suggesting a role for potassium carbonate sesquihydrate in the rumen in the biohydrogenation processes converting linoleic to stearic acid. Cows fed the diet with 2.1% K had greater apparent balance of K, and no effects were noted on the concentration of blood Mg or amount of fecal Mg. The data support the feeding of greater amounts of K in the early lactation cow.

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum , a mix of Entodinium species, Epidinium caudatum , or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25μ M ) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5μ M ). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture×treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

Pelleting cottonseed (CS) improves handling characteristics. Our objectives were to determine whether increasing the particle size of the CS pellet or dilution of a smaller pellet with delinted CS would limit the rate of CS oil release to optimize digestibility of fatty acids (FA) and fiber while maintaining milk fat production. In a 5 x 5 Latin square design with 3-wk periods, 5 rumen-cannulated cows were fed 1) control with CS hulls (CSH) and CS meal plus tallow and Ca soaps of FA, 2) whole CS (WCS), 3) small CS pellets (SP; 0.44-cm die diameter), 4) larger CS pellets (LP; 0.52-cm die diameter), or 5) a blend of 1/2 SP plus 1/2 partially delinted CS (SPD). Diets contained 39.6% concentrate, 14.4% CS, and 46% forage (40:60, alfalfa hay:corn silage) on a DM basis and were balanced to have similar concentrations of CS protein, CS fiber, and total fat. In a production trial, dietary treatments were 1) WCS control, 2) LP, 3) SPD, and 4) SPD fed at 90%. Sixty cows averaging 105 d in milk were fed the WCS diet for 2 wk and then assigned to one of the 4 diets for 12 wk. Total tract digestibility of NDF was unaffected, but N digestibility was lower for SPD than for other treatments. Fatty acid digestibility was higher for SP and LP (82.6 and 82.3%) than for CSH or SPD treatments (78.8 and 75.3%), and WCS was intermediate (81.1%). The trans-11 C18:1 from cows fed SP and LP (6.58 and 6.24% of total milk FA) was greater than that from cows fed CSH, WCS, and SPD (3.23, 3.79, and 3.97%). The trans-10 C18:1 in milk fat from SP and LP (0.508 and 0.511%) was higher than that in WCS and SPD diets (0.316 and 0.295%); CSH was intermediate (0.429%). Using passage rates estimated from the NRC, disappearance of total FA in situ was estimated to be 17.7, 44.2, 46.6, and 35.0% for WCS, SP, LP, and SPD, respectively. In the production trial, a diet x week interaction was explained by a trend for progressively greater milk production for SPD and SPD90 than for WCS or LP. Milk fat was lower for LP (2.74%) and SPD90 (2.85%) than for WCS or SPD (3.07 and 3.08%). The fat yield was lower for LP than for SPD (1.09 and 1.30 kg/d); WCS and SPD90 were intermediate (1.23 and 1.21 kg/d). Although having a lower FA digestibility, SPD appeared to minimize negative effects of free oil from SP in the rumen, explaining higher DMI and milk production compared with WCS or LP.

Accurate estimates of manure excretion are needed for planning manure storage facilities and for nutrient management. Data sets from metabolism studies conducted at several universities were compiled and evaluated for excretion of total manure, N, P, and K. Animal groups included calves weighing up to 204 kg, heifers weighing between 274 and 613 kg, nonlactating cows, and lactating cows. Regression equations were developed to predict excretion of total manure, total dry matter, N, P, and K. Predictors used in the regression equations for lactating cows included milk yield, percentages of protein and fat in milk, dietary concentrations of crude protein and neutral detergent fiber, and intakes of nutrients. The regression equations provide improved predictions of excretion and enable more accurate planning of manure storage and nutrients to be managed at the farm level.

The esterification of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) to isopropanol (HMBi) decreases the rate and extent of its ruminal breakdown. The modes of action of HMB and HMBi appear to be different. The quantification of the production response to HMBi has not been done. The objectives of this study were (1) to determine the lactation response to HMB, (2) to determine the lactation response to HMBi, and (3) to evaluate whether the response to HMBi is affected by HMB in the diet. Sixty-one Holstein cows (24 primiparous, 37 multiparous) were assigned to 1 of 4 dietary treatments 21 to 28 d after calving. The base diet consisted of [on a dry matter (DM) basis] 32.5% corn silage, 17.5% alfalfa hay, 10% whole cottonseed, and 40% of a pelleted concentrate made primarily of ground corn, soybean meal, and blood meal, and was fed for 16 wk as a control diet. To prepare the dietary treatments, the base diet was supplemented with 0.1% of diet DM with HMB (treatment 2), with 0.15% HMBi (treatment 3), or with 0.045% HMB and 0.15% HMBi (treatment 4). Results showed a significant increase in milk yield (2.9 kg/d), protein content (0.15%), protein yield (115 g/d), fat yield (165 g/d), and lactose yield (182 g/d) from HMBi. Supplementation of HMB had small and nonsignificant effects on milk yield and composition. There were no significant interaction effects of HMB with HMBi on any of the production traits measured in this experiment. Plasma free Met as a proportion of essential amino acids was increased by HMBi, but not by HMB. Dietary supplementation of HMBi increased gross N efficiency expressed as the proportion of ingested N secreted in milk. Consequently, HMBi significantly improved N efficiency.

Objectives were to quantify the ruminal effects and flows to the omasum of Met provided as 2-hydroxy-4-(methylthio)-butanoic acid (HMB), the isopropyl ester of HMB (HMBi), and DL-Met. Eight ruminally cannulated cows were used in a replicated 4 x 4 Latin square design. Treatments were 1) no Met (control), 2) HMB at 0.10% of DM, 3) HMBi at 0.13% of DM, and 4) DL-Met at 0.088% of DM. Diets were identical except for type of Met supplement and were based on corn silage and alfalfa hay at 30 and 13% of dietary DM, respectively. Samples of omasal fluid were used to determine the proportion of Met supplements passing out of the reticulorumen. Dry matter intake (20.1 kg/d) was restricted during the week of sampling to a maximum of 95% of ad libitum DMI determined during the first 2 wk of the period. Milk yields (37.7 +/- 0.8 kg/d) and fat concentration (3.42 +/- 0.15%) were not significantly different for control, HMB, HMBi, and DL-Met. Milk protein concentration (2.91, 2.95, 3.02, 2.96 +/- 0.07%, respectively) was significantly increased by the HMBi treatment. Rumen volatile fatty acids profile and NH3 concentrations were similar across treatments. Apparent ruminal digestibility of organic matter and neutral detergent fiber were higher for the three diets supplemented with Met sources than for the control diet. In situ rate of digestibility of CP from alfalfa hay, TMR, and corn silage was affected by Met sources. Passage rates of small particles (0.071/h) and fluid (0.167/h) were not affected by treatments. Protozoal counts in the rumen and omasum were not significantly affected by Met sources. Proportion of omasal N from bacterial N was not different (0.54 +/- 0.03), and bacterial N flow (305 +/- 24.4 g/d) was similar across treatments. The proportion of HMB that passed into the omasum was 5.3 +/- 1.5% of the amount consumed. Only a small amount (2.3%) of HMBi was found as HMB in the omasum. These results indicate that little HMB escapes ruminal degradation through passage to the omasum and that the site of HMBi absorption must be preomasal.

The objectives of this study were: 1) to evaluate the National Research Council equation used to predict microbial N flow to the duodenum in lactating cows, and 2) to determine whether improved equations could be developed by using dietary parameters used in the field. Treatment means from 55 trials with lactating and nonlactating cattle with duodenal cannulas were subjected to the backward elimination procedure of multiple regression. Variation within and among trials was accounted for by weighting the observations and including trial effects in all models. The equations to predict microbial N flow based on net energy for lactation (NE L ) intake were different from the equation based on NE L intake used by the dairy National Research Council. Dry matter intake (DMI) estimated microbial N flow as well as did NE L intake, indicating that DMI drives predictions based on NE L intake. When multiple dietary factors [i.e., DMI; dietary percentages of crude protein, forage, and neutral detergent fiber; and all two-way interactions] were included, the resulting equation [microbial N (grams per day) = 16.1 + 22.9 × DMI (kilograms per day) – 0.365 × DMI 2 – 1.74 × dietary neutral detergent fiber (percentage of dry matter)] tended to fit the data better than the equations based on NE L intake but not better than the equation based on DMI alone. The multiple-factor equation appeared to be the best overall equation for prediction; in contrast to the equation based on DMI, this equation is sensitive to diet composition. An asymptotic multiple-factor equation was developed, which may be more appropriate when extrapolating beyond the data range.

Carbohydrates are the major source of energy for dairy cows and for microbial protein synthesis in the rumen. The prediction of ruminal carbohydrate digestibility and of the flow of microbial protein to the small intestine is difficult because of the variability among various feeds in the kinetics of digestion and passage of neutral detergent fiber and starch. Disappearance of fiber and starch in vitro or in situ and gas production in vitro have been extensively evaluated, improved, and reviewed. Similarly, markers and models to measure ruminal passage rate have been extensively researched and improved. Sources of variation and decreased accuracy for these techniques are discussed. Variation and potential errors also remain for the prediction of microbial protein flow to the duodenum using in vivo procedures. However, when in vivo results were accumulated into a database, microbial N flow to the duodenum over a wide range of conditions could be predicted accurately by intake of net energy for lactation or by dry matter intake and percentage of neutral detergent fiber in the diet. Although evaluation of feeding interactions and specific dietary limitations for microbial protein production in the rumen are possible with some models but not with this regression approach, mechanistic models need further validation and more accurate rate constants for improved accuracy over a wide range of conditions.