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A study was conducted to evaluate whether the risk of acidosis in dairy cows can be lowered by increasing the physically effective fiber (peNDF) concentration of the diet, either through increased theoretical chop length of alfalfa silage or higher proportion of forage in the diet. The experiment was designed as a replicated 4 x 4 Latin square using 8 ruminally cannulated lactating dairy cows. Treatments were arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) forage:concentrate (F:C) ratios [dry matter (DM) basis]. Dietary peNDF concentration (DM basis) was determined from the sum of the proportion of dietary DM retained either on the 2 sieves (8 and 19 mm) or on the 3 sieves (1.18, 8, and 19 mm) of the Penn State Particle Separator multiplied by the neutral detergent fiber concentration of the diet. The dietary peNDF concentrations were altered by changing the F:C or the FPL, and ranged from 10.7 to 17.5% using 2 sieves, or from 23.1 to 28.2% using 3 sieves. Intake of peNDF was increased by increasing FPL but not by increasing F:C ratio because of the reduction of DM intake at the higher F:C ratio. Chewing activity, including number of chews and chewing time, increased with increasing F:C ratio or FPL. Mean ruminal pH was elevated by 0.4 and 0.2 units with increasing F:C ratio and FPL, respectively. Lowering the F:C ratio decreased the duration that ruminal pH was below 5.8 (1.2 vs. 8 h/d). Increased F:C ratio or FPL reduced ruminal volatile fatty acids concentration from 137 to 122 or from 133 to 126 mM, respectively, whereas acetate:propionate ratio was increased from 2.55 to 3.46 with increasing F:C ratio. Dietary peNDF concentration measured using 2 sieves was correlated to chewing time (r = 0.57) and mean ruminal pH (r = 0.75), whereas dietary peNDF concentration measured using 3 sieves was correlated to mean ruminal pH (r = 0.83) and negatively correlated to the time that pH was below 5.8 (r = -0.78). This study shows that the risk of ruminal acidosis is high for cows fed a low F:C diet. Increasing the proportion of forage in the diet helps prevent ruminal acidosis through increased chewing time, a change in meal patterns, and decreased ruminal acid production. Increasing FPL elevates ruminal pH, but in low forage diets, increased FPL does not alleviate subacute acidosis because the fermentability of the diet is high and changes in chewing activity are marginal. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of feeding essential oils from garlic (GAR) and juniper berry (JUN), or monensin (MO) on feed intake, ruminal fermentation, the site and extent of digestion, microbial protein synthesis, milk production, and immune status in dairy cows. Four midlactating Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square design with 21-d periods and 4 treatments: control (no additive), MO (330 mg/cow per d), GAR (5 g/cow per d), and JUN (2 g/cow per d). Cows were fed ad libitum a TMR consisting of 40% forage and 60% barley-based concentrate. Dry matter intake averaged 20.4 kg/d and was not affected by dietary additives. Total tract digestibilities of dry matter, organic matter, fiber, and starch were not affected by experimental treatments. However, ruminal digestibilities of dry matter and organic matter were higher (+13%) for GAR and JUN than for the control diet, mainly because of increased crude protein digestion in the rumen. Feeding GAR and JUN increased ruminal digestion of dietary protein by 11% as compared with the control. In contrast, ruminal digestion of dietary protein was reduced by 11% with MO as compared with the control. Milk fat content was lower for MO (2.68%) than for the GAR (3.46%), JUN (3.40%), and control (3.14%) diets. No effects of GAR, JUN, or MO were observed on milk production, ruminal microbial protein synthesis, ruminal pH, and ruminal concentrations of volatile fatty acids and ammonia N. The total and differential numbers of white blood cells as well as serum amyloid A and haptoglobin were not affected by the treatments, suggesting that additives had no effect on the immune status of cows. Results of this study indicate that supplementing dairy cows with GAR (5 g/d) and JUN (2 g/d) essential oils improved feed digestibility in the rumen, but possibly at the expense of a reduction in the flow of bypass protein to the small intestine. Feeding monensin could be beneficial in terms of increasing bypass protein from the rumen but did not improve feed digestion or milk production under the current experimental conditions [ABSTRACT FROM AUTHOR]

Intake of physically effective neutral detergent fiber (peNDF) of dairy cows was altered by adjusting the proportion of forage in the diet and forage particle length, and effects on nutrient intake, site and extent of digestion, microbial N synthesis, and milk production were measured. The experiment was designed as a triplicated 4 x 4 Latin square using 12 lactating dairy cows, with 4 that were ruminally and duodenally cannulated, 4 that were ruminally cannulated, and 4 that were intact. Thus, the site and extent of digestion, and microbial N synthesis were measured in a single 4 x 4 Latin square. Treatments were arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) forage:concentrate (F:C) ratios (dry matter basis). Dietary peNDF content was determined from the sum of the proportion (dry matter basis) of dietary dry matter retained either on the 2 screens (8- and 19- mm) or on the 3 screens (1.18-, 8-, and 19-mm) of the Penn State Particle Separator multiplied by the neutral detergent fiber content of the diet. An increased F:C ratio reduced intakes of dry matter and starch by 9 and 46%, respectively, but increased intake of fiber from forage sources by 53%. Digestibility of dry matter in the total tract was not affected, whereas total digestion of fiber and N was improved by increasing the F:C ratio. Improved total fiber digestion resulted from higher ruminal digestion, which was partially due to a shift in starch digestion from the rumen to the intestine with the increased F:C ratio. Actual milk yield was decreased but production of 4% fat-corrected milk was similar between the low and high F:C diets because of increased milk fat content. Increased FPL increased intake of peNDF, especially when the high F:C diet was fed. However, nutrient intakes, N metabolism in the digestive tract, and milk production were not affected. Digestibility of neutral detergent fiber in the total tract was increased because of improved fiber digestion in the rumen with increased FPL. These results indicate that feeding dairy cows a low F:C diet is beneficial in terms of increasing feed intake, microbial N synthesis, and milk production. However, low F:C diets do not maximize feed digestion and production efficiency because of the effects of subacute ruminal acidosis. Increased FPL improves fiber utilization with minimal effects on the digestion of other nutrients and milk production. Increasing dietary peNDF, through an increased proportion of forage or increased FPL, improves fiber digestion because of improved rumen function. [ABSTRACT FROM AUTHOR]

Alfalfa silages varying in theoretical chop length and diets high and low in forage proportion were used to evaluate whether increasing the physically effective (pe) neutral detergent fiber (NDF) content of dairy cow diets reduces the risk of acidosis. The experiment was designed as a replicated 4 x 4 Latin square using 8 ruminally cannulated lactating dairy cows. Treatments were arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) forage:concentrate (F:C) ratios [dry matter (DM) basis]. Dietary peNDF content (DM basis) was determined from the sum of the proportion of dietary DM retained on either the 2 sieves (8 and 19 mm) or the 3 sieves (1.18, 8, and 19 mm) of the Penn State Particle Separator multiplied by the NDF content of the diet. The dietary peNDF contents ranged from 9.6 to 19.8% using 2 sieves, or from 28.6 to 34.0% using 3 sieves. Intake of peNDF was increased by increasing both the F:C ratio and the FPL of the diets. However, F:C ratio and FPL affected chewing activity differently; increasing F:C ratio increased chewing time but increasing FPL only increased chewing when a high-forage diet was fed. Mean ruminal pH was increased by 0.5 and 0.2 units with increasing F:C ratio and FPL, respectively. Cows fed the low F:C diet had >10 or 7 h daily in which ruminal pH was below 5.8 or 5.5, respectively, compared with 1.2 and 0.1 h for cows fed the high F:C ratio diet. Increased F:C ratio reduced ruminal VFA concentration from 135 to 121 mM but increased the acetate:propionate ratio from 1.82 to 3.13. Dietary peNDF content when measured using 2 sieves was positively correlated to chewing time (r = 0.61) and mean ruminal pH (r = 0.73), and negatively correlated to the time that pH was below 5.8 or 5.5 (r = -0.46). This study shows that the risk of ruminal acidosis is high for cows fed a low F:C diet, particularly when finely chopped silage is used. Intake of dietary peNDF is a good indicator of ruminal pH status of dairy cows. Increasing the proportion of forage in the diet helps prevent ruminal acidosis through increased chewing time, a change in meal patterns, and decreased ruminal acid production. Increasing FPL elevates ruminal pH, but in low-forage diets increased FPL does not completely alleviate subacute acidosis because the fermentability of the diet is high and changes in chewing activity are marginal. [ABSTRACT FROM AUTHOR]

Barley silages varying in theoretical chop length were used to evaluate the effects of physically effective (pe) neutral detergent fiber (NDF) content of dairy cow diets on nutrient intakes, site and extent of digestion, microbial protein synthesis, and milk production. The experiment was designed as a replicated 3 x 3 Latin square using 6 lactating dairy cows with rum(hal and duodenal cannulas. During each of 3 periods, cows were offered 1 of 3 diets (low, medium, and high peNDF) obtained using barley silage that varied in particle length: fine (theoretical chop length of 4.8 mm), medium (equal proportions of long and fine silages), and long I theoretical chop length of 9.5 mm). The peNDF contents were determined by multiplying the proportion (dry matter basis) of feed retained on the 2 screens (8 and 19 mm) of the Penn State Particle Separator by the NDF content of the diet, and were 10.5, 11.8, and 13.8% for the low, medium, and high diets, respectively. Increased forage particle length linearly increased intake of peNDF but intakes of dry matter, organic matter, starch, and N were highest for cows fed the medium peNDF diet. Digestibilities of organic matter, NDF, and acid detergent fiber in the total tract were linearly decreased with increasing dietary peNDF, although total digestibility of starch and N was not affected by the treatments. Nevertheless, decreased digestibility due to increased dietary peNDF did not reduce milk production or milk composition because the cows were in mid to late lactation. Ruminal microbial protein synthesis and microbial efficiency were numerically higher with the low peNDF than with the medium or high peNDF diets. These results indicate that increasing the peNDF content of a diet containing barley silage decreases fiber digestibility in the total tract and lowers microbial efficiency. Therefore, the benefits of increasing dietary particle size, expressed as peNDF, on reducing the risk of ruminal acidosis should be weighed against potentially negative effects on efficiency of feed use. [ABSTRACT FROM AUTHOR]

A study was conducted to investigate the effects of physically effective neutral detergent fiber (peNDF) content of dairy cow diets containing corn silage as the sole forage on intake, chewing, ruminal pH, microbial protein synthesis, digestibility, and milk production. A second objective was to compare current methods of measuring peNDF to determine the most suitable approach for use in ration formulation. The experiment was designed as a replicated 3 x 3 Latin square using 6 lactating dairy cows with ruminal cannulas. Diets varied in peNDF content (high, medium, and low) by altering the particle length of corn silage. The physical effectiveness factors (pef) and peNDF contents of the corn silage and diets were determined based on the original (19- and 8-mm sieves) and new Penn State Particle Separator (PSPS; 19-, 8-, and 1.18-mm sieves). A dry-sieving technique that measures the proportion of particles retained on a 1.18-mm sieve was also used. The new PSPS and the 1.18-mm sieve produced similar estimates of pef and peNDF of diets but gave higher values than the original PSPS. There was a much smaller range in pef of corn silage when 3 sieves, rather than 2, were used with the PSPS (range of 0.93 to 0.96 vs. 0.41 to 0.72, respectively). Consequently, increased forage particle length in the diets increased dietary peNDF content and its intake when using the original PSPS; however, the new PSPS and the 1.18-ram sieve failed to detect changes in dietary peNDF and peNDF intake. The peNDF values estimated based on fractional NDF rather than the total NDF content were higher, but the ranking of diets was not changed. Increased intake of peNDF linearly increased digestibility of CP and tended to linearly increase digestibility of fiber in the total tract. As a result, milk yield tended to linearly increase with no effect on milk composition. Ruminal microbial protein synthesis and microbial efficiency were higher with the medium peNDF than with the high or low peNDF diets. Total chewing time and ruminating time were linearly increased with increasing dietary peNDF, whereas influence of dietary peNDF on ruminal pH and fermentation was minimal. This study showed that increasing peNDF content of diets improved fiber digestion. Of the systems used to measure peNDF, the original PSPS provided a good description of dietary particle length and its effects on chewing time and rumen pH, whereas the new PSPS provided a more consistent chewing index, the ratio of total chewing activity to peNDF, across diets varying in chop length of corn silage. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of physically effective neutral detergent fiber (peNDF) content of dairy cow diets containing barley silage as the sole forage source on feed intake, chewing activity, and ruminal pH. The experiment was designed as a replicated 3 x 3 Latin square using 6 lactating dairy cows with ruminal cannulas. Cows were offered 1 of 3 diets (high, medium, and low peNDF) obtained using barley silage that varied in particle length: long (theoretical cut length of 9.5 mm), medium (equal proportions of long and fine silages), and fine (theoretical cut length of 4.8 mm). The peNDF contents were determined using the Penn State Particle Separator and were 13.8, 11.8, and 10.5%, for the high, medium, and low diets, respectively. The physical effectiveness factors (defined as proportion retained on 19- and 8-mm screens) for the long and fine silages were 0.84 and 0.68, respectively. Increased forage particle size increased intake of peNDF but did not affect intake of DM and NDF. Ruminating and total chewing time were linearly increased with increasing dietary peNDF. Mean ruminal pH, area between the curve and a horizontal line drawn at pH 5.8 or 5.5, and time that pH was below 5.8 or 5.5 were not affected by peNDF content. Intake of peNDF was not correlated to any chewing activity but proportion of long particles on the 19-mm sieve tended to be correlated to ruminating chews (r = 0.36) and ruminating time (r = 0.36). These results indicate that increasing the peNDF content of diets increases chewing time. However, increased chewing time does not always improve ruminal pH status. Increasing chewing time and thus increasing salivary secretion may not fully overcome the effects of feed digestion and the production of fermentation acids that lower rumen pH. The results suggest that dietary peNDF and fermentable OM intake are critical in regulating rumen pH. Dietary particle size, expressed as peNDF, was a reliable indication of chewing activity. [ABSTRACT FROM AUTHOR]

A study was conducted to investigate the effects of physically effective (pe) neutral detergent fiber (NDF) content of dairy cow diets containing corn silage as the sole forage type on feed intake, meal patterns, chewing activity, and rumen pH. The experiment was designed as a replicated 3 x 3 Latin square using 6 lactating dairy cows with ruminal cannulas. Diets were chemically similar but varied in peNDF content (high, medium, and low) by altering corn silage particle length. The physical effectiveness factors for the long (original), medium (rechopped once), and fine (rechopped twice) silages were determined using the Penn State Particle Separator and were 0.84, 0.73, and 0.67, respectively. The peNDF contents of the diets were 11.5, 10.3, and 8.9%, for the high, medium, and low diets, respectively. Increased forage particle length increased intake of peNDF but did not affect intake of DM or NDF. Number of chews (chews/d) and chewing time, including eating and ruminating time, were linearly increased with increasing dietary peNDF. Meal patterns were generally similar for all treatments, except that number of meals was quadratically increased with increasing dietary peNDF. Mean ruminal pH, area between the curve and a horizontal line at pH 5.8 or 5.5, and time that pH was below 5.8 or 5.5 were not affected by peNDF content. Dietary peNDF content was moderately correlated to number of chews during eating (r = 0.41) and to total chewing time (r = 0.37). The present study demonstrates that increasing the peNDF content of diets increased chewing time, but increased chewing time did not necessarily reduce ruminal acidosis. Models that predict rumen pH should include both peNDF and fermentable OM intake. Dietary particle size, expressed as peNDF, was a reliable indicator of chewing activity. [ABSTRACT FROM AUTHOR]

The objectives of this study were to evaluate effects of dietary factors that alter ruminal fermentability on rumen N degradation, microbial protein synthesis, duodenal flows, and digestibility of amino acids (AA) in the intestines and the total tract. The experiment was a double 4 x 4 quasi-Latin square with a 2³ factorial arrangement of treatments. The dietary factors were extent of barley grain processing, coarse (processing index; PI = 75.5%) or flat (PI = 60.2%); forage-to-concentrate (F:C) ratio, low (35:65) or high (55:45) on a DM basis; and forage particle length (FPL), long (7.59 mm) or short (6.08 mm). Eight lactating cows with ruminal and duodenal cannulas were offered ad libitum access to a total mixed diet. There were no significant interactions between dietary treatments for ruminal N degradation or its duodenal flow and digestibility in the intestines. Passage of microbial protein to the duodenum was improved with increased F:C ratio of the diet but was not affected by grain processing or FPL. Ruminal digestibility of N was increased with increased F:C ratio (49 vs. 60%) and with reduced FPL (59 vs. 50%). Increased grain processing improved N digestibility both in the intestine (15%) and in the total tract (8%). Reduction in the FPL of the diets reduced intestinal N digestion by 14% without affecting the N digestion in the total tract. Increased extent of grain processing tended to enhance duodenal flows of AA. In contrast, reducing FPL lowered flows of dietary AA to the duodenum because of lowered flows of feed plus endogenous N. Increased F:C ratio of the diet did not change the flow of total AA, but there was a reduced flow of dietary AA and increased flow of microbial AA. Flows of several individual AA were increased by feeding flatly rolled barley with limited effects of F:C ratio or FPL. An interaction between grain processing and FPL was detected for flows of some AA. Diets formulated with flatly rolled barley plus long FPL ... [ABSTRACT FROM AUTHOR]

Dried distillers grains with solubles (DDGS) has been commonly used as a dietary protein source for lactating dairy cows. However, there is a paucity of data evaluating the use of DDGS as a partial replacement of forage or grain. The objective of this study was to determine the effects of partially replacing barley silage or barley grain with corn/wheat-based DDGS on dry matter intake (DMI), chewing activity, rumen fermentation, and milk production. Six ruminally cannulated lactating Holstein cows were used in a replicated 3 × 3 Latin square design with 21-d periods. Cows were fed the control diet (CON: 45% barley silage, 5% alfalfa hay, and 50% concentrate mix), a low forage (LF) diet or a low grain (LG) diet, in which barley silage or barley grain was replaced by DDGS at 20% of dietary dry matter, respectively. All diets were formulated to contain 18% crude protein and fed as total mixed rations. Compared with CON, cows fed the LF diet had greater DMI (26.0 vs. 22.4kg/d), yields of milk (36.4 vs. 33.0kg/d), milk protein (1.18 vs. 1.05kg/d), and milk lactose (1.63 vs. 1.46kg/d), but milk fat yield was not affected. The LF diet decreased chewing time compared with the CON diet (29.7 vs. 39.1 min/kg of DMI), but did not affect rumen pH and duration of rumen pH below 5.8. Compared with CON, feeding the LG diet tended to increase minimum and maximum rumen pH, but did not affect DMI, milk yield, and milk composition in this study. These results indicate that a partial replacement of barley silage with DDGS can improve the productivity of lactating dairy cows without negatively affecting rumen fermentation and milk fat production. Barley grain can also be partially replaced by DDGS in diets for lactating dairy cows without causing negative effects on productivity. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of 2 lots of barley grain cultivars differing in expected ruminal starch degradation on dry matter (DM) intake, ruminal fermentation, ruminal and total tract digestibility, and milk production of dairy cows when provided at 2 concentrations in the diet. Four primiparous ruminally cannulated (123 ± 69 d in milk; mean ± SD) and 4 multiparous ruminally and duodenally cannulated (46 ± 14 d in milk) cows were used in a 4 x 4 Latin Square design with a 2 x 2 factorial arrangement of treatments with 16-d periods. Primiparous and multiparous cows were assigned to different squares. Treatments were 2 dietary starch concentrations (30 vs. 23% of dietary DM) and 2 lots of barley grain cultivars (Xena vs. Dillon) differing in expected ruminal starch degradation. Xena had higher starch concentration (58.7 vs. 50.0%) and greater in vitro 6-h starch digestibility (78.0 vs. 73.5%) compared with Dillon. All experimental diets were formulated to supply 18.3% crude protein and 20.0% forage neutral detergent fiber. Dry matter intake and milk yield were not affected by treatment. Milk fat concentration (3.55 vs. 3.29%) was greater for cows fed Dillon compared with Xena, but was not affected by dietary starch concentration. Ruminal starch digestion was greater for cows fed high-starch diets compared with those fed low-starch diets (4.55 vs. 2.49 kg/d), and tended to be greater for cows fed Xena compared with those fed Dillon (3.85 vs. 3.19 kg/d). Ruminal acetate concentration was lower, and propionate concentration was greater, for cows fed Xena or high-starch diets compared with cows fed Dillon or low-starch diets, respectively. Furthermore, cows fed Xena or high-starch diets had longer duration that ruminal pH was below 5.8 (6.6 vs. 4.0 and 6.4 vs. 4.2 h/d) and greater total tract starch digestibility (94.3 vs. 93.0 and 94.3 vs. 93.0%) compared with cows fed Dillon or low-starch diets, respectively. These results demonstrate that selection of barley grain can affect milk fat production and rumen fermentation to an extent at least as great as changes in dietary starch concentration. [ABSTRACT FROM AUTHOR]

Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 µg/mL of insulin, 1 µg/mL of hydrocortisone, 2 µg/mL of prolactin, 1 µg/mL of progesterone, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferatoractivated receptor gamma (PPARG), CCAAT/enhancer- binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPa) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the effects of dietary factors that alter ruminal fermentability on intake, duodenal flows and intestinal digestibility of individual amino acids (AA) in lactating dairy cows. The experiment was designed as a 4 x 4 Latin square using 4 ruminally and duodenally cannulated lactating dairy cows. Treatments were arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) forage-to-concentrate ratio (F:C; dry matter basis). Four diets were formulated using 2 cuts of alfalfa silage [short (7.9 mm) and long (19.1 mm)], combined with 2 ratios of forage to barley grain concentrate (35:65 and 60:40). Overall, the interactions between dietary F:C and FPL on intake, duodenal flows, and intestinal digestibility of AA were marginal. Intakes of total AA and nonessential AA were not different between low- and high-F:C diets, whereas that of essential AA tended to be less with high-F:C diet as a result of lower intakes of Met, Phe, Arg, and His. The flows of total AA and microbial AA were reduced by 22 and 19%, respectively, with increasing F:C ratio in the diets due to consistently reduced flows of individual AA, whereas AA profiles (% of AA-N) of the duodenal protein were not different. Altering F:C from 35:65 to 60:40 decreased the intestinal digestibility of Ile, Leu, Thr, Val, Ala, Cys, and Ser, and consequently, tended to decrease the digestibility of total AA, essential AA, and nonessential AA. Intakes of total AA, essential AA, and nonessential AA were overall not affected by dietary FPL so FPL did not affect the flows or intestinal digestibility of AA. These results indicate that increasing dietary F:C ratio decreased overall AA supply because flow to the duodenum and intestinal digestibility of AA were decreased. However, increasing FPL had no effect on AA supply. The measured duodenal flows of AA were consistent with the predictions of the Cornell Net Carbohydrate and Protein System model for the low-forage diet, and were consistent with the National Research Council model for the high-forage diet. Furthermore, the digestibility of individual AA in the intestine varied considerably, regardless of dietary treatment. The results revealed the necessity to consider the both flows and digestibility of individual AA when optimizing ration formulation to meet AA requirements of dairy cows. [ABSTRACT FROM AUTHOR]

Feed intake and feeding behavior of dairy cows fed diets that varied in fat supplementation and forage:concentrate (F:C) ratio were investigated. Eight multiparous Holstein dairy cows were used in a replicated 4 × 4 Latin square experiment with 21-d periods. Treatments were 1) no supplemental fat and 34:66 F:C ratio; 2) 2% hydrogenated palm oil and 34:66 F:C ratio; 3) 2% yellow grease and 34:66 F:C ratio; and 4) 2% yellow grease and 45:55 F:C ratio. Cows were fed ad libitum twice daily as total mixed ration with free access to water. Dry matter intake (DMI) was not affected by fat supplementation regardless of fat source, whereas increased F:C ratio (from 34:66 to 45:55) lowered DMI by 7.5%. Meal interval, eating rate, and meal size were lower for cows fed yellow grease, and eating rate was less for cows fed the 45:55 F:C ratio diet. Chewing activity was not affected by fat supplementation, but was greater for cows fed the 45:55 F:C ratio diet. Results suggest that supplementation of 2% hydrogenated palm oil or 2% yellow grease had little effect on DMI and chewing behavior of Holstein dairy cows fed a 34:66 F:C ratio diet. The 2 fat sources can replace each other, depending on the availability or cost. Results also showed that DMI and chewing activity can be effectively manipulated by changing the F:C ratio of diet. [ABSTRACT FROM AUTHOR]

The objectives of the present in vivo and in situ trials were to evaluate whether feeding barley grain steeped in lactic acid (LA) would affect rumen fermentation patterns, in situ dry matter (DM) degradation kinetics, and milk production and composition in lactating dairy cows. The in vivo trial involved 8 rumen-fistulated Holstein cows fed once daily a total mixed ration containing rolled barley grain (27% in DM) steeped for 48 h in an equal quantity of tap water (CTR) or in 0.5% LA (TRT) in a 2 × 2 crossover design. The in situ trials consisted of incubation of untreated rolled barley grain in cows fed CTR or TRT diets and of incubation of 3 different substrates including CTR or barley grain steeped in 0.5% or 1.0% LA (TRT1 and TRT2, respectively) up to 72 h in the rumen. Results of the in vivo trial indicated that cows fed the TRT diet had greater rumen pH during most intensive fermentation phases at 10 and 12 h post-feeding. The latter effect was associated with a shorter duration in which rumen pH was below 5.8 for cows fed the TRT diet (2.4 h) compared with CTR diet (3.9 h). Furthermore, cows fed the TRT diet had lower concentrations of volatile fatty acids at 2 and 4 h post-feeding. In addition, concentrations of preprandial volatile fatty acids were lower in the rumen fluid of cows fed the TRT diet. Results also showed that molar proportion of acetate was lower, whereas propionate tended to increase by feeding cows the TRT diet. Cows fed the TRT diet demonstrated greater rumen in situ lag time of substrate DM degradation and a tendency to lower the fractional degradation rate. Other in situ results indicated a quadratic effect of LA on the effective rumen degradability of substrates whereby the latter variable was decreased from CTR to TRT1 but increased for TRT2 substrate. Although the diet did not affect actual milk yield, fat-corrected milk, percentages The objectives of the present in vivo and in situ trials were to evaluate whether feeding barley grain steeped in lactic acid (LA) would affect rumen fermentation patterns, in situ dry matter (DM) degradation kinetics, and milk production and composition in lactating dairy cows. The in vivo trial involved 8 rumen-fistulated Holstein cows fed once daily a total mixed ration containing rolled barley grain (27% in DM) steeped for 48 h in an equal quantity of tap water (CTR) or in 0.5% LA (TRT) in a 2 × 2 crossover design. The in situ trials consisted of incubation of untreated rolled barley grain in cows fed CTR or TRT diets and of incubation of 3 different substrates including CTR or barley grain steeped in 0.5% or 1.0% LA (TRT1 and TRT2, respectively) up to 72 h in the rumen. Results of the in vivo trial indicated that cows fed the TRT diet had greater rumen pH during most intensive fermentation phases at 10 and 12 h post-feeding. The latter effect was associated with a shorter duration in which rumen pH was below 5.8 for cows fed the TRT diet (2.4 h) compared with CTR diet (3.9 h). Furthermore, cows fed the TRT diet had lower concentrations of volatile fatty acids at 2 and 4 h post-feeding. In addition, concentrations of preprandial volatile fatty acids were lower in the rumen fluid of cows fed the TRT diet. Results also showed that molar proportion of acetate was lower, whereas propionate tended to increase by feeding cows the TRT diet. Cows fed the TRT diet demonstrated greater rumen in situ lag time of substrate DM degradation and a tendency to lower the fractional degradation rate. Other in situ results indicated a quadratic effect of LA on the effective rumen degradability of substrates whereby the latter variable was decreased from CTR to TRT1 but increased for TRT2 substrate. Although the diet did not affect actual milk yield, fat-corrected milk, percentages [ABSTRACT FROM AUTHOR]

Mechanical processing of whole crop barley before ensiling may be useful for improving nutrient use by dairy cattle. The objective of this study was to assess the effects of feeding mechanically processed barley silage as the main forage source on lactational performance. Twenty-four Holstein cows, 16 primiparous (187 ±52 days in milk) and 8 multiparous (87 ±69 days in milk) cows, were used in a completely randomized design with a 2-wk covariate period and a 6-wk treatment period. The 2 treatments were: 1) total mixed ration (TMR) containing regular barley silage (RBS-TMR), and 2) TMR containing mechanically processed barley silage (MPBS-TMR). Barley silage and alfalfa hay supplied 41 and 5% of the dietary dry matter (DM), respectively. Intake, body weight, and milk production were measured during the covariate and treatment periods. In addition, 2 multiparous cows were used for in situ measurements of the ruminal DM and fiber degradation kinetics of the barley silages and TMR. Data were analyzed with repeated measurements using a mixed model that included the covariate adjustment. Feeding MPBS-TMR had no significant effects on DM intake (DMI; 21.7 kg/d), milk yield (33.9 kg/d), or milk composition, with only 4% FCM (fat-corrected milk) yield (29.7 vs. 31.7 kg/d) and milk fat concentration (3.30 vs. 3.57%) showing a numerical improvement. Apparent digestibilities of DM and nutrients were not affected by feeding MPBS-TMR, with the exception of starch digestibility, which tended to increase. Dairy efficiencies calculated as milk yield/DMI or FCM/DMI were not different between treatments. Body weight and body condition score were not affected by treatments. Effective ruminal degradability of DM was similar for both barley silages, indicating that when the silages were ground to remove the effects of mechanical processing, the potential digestion was similar. Mechanical processing of barley silage harvested at a mid-dough stage of maturity resulted in... [ABSTRACT FROM AUTHOR]

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, non-ruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated there are functional differences among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The purpose of this study was to investigate the effects of early castration and eucalyptus oil (EUC) supplementation on dry matter intake (DMI), growth performance, and immune response of Holstein calves. Fifty-six male Holstein calves 52 d old and with an initial body weight (BW) of 63.5 ± 5.27 kg were used. The animals were blocked by BW and randomly assigned into 1 of the 4 treatment groups in a randomized complete block design with a 2 (no castration vs. castration) × 2 (without vs. with EUC) factorial arrangement of treatments. The treatments were (1) uncastrated calves fed without EUC, (2) uncastrated calves fed 0.5 g/d EUC (EUC group), (3) castrated calves (steers) fed without EUC (castrated group), and (4) steers fed with 0.5 g/d EUC (castrated + EUC). The experiment was 8 wk long, including pre- and postweaning (weaned at 72 d). The EUC × castrated interactions were not significant for DMI, growth performance, nutrient digestibility, and immune response. Castration did not affect the DMI, final BW, average daily gain (ADG), or feed efficiency, except that the ADG was greater for bull calves than for steers at postweaning. Supplementation with EUC increased DMI pre- and postweaning and increased the ADG of weaned calves. Digestibility in the total digestive tract was not affected by castration (except for organic matter digestibility), whereas adding EUC improved the digestibility of dry matter, acid detergent fiber, and crude protein. Blood concentration of IL-6 at d 94 was decreased by feeding EUC. These results indicate that the EUC could be fed to either intact or castrated dairy calves to promote growth and health postweaning; castration before weaning may reduce ADG and cause inflammatory stress without affecting feed intake or feed efficiency., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca 2+ entry (SOCE)-related molecules. It is well established in nonruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca 2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through quantitative reverse-transcription PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII), and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 (ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 [STIM1], and STIM2). Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin β9 (ITGA9), and integrin β3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 μM HKII inhibitor benserazide-d3 or 1 μM fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGA9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGA9, and CD36 in the HYP group. Finally, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 μM SOCE activator thapsigargin or 50 μM inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGA9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca 2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism-related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum β-hydroxybutyrate concentration <0.6 mM) and cows diagnosed with clinical ketosis (serum β-hydroxybutyrate concentration >3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid-mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35-45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

High circulating concentrations of fatty acids cause triacylglycerol (TAG) accumulation in hepatocytes of dairy cows, a common metabolic disorder after calving. Low secretion of apolipoprotein B (APOB) and very low density lipoprotein (VLDL) are thought to be the major factors for TAG accumulation in hepatocytes. Recent data in nonruminant models revealed that sortilin 1 (SORT1) is a key regulator of VLDL secretion in part due to its ability to bind APOB. Thus, SORT1 could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights in vivo and in vitro, we performed experiments using liver biopsies or isolated primary hepatocytes. For the in vivo study, blood and liver samples were collected from healthy multiparous dairy cows (n = 6; 9.0 ± 2.1 d in milk) and cows with fatty liver (n = 6; 9.7 ± 2.2 d in milk). In vitro, hepatocytes isolated from 4 healthy female calves (1 d old, 42-51 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with empty adenovirus vectors (Ad-GFP) or SORT1 overexpressing adenovirus (Ad-SORT1) for 6 h, or SORT1 inhibitor for 2 h followed by a challenge with (Ad-GFP + fatty acids, Ad-SORT1 + fatty acids, or SORT1 inhibitor + fatty acids) or without (Ad-GFP, Ad-SORT1, or SORT1 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data from liver biopsies were compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocytes were analyzed by one-way ANOVA. Data revealed that both fatty liver and in vitro challenge with fatty acids were associated with greater concentrations of TAG and mRNA and protein abundance of SORT1, SREBF1, FASN, and ACACA. In contrast, mRNA and protein abundance of CPT1A and APOB, and mRNA abundance of MTTP were markedly lower. Compared with fatty acid challenge alone, SORT1 overexpression led to greater concentration of TAG and mRNA abundance of SREBF1, FASN, ACACA, DGAT1, and DGAT2, and protein abundance of SREBF1, FASN, and ACACA. In contrast, concentration of secreted VLDL-APOB and mRNA abundance of APOB and MTTP, and protein abundance of CPT1A, APOB, and MTTP were lower. Compared with fatty acid challenge alone, SORT1 inhibitor + fatty acids led to lower concentrations of TAG and mRNA abundance of SREBF1, FASN, and DGAT2, and protein abundance of FASN, ACACA, and DGAT1. Concentrations of secreted VLDL-APOB and mRNA abundance of CPT1A and protein abundance of CPT1A and APOB were greater. Overall, in vitro data suggested that greater SORT1 abundance induced by exogenous fatty acids caused a reduction in VLDL-APOB secretion and increased hepatocyte TAG synthesis. Such mechanism was also apparent in tissue from cows with fatty liver. Thus, targeted downregulation of hepatic SORT1 could represent a viable mechanism to unload lipid during conditions where the influx of fatty acids increases markedly., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Fatty acid accumulation in hepatocytes induced by high concentrations of fatty acids due to lipolysis and the associated oxidative damage they cause occur most frequently after calving. Because of their role in esterification of fatty acids, diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights, we performed in vivo and in vitro analyses using liver biopsies or isolated primary hepatocytes. The in vivo study (n = 5 cows/group) involved healthy cows [average liver triacylglycerol (TAG) = 0.78%; 0.58 to 0.93%, ratio of triglyceride weight to wet liver weight] or cows diagnosed with fatty liver (average TAG = 7.60%; 5.31 to 10.54%). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old, 44 to 53 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with DGAT1 inhibitor or DGAT2 inhibitor for 2 h followed by a challenge with (DGAT1 inhibitor + fatty acids or DGAT2 inhibitor + fatty acids) or without (DGAT1 inhibitor or DGAT2 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data analysis of liver biopsies was compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocyte treatment comparisons were assessed by one-way ANOVA, and multiplicity for each experiment was adjusted by the Holm's procedure. Data indicated that both fatty liver and in vitro challenge with fatty acids were associated with greater mRNA and protein abundance of SREBF1, FASN, DGAT1, and DGAT2. In contrast, mRNA and protein abundance of CPT1A and very low-density lipoprotein synthesis-related proteins MTTP and APOB were markedly lower. However, compared with fatty acid challenge alone, DGAT1 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A and APOB, and greater mRNA abundance of SREBF1 and MTTP. Furthermore, this treatment led to lower mRNA abundance of FASN and DGAT2 and TAG concentrations. Compared with fatty acid challenge alone, DGAT2 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A, MTTP, and APOB, and lower mRNA and protein abundance of SREBF1 and FASN. In addition, compared with control and fatty acids, there was greater protein abundance of GRP78 and PERK in both DGAT1 and DGAT2 inhibitor with or without fatty acids. Furthermore, compared with control and fatty acids, reactive oxygen species concentrations in the DGAT1 inhibitor with or without fatty acid group was greater. Overall, data suggested that DGAT1 is particularly relevant in the context of hepatocyte TAG synthesis from exogenous fatty acids. Disruption of both DGAT1 and DGAT2 altered lipid homeostasis, channeling fatty acids toward oxidation and generation of reactive oxygen species. Both DGAT isoforms play a role in promoting fatty acid storage into TAG and lipid droplets to protect hepatocytes from oxidative damage., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Hypocalcemia in dairy cows is associated with decreased neutrophil phagocytosis, adhesion capacity, migration, and reactive oxygen species (ROS) production through alterations in ORAI calcium release-activated calcium modulator 1 (ORAI1). Neutrophils can resist the invasion of pathogenic microorganisms by releasing neutrophil extracellular traps (NET). However, the mechanisms controlling NET formation during hypocalcemia are unknown. To address the role of ORAI1 in NET formation, neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (n = 10 per group) diagnosed as clinically healthy (control) or with plasma concentrations of Ca 2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (SCH). A series of ex vivo experiments were conducted as follows: first, neutrophils isolated from both groups of cows were treated with phorbol 12-myristate 13-acetate (PMA) to stimulate NET formation; second, neutrophils from control and SCH were pretreated with or without the ROS scavenger N-acetylcysteine (NAC), the sarcoendoplasmic Ca 2+ ATPase inhibitor thapsigargin, or ORAI1 blocker 2APB and then treated with PMA to stimulate NET formation; and third, neutrophils were transfected with small interfering (si)ORAI1 or nontarget siRNA (siNEG) and then stimulated with PMA to induce formation of NET. A one-way ANOVA was used for statistical analysis of individual experiments. In the first experiment, neutrophils from SCH cows formed NET with fewer DNA filaments, more diffused nuclei, and reduced translocation of myeloperoxidase (MPO) and neutrophil elastase (NE) to the nucleus. Neutrophils from SCH cows stimulated with PMA had a lower mitochondrial permeability, the state of mitochondrial permeability transition pore was open, ROS production was lower and there was increased mitochondrial damage. In the second experiment, in both control and SCH-PMA stimulated neutrophils, exogenous NAC decreased NET formation (assessed via Hoechst 33342 dye; Beyotime). Furthermore, following the challenge with PMA, thapsigargin increased NET formation and ROS production, but blocking ORAI1 with 2APB decreased NADPH oxidase activation, ROS production, and NET formation. In the third experiment, neutrophils transfected with siORAI1 before stimulation with PMA had lower intracellular concentrations of Ca 2+ , NET formation, and ROS production. Overall, the data indicated that SCH reduces NET formation in neutrophils partly due to damaged mitochondria. The reduction in ORAI1 abundance in neutrophils of dairy cows with hypocalcemia also decreases ROS production., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to investigate the effects of partial substitution of corn silage (CS) with sweet sorghum silage (SS) in the diets of lactating dairy cows on dry matter (DM) intake, milk yield and composition, blood biochemistry, and ruminal fermentation and microbial community. Thirty mid-lactation Holstein dairy cows [mean ± standard deviation; 639 ± 42.0 kg of body weight; 112 ± 24.0 d in milk (DIM)] were assigned to 3 groups (n = 10/treatment) by considering parity, milk yield, and DIM. The cows were fed ad libitum total mixed rations containing 55% forage and 45% concentrate, with only the proportion of CS and SS varying in 3 treatments (DM basis): SS0 (0% substitution of CS), 40% CS and 0% SS; SS25 (25% substitution of CS) , 30% CS and 10% SS; and SS50 (50% substitution of CS) , 20% CS and 20% SS. Dry matter intake and milk protein concentration tended to linearly decrease with increasing proportion of SS in the diet. Yields of milk (mean ± standard deviation, 30.9 ± 1.12 kg/d), 4% fat-corrected milk (30.0 ± 0.81 kg/d), energy-corrected milk, milk protein, lactose, and total solids, concentrations of milk fat, lactose, somatic cell counts, and milk efficiency did not differ among diets. The concentrations in blood of urea nitrogen, phosphorus, aspartate aminotransferase, and malondialdehyde linearly increased with increasing SS proportion. Blood IgA decreased with increasing SS substitution rate, but blood IgG and IgM were not different among diets. Ruminal pH did not differ among diets, whereas ruminal NH 3 -N concentration quadratically changed such that it was greater for SS50 than for SS0 and SS25. Molar proportions of propionate and acetate to propionate ratio were less for SS25 than for SS0. Although the diversity and general ruminal microbial community structure were not altered by partially replacing CS with SS, the relative abundances of predominant bacteria were affected by diets at the phylum and genus levels. Firmicutes and Bacteroidetes were dominant phyla in the ruminal bacterial community for all diets, and their relative abundance linearly decreased and increased, respectively, with increasing SS substitution rate. Prevotella&#95;1 and Ruminococcaceae&#95;NK4A214&#95;group were detected as the most and the second most abundant genera, with their relative abundance linearly increased and decreased, respectively, with increasing SS substitution rate. The relative abundance of Fibrobacter linearly increased with increasing dietary SS proportion, with greater abundance observed for SS25 and SS50 than for SS0. These results suggest that substitution of CS with SS altered the relative abundances of some predominant bacteria; however, these changes had little effect on ruminal fermentation and milk yield. Under the current experimental conditions, substituting up to 50% of CS with SS had no negative effects on milk yield, indicating that SS can partially replace CS in the diets of high-producing lactating dairy cows without adding extra grain, when diets are fed for a short time. As the effects of substituting CS with SS depend upon the chemical composition and digestibility of these silages and the nutrient requirements of the cows, additional grain may be required in some cases to compensate for the lower starch content of SS., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The store-operated Ca 2+ entry (SOCE) moiety ORAI calcium release-activated calcium modulator 1 (ORAI1) located in the endoplasmic reticulum (ER) participates in key cellular functions such as protein folding, transport, and secretion, and lipid metabolism. We used an in vitro approach to test whether exogenous fatty acids alter ORAI1 signaling and to explore potential consequences on mitochondrial dysfunction and ER stress. First, hepatocytes isolated from 4 healthy female calves (1 d old, 40-50 kg) were challenged with a 1.2 mM mixture of oleic, linoleic, palmitic, stearic, and palmitoleic acids for 0.5, 1, 3, 6, 9, and 12 h to measure oxidative stress [intracellular reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and hydrogen peroxide] and ER stress (protein abundance of PERK, IRE, ATF6, and GRP78). Concentrations of GSH and SOD decreased at 0.5 h, and MDA and hydrogen peroxide increased at 1 h; ER stress proteins increased at 6 h. To determine whether ER stress was caused by oxidative stress, primary calf hepatocytes were treated with the same 1.2 mM fatty acid mix or the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) for 6 h. We found that NAC prevented an increase in ER stress protein abundance. Next, the role of ORAI1 on ER stress was measured by transfecting hepatocytes with small interfering (si)ORAI1 or the ORAI1 inhibitor BTP2, followed by a challenge with 1.2 mM fatty acids for 3 h. Without inhibiting ORAI1, exogenous fatty acids upregulated ORAI1 mRNA and protein abundance, oxidative stress, ER stress proteins, and protein abundance of marker indicators of an opened mitochondrial permeability transition pore (mPTP). Inhibition with BPT2 or silencing via siORAI1 abrogated oxidative stress, including increased GSH concentration and SOD activity, decreased MDA, hydrogen peroxide, and ROS concentration; ER stress protein abundance was downregulated, and mitochondrial function was restored. Last, changes in markers of mPTP opening were evaluated by culturing hepatocytes for 6 h with the sarcoendoplasmic Ca 2+ ATPase inhibitor thapsigargin or the calcium ionophore ionomycin. We detected an increase in VDAC1, CLPP, and CypD protein abundance, all of which indicated opening of the mPTP. Overall, data from these in vitro studies suggest that ORAI1 mediates ER stress induced by high concentrations of fatty acids, in part through alleviating mitochondrial dysfunction caused by oxidative stress., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

A study was conducted to evaluate the effects of forage-to-concentrate (F:C) ratio and forage particle length (FPL) on intake, duodenal flow, and digestibility of individual AA in the intestine of lactating dairy cows. The experiment was designed as a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments using 4 lactating dairy cows (parity 2) with ruminal and duodenal cannulas. Low (35:65) and high (60:40) F:C ratios (dry matter basis) were combined with 2 FPL of alfalfa silage (short vs. long; 7.9 vs. 19.1 mm). Few interactions between F:C and FPL for duodenal flow and intestinal digestibility of AA occurred, but interactions were detected for intakes of several AA. Intake of essential AA and nonessential AA decreased with increasing F:C, and the intake of several individual AA increased or decreased with increasing FPL. Increasing F:C decreased duodenal flows of essential AA, nonessential AA, and microbial AA due to consistent decreased flows of most individual AA (except Glu). Degradability of most individual AA in the rumen was not affected by F:C ratio or FPL except that the degradability of His was greater with high than low F:C diets, and the degradability of Ser was greater with long versus short FPL diets. However, the degradability of individual AA within diet varied considerably. Overall, F:C ratio and FPL did not affect intestinal digestibility of AA and rumen undegradable protein AA, whereas the digestibility of individual AA in the intestine varied considerably regardless of dietary treatment. These results indicate that increasing F:C ratio decreased AA supply due to decreased flow of AA to the duodenum but altering FPL did not affect AA supply. The results also revealed the necessity to consider both the flows and digestibility of individual AA when optimizing ration formulation to meet AA requirements of dairy cows., (Crown Copyright © 2020. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. All rights reserved.)

The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca 2+ entry mechanism regulating cellular Ca 2+ balance in nonruminants. Alterations in ORAI1 abundance have been associated with endoplasmic reticulum (ER) stress and changes in lipid metabolism in hepatocytes, an important lipogenic organ in nonruminants. Objectives were to (1) determine abundance of ORAI1 and components of the ER stress response in mammary tissue of ketotic cows, and (2) the potential role of ORAI1 on mammary cell responses to high levels of β-hydroxybutyrate (BHB). Healthy (n = 6, plasma BHB < 0.60 mmol/L) and clinically ketotic (n = 6, plasma BHB > 2.0 mmol/L) Holstein cows (days in milk = 10.13 ± 1.90) were used for mammary gland tissue and blood sample collection. Although milk production (22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) and dry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) were lower in ketotic cows, abundance of ORAI1 protein was greater and was associated with greater mRNA abundance of ER stress proteins (PERK, IRE1, ATF6, and GRP78) and lipogenic genes (FASN, SREBP1, and ACACA). Cellular mechanisms to establish links between BHB and mammary cell responses were evaluated using the immortalized cell line bovine mammary epithelial cells (MAC-T). First, a dose response study was performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHB for 24 h. The mRNA abundance of FASN, SREBP1, and ACACA and lipid droplet formation peaked at 1.2 mM BHB. A subsequent study involved transfecting MAC-T with small interfering Orai 1 (siORAI1) or the ORAI1 inhibitor BTP 2 for 24 h followed by a challenge with 1.2 mM BHB for 24 h. Transcription and protein abundance of FASN, SREBP1, ACACA, and ER stress proteins returned to basal levels when ORAI1 was silenced or inhibited. Furthermore, the Ca 2+ ionophore ionomycin (raises the intracellular level of Ca 2+ ) also increased abundance of ORAI1, FASN, SREBP1, ACACA, and ER stress proteins. Data suggest that the mammary gland experiences ER stress during ketosis, partly due to the greater supply of BHB originating from ketogenesis in the liver. Intracellular Ca 2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Physically effective neutral detergent fiber (peNDF) content of dairy cow diets was modified by varying the theoretical chop length of alfalfa silage and forage:concentrate (F:C) ratio, and effects on nutrient intakes, ruminal fermentation, site and extent of digestion, microbial protein synthesis, and milk production were evaluated. Estimates of dietary peNDF contents were compared with recommendations, and predictions of ruminal pH from peNDF and the recently developed physically adjusted neutral detergent fiber (paNDF) system were compared with observed pH. The experiment was designed as a triple 4 × 4 Latin square using 12 mid-lactating dairy cows with 4 intact, 4 ruminally cannulated, and 4 ruminally and duodenally cannulated cows. Site and extent of digestion and microbial protein synthesis were measured in a single 4 × 4 Latin square. Treatments were a 2 × 2 factorial arrangement; 2 forage particle lengths (FPL) of alfalfa silage (short and long) were combined with low (35:65) and high (60:40) F:C ratios [dry matter (DM) basis]. The peNDF contents were determined by multiplying the proportion (DM basis) of total mixed ration retained on 2 (8 and 19 mm; peNDF 8.0 ) or 3 (1.18, 8, and 19 mm; peNDF 1.18 ) sieves of the Penn State Particle Separator by the neutral detergent fiber content of the diet. The dietary peNDF contents ranged from 10.7 to 17.5% for peNDF 8.0 or from 23.1 to 28.2% for peNDF 1.18 . Interactions between F:C ratio and FPL content were few. Increasing peNDF content of diets by increasing F:C ratio decreased DM intake, milk yield, and milk protein yield, whereas apparent total-tract DM digestibility and milk efficiency improved. Increasing F:C ratio improved ruminal pH status but decreased total volatile fatty acid concentration and microbial protein synthesis. Increasing peNDF content of diets via dietary FPL increased mean ruminal pH, but did not affect DM intake, total-tract digestibility, or milk production. The results indicate that feeding dairy cows a low F:C diet helps increase DM intake, milk production, and microbial protein synthesis, but may adversely affect feed digestibility and milk efficiency due to increased risk of subacute ruminal acidosis. Increased FPL improved ruminal pH status, but had minimal effects on feed intake, ruminal fermentation, nutrient digestibility, and milk production. The results indicate a trade-off between reducing the risk of subacute ruminal acidosis and maximizing ruminal fermentation, feed digestibility, and milk production of dairy cows. The paNDF model showed improvement in the predictability of ruminal pH over the peNDF model, but the accuracy of predictions varied depending upon the diet and ruminal fermentation variables considered in the equations., (Crown Copyright © 2020. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. All rights reserved.)

This study was conducted to reveal the prevalence, molecular characterization, and antibiotic susceptibility of Bacillus cereus isolated from dairy products including powdered infant formula, raw milk, pasteurized milk, ultra-high-temperature milk, and cheese. Five hundred samples collected from 5 provinces in China were analyzed in overall experiments. Multilocus sequence typing, distribution of toxin genes, and antibiotic susceptibility of the isolates were analyzed. Fifty-four B. cereus strains were detected; of these, 13 isolates (26%) were from raw milk, 12 isolates (12%) from pasteurized milk, 10 isolates (10%) from cheese, 12 isolates (8%) from ultra-high-temperature milk, and 7 isolates (7%) from powdered infant formula. These isolates were divided into 24 sequence types (ST); among them, ST24, ST26, ST82, ST142, ST377, ST857, and ST1046 were the main dominant ST. The results of detection of toxin genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM, bceT, hlyII, and cesB) showed that 94.4% isolates carried nheABC genes, whereas only 11.1% of the isolates contained the hblACD gene cluster. In addition, detection rates of cytK, bceT, entFM, hlyII, and cesB genes were 75.9, 77.8, 85.2, 53.7, and 11.1%, respectively. The antibiotic susceptibility test indicated that most of B. cereus isolates were resistant to ampicillin, penicillin, cefepime, cephalothin, and oxacillin, and were susceptible to gentamicin, chloramphenicol, imipenem, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, erythromycin, kanamycin, and cefotetan. Therefore, this study revealed the prevalence and characteristics of B. cereus isolated from dairy products in China, indicating the potential risk and contributing to the effective prevention and control of this pathogen., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objectives of this study were to evaluate the effects of pretreating dairy cow rations with a fibrolytic enzyme derived from Trichoderma reesei (FETR; mixture of xylanase and cellulase; AB Vista, Wiltshire, UK) on lactation performance, digestibility, and feeding behavior in response to feeding a barley silage-based diet. Before starting the dairy trial, in vitro incubations were conducted to determine whether the addition of FETR would have an effect on these animal performance characteristics when applied to a barley silage-based diet for dairy cows. The dairy trial was performed using 8 Holstein dairy cows. The cows were blocked by parity and assigned randomly to 1 of 4 treatments: 0, 0.5, 0.75, and 1 mL of FETR/kg of dry matter (DM) diet in a replicated Latin square design. The pretreatment was applied to the complete diet during the mixing process. The experimental period continued for 22 d, with each experimental period consisting of a 16-d adaptation period and a 6-d sampling period. The daily feed intake of each individual cow was monitored using Insentec feed bins (RIC system, Insentec, Marknesse, the Netherlands). Feeding behavior characteristics were measured during the entire sampling period using the feed bin attendance data. Milk samples were collected in the last 3 d of each experimental period. The addition of FETR linearly increased the in vitro DM digestibility and tended to improve the in vitro digestibility of barley silage. There was a cubic effect of the enzyme levels on the total-tract DM and neutral detergent fiber digestibility. Maximal digestibility was reached at 0.75 mL of FETR/kg of TMR. The milk fat yield, fat-corrected milk, and energy-corrected milk quadratically responded to the incremental levels of FETR. The milk protein percentage linearly improved in response to FETR. Increasing FETR levels resulted in a quadratic effect on feed efficiency. There was no effect of FETR level on feeding behavior. In conclusion, pretreating dairy cow barley silage-based diet with 0.75 mL of FETR/kg of TMR increased the milk production efficiency of dairy cows fed diet containing 34% barley silage (DM basis). The positive effect of adding FETR could benefit the dairy industry in western Canada, where barley silage-based diets are common., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Effects of physically effective (pe) neutral detergent fiber (NDF) content of dairy cow diets on nutrient intakes, site and extent of digestion, microbial protein synthesis and milk production were evaluated in a double 3 x 3 Latin square design using 6 lactating dairy cows with ruminal and duodenal cannulas. During each of 3 periods, cows were offered 1 of 3 diets that were chemically similar but varied in peNDF content (high, medium, and low) by altering corn silage particle length. The peNDF contents were determined using the Penn State Particle Separator and were 11.5, 10.3, and 8.9%, for the high, medium, and low diets, respectively, and the physical effectiveness factors for the long, medium, and fine silages were 84.1, 72.6, and 67.2%, respectively. Increased forage particle length increased intake of peNDF but did not affect intakes of nutrients including dry matter, NDF, starch, and nitrogen. Except for starch, apparent digestibilities of nutrients in the total tract were linearly increased with increasing dietary peNDF. Fiber digestion was affected by dietary peNDF to a greater extent than were the other nutrients. However, increased digestibility due to increased dietary peNDF did not significantly improve milk production or milk composition. Increased dietary peNDF also increased numerically rumen microbial protein synthesis due to increased amount of organic matter fermented in the rumen. These results indicate that increasing the peNDF content of a corn silage based diet improves digestibility, especially digestibility of fiber, in the total tract. Dietary particle size, expressed as peNDF, is positively associated with nutrient digestibility when level of peNDF in the diet is low.

Effects offorage particle size measured as physically effective NDF and ratio of alfalfa silage to alfalfa hay of diets on feed intake, chewing activity, particle size reduction, salivary secretion, ruminal fermentation, and milk production of dairy cows were evaluated using a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. The diets consisted of 60% barley-based concentrate and 40% forage, comprised either of 50:50 or 25:75 of alfalfa silage:alfalfa hay, and alfalfa hay was either chopped or ground. Various methods were used to determine physically effective NDF content of the diets. Cows surgically fitted with ruminal and duodenal cannulas were offered ad libitum access to these total mixed diets. The physically effective NDF content of the diets was significantly lower when measured using the Penn State Particle Separator than when measured based on particles retained on 1.18-mm screen. Intake of DM was increased by increasing the ratio of silage to hay but was not affected by physically effective NDF content of diets. Eating time (hours per day) was not affected by the physically effective NDF content of diets, although cows spent more time eating per unit of DM or NDF when consuming high versus low alfalfa hay diets. Ruminating time (hours per day) was increased with increased physically effective NDF content of the diets. Rumen pH was affected more by changing dietary particle size than altering the ratio of silage to hay. Feeding chopped hay instead of ground hay improved ruminal pH status: time during which ruminal pH was above 6.2 increased and time during which ruminal pH was below 5.8 decreased. Milk production was increased by feeding higher concentrations of alfalfa silage due to increased DM intake, but was not affected by dietary particle size. Feed particle size, expressed as mean particle length or physically effective NDF was moderately correlated with ruminating time but not with eating time. Although physically effective NDF and chewing time were not correlated to mean rumen pH, they were negatively correlated to the area between the curve and pH 5.8, indicating a positive effect on reducing the risk of acidosis. Milk fat content was correlated to rumen pH but not to physically effective NDF or chewing activity. These results indicate that increasing physically effective NDF content of the diets increased chewing activity and improved rumen pH status but had limited effect on milk production and milk fat content.

Effects of ratio of alfalfa silage to alfalfa hay and forage particle size on nutrient intakes, site of digestion, rumen pools, and passage rate of ruminal contents were evaluated in a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. The diets consisted of 60% barley-based concentrate and 40% forage made up either of 50:50 or 25:75 of alfalfa silage:alfalfa hay and alfalfa hay was either chopped or ground. Lactating dairy cows surgically fitted with ruminal and duodenal cannulas were used and offered ad libitum access to a total mixed ration. Intakes of nutrients were increased by increasing ratio of silage to hay but were not affected by particle size of forage. Change in ratio of silage to hay of diets did not affect site and extent of digestion. However, increased forage particle size of the diets improved digestibility of fiber and N in the total tract, and as well as digestibility of organic matter, starch, and acid detergent fiber in the intestine. There was a shift of starch digestion from the rumen to the intestine when forage particle size was increased, although total digestion of starch was not changed. Ruminal microbial protein synthesis and microbial efficiency also improved with increasing forage particle size. Cows fed ground hay versus chopped hay had significantly lower rumen wet mass regardless of the ratio of silage to hay. Reduced forage particle size also lowered ruminal nutrient pool size for cows fed the high silage diet. Ruminal passage rates of liquid and solid were decreased by reducing the ratio of silage to hay, and retention time of solids in the total tract was shortened by reducing forage particle size. These results indicate that manipulating ratio of silage to hay in the diets of dairy cows changed feed intake but had little effect on digestion. In contrast, increased forage particle size in dairy cow diets improved fiber digestion and microbial protein synthesis in the rumen, and shifted starch digestion from the rumen to the intestine. Dietary particle size, expressed as physically effective neutral detergent fiber, was a reliable indication of ruminal microbial protein synthesis and nutrient digestion.

Dietary factors that alter fermentability, NDF content, or particle size of the diet were evaluated for their effects on chewing behavior and distribution and passage of feed particles in the digestive tract of dairy cows. A double 4 x 4 quasi-Latin square design with a 2(3) factorial arrangement of treatments was used. The dietary factors were: extent of barley grain processing, coarse (1.60 mm) or flat (1.36 mm); forage-to-concentrate ratio (F:C), low (35:65) or high (55:45) (dry matter basis); and forage particle length, long (7.59 mm) or short (6.08 mm). Eight lactating cows with ruminal and duodenal cannulas were offered ad libitum access to total mixed diets. Chewing time, expressed as minutes per day or per kilogram of dry matter or neutral detergent fiber (NDF), was increased with high F:C diets due to increased eating and ruminating times but was decreased when expressed per kilogram of NDF intake from forage. The influence of forage particle length or grain processing on chewing activity was less pronounced than F:C ratio. Chewing activity was positively correlated to proportion of long forage particles in the diet but not to particle length of the diets. Influence of feed particle size on particle size distribution in different sites of the digestive tract was minimal. Particle size distributions of duodenal digesta and feces differed; the proportion of particles retained on the 3.35- or 1.18-mm screens was higher, but proportion of particles that passed through the 1.18-mm screen was lower in duodenal digesta than in feces. Relationships between chewing activities and ruminal pH or fractional passage rate of rumen contents were not significant. These results indicate that particle size of barley-based diets was not a reliable indicator of chewing activity. Forage particle size and NDF content of the diets were more reliable indicators of chewing activity than was the NDF content of forage. Fecal particle size was not an appropriate means of estimating the size of particles exiting the reticulorumen, at least for barley-based diets. Breakdown of coarse particles was necessary, but not a rate-limiting step for particles exiting the rumen. Passage rate of the rumen contents was affected by numerous factors including chewing activity.

Dietary factors that alter the intake of effective fiber were evaluated for their effects on rumen fermentation, digestion, and milk production using a double 4 x 4 quasi-Latin square design with a 2(3) factorial arrangement of treatments. The dietary factors were extent of barley grain processing, coarse (1.60 mm) or flat (1.36 mm); forage-to-concentrate (F:C) ratio, low (35:65) or high (55:45) (dry matter basis); and forage particle length, long (7.59 mm) or short (6.08 mm). Eight lactating cows with ruminal and duodenal cannulas were offered ad libitum access to a total mixed diet and milked twice daily. Dry matter intake was increased by increasing the extent of grain processing. Mean rumen pH was lower for cows fed flatly rolled barley than for cows fed coarsely rolled barley, whereas F:C ratio or forage particle size had no effect on rumen pH. Rumen pH was not correlated with effective NDF intake but tended to be correlated with digestibility of starch in the rumen. Total tract digestibilities of dry matter, organic matter, starch, and neutral detergent fiber were increased by feeding flatly rolled barley or low F:C ratio diets. Milk yield and milk protein content were higher in cows fed flatly rolled barley or low F:C ratio diets. Milk fat content tended to increase with high F:C ratio or long forage particle length but was reduced by feeding flatly rolled barley. In this study, extent of grain processing and intake of ruminal available starch were the most influential factors affecting milk production. Reducing the ratio of F:C improved total digestion and actual milk production. Forage particle length had minimal impact on digestibility and milk production.

Holstein cows (n = 43) in early lactation were used to investigate effects of method of adding fibrolytic enzymes to diets on feed intake, milk production, and digestibility. Cows were blocked according to parity and calving date and randomly assigned to three treatments: control, enzymes applied to the total mixed ration (E-TMR), or enzymes added to the barley-based concentrate (E-Conc). The enzyme product used was a proprietary blend that contained relatively high xylanase and low cellulase activities (Biovance Technol. Inc., Omaha, NE). An enzyme solution (50 mg of enzyme powder dissolved into 20 ml of water) was sprayed onto each kilogram of total mixed ration (dry matter basis) before feeding. Alternatively, 73 g of enzyme powder, dissolved in 20 L of water, was added per ton of concentrate (50 mg of enzyme/kg of diet dry matter). The total mixed rations contained 24% corn silage, 14% alfalfa hay, and 62% concentrate (dry matter basis) and were offered ad libitum. In vitro gas production assays and two experiments using sheep were conducted to measure the effects of enzyme treatment on digestibility. Dry matter intake (mean: 19.8 kg/d) was not affected by enzyme supplementation. Milk yield (kg/d) was higher for cows fed E-Conc (37.4) than for cows fed control (35.3) or E-TMR (35.2) with no effects on milk composition. Total tract digestibility (%) of dry matter was higher for E-Conc (66.6%) than for the control diet (63.9%) and intermediate for E-TMR (65.7%) when measured in dairy cows. However, the digestibility of the diets was substantially higher when measured in sheep than in dairy cows, with no effects of enzyme supplementation. The results indicate that fibrolytic enzymes have the potential to increase digestibility and milk production in dairy cows because digestion is low relative to potential digestibility. When digestion is higher, as was observed in lambs or in vitro, no improvement in digestibility occurs. Method of enzyme delivery must also be considered to maximize the benefits of using fibrolytic enzymes in dairy cow diets.

The mechanism by which enzyme additives improve feed digestion in ruminants is not fully understood. Direct hydrolysis of feed in the rumen is a potential mode of action, but the importance of this mode needs to be quantified because of the relatively low exogenous hydrolase activity added compared with the total activity present in the rumen. We examined the interactions between ruminal and exogenous enzymes on fiber degradation using a completely randomized experimental design, with an 11 (enzyme preparations and their combinations) x 5 (assay pH) arrangement of treatments. Ruminal enzymes were extracted from cattle receiving high fiber or high concentrate diets and exogenous enzymes were Trichoderma longibrachiatum preparations containing different proportions of xylanase and cellulase activities. Ruminal and exogenous enzyme preparations and their combinations were tested for the ability to degrade soluble cellulose, xylan, and corn silage over a range of pH from 4.5 to 6.5 at 39 degrees C. T. longibrachiatum enzymes acted synergistically with enzymes from mixed rumen microorganisms in degrading soluble cellulose, xylan, and corn silage. Hydrolysis increased by up to 35, 100, and 40% for soluble cellulose, xylan, and corn silage, respectively, and was most evident at a pH range between 5.0 and 6.0. The synergistic effect between ruminal and exogenous enzymes increases the hydrolytic potential within the rumen environment and is likely a significant mechanism by which enzyme additives improve feed digestion.

Effects of barley processing on site and extent of digestion and milk production in dairy cows were evaluated in a 4 x 4 Latin square design with four lactating cows with ruminal and duodenal cannulas. Barley grain was steam-rolled to four thicknesses: coarse, medium, medium-flat, and flat. The processing index (PI), measured as volume weight of barley after processing expressed as a percentage of its volume weight before processing, was 81.0, 72.5, 64.0, and 55.5% for the four treatments, respectively. Diets consisted of 53% concentrate (dry matter basis) containing one of the four processed barleys. Cows were offered ad libitum access to a total mixed ration three times daily. Dry matter intake was quadratically increased with decreasing PI, with maximum intake for cows fed medium-flat barley. Although ruminal digestibilities of organic matter, starch, and crude protein were not affected by grain processing, intestinal and total tract digestibilities were linearly increased as PI of barley was reduced. Milk yield was quadratically increased (25.6, 28.1, 30.8, and 29.0 kg/d) with decreasing PI, and maximum milk yield was for cows fed medium-flat barley. Milk fat and lactose contents were similar, but milk protein content was increased with decreasing PI. These results indicate that the optimal extent of barley processing for dairy cows fed diets supplying adequate fiber was medium-flat, corresponding to a processing index of about 64%. Coarsely or flatly rolled barley is not recommended, because extensive processing did not further improve intake of digestible nutrients, and coarsely processed barley resulted in the lowest intake of digestible organic matter; hence, lowest milk production. Processing index is a reliable and practical method to quantitatively measure extent of steam rolling.

Twenty multiparous lactating Holstein cows in early lactation were used to investigate effects of exogenous fibrolytic enzyme supplementation on dry matter intake, milk production, and digestibility. Cows were blocked according to parity, expected calving date, and milk yield in the previous lactation, and then randomly assigned after calving to two treatments: control or enzyme. The enzyme mixture, which contained mainly xylanase and cellulase activities (Pro-Mote, Biovance Technol. Inc., Omaha, NE), was added to the concentrate to supply 1.3 g/kg of total mixed ration (dry matter basis). The total mixed rations contained 24% corn silage, 15% alfalfa hay, and 61% barley concentrate (dry matter basis) and were offered for ad libitum intake. Enzyme addition did not affect dry matter intake. However, total digestibility of nutrients, determined using Cr2O3, was dramatically increased by enzyme treatment (dry matter, 61.7 vs. 69.1%; neutral detergent fiber, 42.5 vs. 51.0%; acid detergent fiber, 31.7 vs. 41.9%; crude protein, 61.7 vs. 69.8%). Consequently, milk yield tended to increase (35.9 vs. 39.5 kg/d). Percentage of milk fat was lower, and percentages of milk protein tended to be lower for cows fed a diet supplemented with enzymes, such that component yields were similar for cows fed either diet. Energy deficiency was numerically lower for cows fed a diet supplemented with enzymes than for cows fed the control diet (-3.62 vs. -3.33 Mcal/d). Supplementing dairy cow diets with a fibrolytic enzyme mixture has the potential to enhance milk yield and nutrient digestibility of cows in early lactation without changing feed intake.

Four lactating, cannulated Holstein cows were used in a 4 x 4 Latin square design to investigate the effects of grain source and fibrolytic enzyme supplementation on ruminal fermentation, nutrient digestion in the rumen and in the intestine, and milk production. A 2 x 2 factorial arrangement was used; two grains (barley and hull-less barley) were combined with and without enzyme. The enzyme supplement (Pro-Mote; Biovance Technologies Inc., Omaha, NE) contained primarily cellulase and xylanase activities and was applied daily to the total mixed diet. Dry matter intake was not affected by diet, but starch intake was greatest when hull-less barley was fed. Starch from hull-less barley was more digestible in the rumen and in the total tract than was starch from barley, but opposite results occurred for fiber digestion, indicating that hull-less barley depressed fiber digestion. As a result, cows fed the hull-less barley diets tended to produce more milk with a higher milk lactose content than did cows fed the barley diets. Enzyme supplementation had minimal effects on ruminal digestion but increased nutrient digestibility in the total tract and the proportion of microbial N in nonammonia N. Consequently, cows fed diets supplemented with enzyme had a higher milk protein content and tended to produce more 4% fat-corrected milk than did control cows. These results indicate that the use of hull-less barley rather than barley increased the digestible energy intake of dairy cows, resulting in higher milk production. The use of a fibrolytic enzyme mixture enhanced feed digestibility and milk production.

A study was conducted using lactating Holstein cows with ruminal and duodenal cannulas in a 4 x 4 Latin square design to investigate fibrolytic enzyme supplementation on site and extent of nutrient digestion. The four diets consisted of 45% concentrate, 10% barley silage, and 45% cubed alfalfa hay (dry matter basis) and differed in enzyme supplementation: 1) control cubes, 2) cubes treated with 1 g of enzyme mixture/kg of cubes, 3) cubes treated with 2 g of enzyme mixture/kg of cubes, and 4) both concentrate and cubes treated with 1 g of enzyme mixture/kg of dry matter. The enzyme supplement contained primarily cellulase and xylanase activities. Digestion of organic matter and neutral detergent fiber in the total tract was higher for cows fed the high dosage of enzyme than for cows fed the control cubes. Ruminal digestibility of crude protein was higher, but that of organic matter and neutral detergent fiber was only numerically higher, for cows fed the high dosage of enzyme compared with that of cows fed the control cubes. Higher ruminal digestibility associated with the high dosage of enzyme resulted in more microbial protein synthesis. Milk production increased for cows fed the high dosage of enzyme compared with cows fed the control cubes and effects of the addition of enzyme on milk composition were minimal. The results demonstrated the benefits of using a fibrolytic enzyme additive to enhance feed digestion and milk production by dairy cows. The response to enzyme supplementation was affected more by amount of enzyme than by whether the enzyme was added to forage or concentrate.

Twelve multiparous and 12 primiparous lactating Holstein cows were used to compare the effects of hull-less barley with barley and corn on dry matter intake (DMI), digestibility, and milk production. Three concentrates were formulated using steam-rolled grains: barley, hull-less barley, or corn. During three 21-d periods, cows received a total mixed diet consisting of 60% concentrate, 30% barley silage, and 10% cubed alfalfa hay [dry matter (DM) basis]. Milk production and DMI were higher for cows fed the corn diet than for cows fed the barley or hull-less barley diets; no interaction with parity was detected. The DMI of cows fed the hull-less barley and barley diets were similar. Despite the higher estimated energy density of the hull-less barley diet, milk production was similar for cows fed the hull-less barley and barley diets because of the lower digestibility of the hull-less barley. Results of an in situ study showed that, for steam-rolled grains, DM and starch from hull-less barley were less degradable than were DM and starch from barley, although the opposite result was observed for ground grains. For steam-rolled hull-less barley, low ruminal degradabilities of DM and starch were apparently not compensated by high intestinal digestibility because total tract digestibility and milk production were lower than expected. Although the net energy for lactation value of hull-less barley is higher than that for barley, milk production by cows might be limited unless hull-less barley is adequately processed to ensure high ruminal and total tract digestibilities.

Six lactating, cannulated Holstein cows were used in a double 3 x 3 Latin square design to compare the effects of hull-less barley with barley and corn on ruminal fermentation, rate of passage, flow of nutrients to the duodenum, and milk production. Diets consisted of 60% concentrate, 30% barley silage, and 10% alfalfa hay (dry matter basis). Concentrates contained steam-rolled grains: hull-less barley, barley, or corn. Dry matter intake was unaffected by grain source, but starch intake tended to be greatest when hull-less barley or corn was fed. The barley diet was more degradable in the rumen than was the hull-less barley or corn diet, and, therefore, flow of microbial organic matter to the duodenum was greatest for cows fed the barley diet. Flow of microbial N to the duodenum was greater (50 g/d) for cows fed the barley diet than for cows fed the other diets, and the flow of ruminally undegradable N was greater (43 and 28 g/d) for cows fed the hull-less barley and corn diets, respectively, than for cows fed the barley diet. As a result, flow of nonammonia N to the duodenum was unaffected by grain source. Total tract apparent digestibility was highest for cows fed the barley and corn diets. Despite its low digestibility, cows fed the hull-less barley diet produced a similar amount of milk as did cows fed the barley and corn diets. Further studies are needed to evaluate the effects of processing hull-less barley on its utilization by dairy cows.

Five primiparous and 5 multiparous Holstein cows were used in an experiment with a double 5 x 5 Latin square design to evaluate the effects of lowering the concentration of nonstructural carbohydrates in high grain diets on intake, chewing activities, digestibility, and yield and composition of milk. Cows received diets consisting of 30% barley silage (dry matter basis) and one of five isocaloric concentrates containing corn, hull-less barley, or barley, ranging in the percentage of nonstructural carbohydrates from 24 to 42%. Diet did not affect the dry matter intake (DMI) of primiparous cows. However, for multiparous cows, as the concentration of nonstructural carbohydrates in the corn diets was lowered from 34 to 30%, DMI increased from 16.3 to 18.9 kg/d (2.66 to 3.07% of body weight), and, as the concentration of nonstructural carbohydrates in the barley diets was lowered from 29 to 22%, DMI increased from 19.6 to 21.0 kg/d (3.09 to 3.19% of body weight). For both groups of cows, a reduction in the nonstructural carbohydrates in either the corn or barley diets had minimal effects on milk yield, fat-corrected milk yield, fat content, and protein content, except that the fat content of milk from younger cows fed barley diets was lowered (2.70% vs. 2.30%). For diets formulated at similar concentrations of nonstructural carbohydrates, grain source had no effect on DMI or digestible DMI, but milk yield was lower for cows fed the diet containing hull-less barley (25.2 kg/d) than for cows fed the diets containing corn (26.8 kg/d) or barley (28.1 kg/d). When the concentrations of nonstructural carbohydrates in high grain diets were reduced, feed intake was enhanced, but the relationship between the concentration of nonstructural carbohydrates and performance was not consistent. Differences in ruminal availability of carbohydrates need to be accounted for when diets are formulated on the basis of nonstructural carbohydrates using a range of cereal grains.

Two studies were conducted to evaluate the effects of Cr supplementation on blood metabolite and hormonal responses of Holstein cows to glucose challenges during late pregnancy and early lactation and to propionate challenges during early lactation. Eight multiparous and 4 primiparous cows (Experiment 1) and 12 primiparous cows (Experiment 2) were assigned to one of two treatments: control and 0.5 ppm of supplemental Cr. The glucose challenges were performed at 2 wk prepartum and at 2 wk postpartum, and the propionate challenges were conducted at wk 2 and 6 postpartum. During glucose tolerance tests, Cr supplementation reduced the ratio of insulin to glucose and reduced plasma concentrations of insulin and triglycerides of primiparous cows during the prepartum period. Chromium supplementation decreased plasma Cr of primiparous cows following glucose challenge. With supplemental Cr, insulin sensitivity was reduced postpartum, particularly for primiparous cows, but insulin sensitivity was increased prepartum. Results of this study suggested that primiparous cows experienced Cr deficiency during late pregnancy and possibly during early lactation. Following propionate infusion, Cr supplementation increased the serum glucose peak, increased the area under the response curve for serum glucose, and tended to increase IGF-I concentrations. Chromium supplementation tended to reduce the ratio of insulin to glucagon. Supplementation might have enhanced gluconeogenesis or glycogenolysis. Supplemental Cr also resulted in reduced variability of most parameters during both experiments.