Plasma and milk metabolomics revealed changes in amino acid metabolism in Holstein dairy cows under heat stress.

Our understanding of metabolic alterations triggered by heat stress is incomplete, which limits the designing of nutritional strategies to mitigate negative productive and health effects. Thus, this study aimed to explore the metabolic responses of heat-stressed dairy cows to dietary supplementation with vitamin D3/Ca and vitamin E/Se. Twelve multiparous Holstein cows were enrolled in a split-plot Latin square design with two distinct vitamin E/Se supplementation levels, either at a low (ESe-, n = 6, 11.1 IU/kg vitamin E and 0.55 mg/kg Se) or a high dose (ESe+, n = 6 223 IU/kg vitamin E and 1.8 mg/kg Se) as the main plot. Treatment subplots, arranged in a replicated 3 × 3 Latin square design, comprised heat challenge (Temperature Humidity Index, THI: 72.0-82.0) supplemented with different levels of vitamin D3/Ca: either low (HS/DCa-, 1 012 IU/kg and 0.73%, respectively) or high (HS/DCa+, 3 764 IU/kg and 0.97%, respectively), and a pair-fed control group in thermoneutrality (THI = 61.0-64.0) receiving the low dose of vitamin D3/Ca (TN). The liquid chromatography-mass spectrometry-based metabolome profile was determined in blood plasma and milk sampled at the beginning (day 0) and end (day 14) of each experimental period. The results were analyzed for the effect of (1) TN vs. HS/ESe-/DCa-, and (2) the vitamin E/Se and vitamin D3/Ca supplementation. No group or group × day effects were detected in the plasma metabolome (false discovery rate, FDR > 0.05), except for triglyceride 52:2 being higher (FDR = 0.03) on day 0 than 14. Taurine, creatinine and butyryl-carnitine showed group × day interactions in the milk metabolome (FDR ≤ 0.05) as creatinine (+22%) and butyryl-carnitine (+190%) were increased (P < 0.01) on day 14, and taurine was decreased (-65%, P < 0.01) on day 14 in the heat stress (HS) cows, compared with day 0. Most compounds were unaffected by vitamin E/Se or vitamin D3/Ca supplementation level or their interaction (FDR > 0.05) in plasma and milk, except for milk alanine which was lower (-69%, FDR = 0.03) in the E/Se+ groups, compared with E/Se-. Our results indicated that HS triggered more prominent changes in the milk than in the plasma metabolome, with consistent results in milk suggesting increased muscle catabolism, as reflected by increased creatinine, alanine and citrulline levels. Supplementing with high levels of vitamin E/Se or vitamin D3/Ca or their combination did not appear to affect the metabolic remodeling triggered by HS.

Dietary supplementation of vitamin D3 and calcium partially recover the compromised time budget and circadian rhythm of lying behavior in lactating cows under heat stress.

Heat stress (HS) impedes cattle behavior and performance and is an animal comfort and welfare issue. The objective of this study was to characterize the time budget and circadian rhythm of lying behavior in dairy cows during HS and to assess the effect of dietary supplementation of vitamin D3 and Ca. Twelve multiparous Holstein cows (42.2 ± 5.6 kg milk/d; 83 ± 27 d in milk) housed in tiestalls were used in a split-plot design with the concentration of dietary vitamin E and Se as main plots (LESe: 11.1 IU/kg and 0.55 mg/kg, and HESe: 223 IU/kg and 1.8 mg/kg, respectively). Within each plot cows were randomly assigned to (1) HS with low concentrations of vitamin D3 and Ca (HS, 1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D3 and Ca (HS+D3/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) thermoneutral pair-fed (TNPF) with low concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73%, respectively) in a Latin square design with 14-d periods and 7-d washouts. Lying behavior was measured with HOBO Loggers in 15-min intervals. Overall, cows in HS spent less time lying per day relative to TNPF from d 7 to 14. Daily lying time was positively correlated with milk yield, energy-corrected milk yield, and feed efficiency, and was negatively correlated with rectal temperature, respiratory rate, fecal calprotectin, tumor necrosis factor-α, and C-reactive protein. A treatment by time interaction was observed for lying behavior: the time spent lying was lesser for cows in HS than in TNPF in the early morning (0000-0600 h) and in the night (1800-2400 h). The circadian rhythm of lying behavior was characterized by fitting a cosine function of time into linear mixed model. Daily rhythmicity of lying was detected for cows in TNPF and HS+D3/Ca, whereas only a tendency in HS cows was observed. Cows in TNPF had the highest mesor (the average level of diurnal fluctuations; 34.2 min/h) and amplitude (the distance between the peak and mesor; 17.9 min/h). Both the mesor and amplitude were higher in HS+D3/Ca relative to HS (26.6 vs. 25.2 min/h and 3.91 min/h vs. 2.18 min/h, respectively). The acrophase (time of the peak) of lying time in TNPF, HS, and HS+D3/Ca were 0028, 0152, and 0054 h, respectively. Lastly, a continuous increase in daily lying time in TNPF was observed during the first 4 d of the experimental period in which DMI was gradually restricted, suggesting that intake restrictions may shift feeding behavior and introduce biases in the behavior of animals. In conclusion, lying behavior was compromised in dairy cows under HS, characterizing reduced daily lying time and disrupted circadian rhythms, and the compromised lying behavior can be partially restored by supplementation of vitamin D3 and Ca. Further research may be required for a more suitable model to study behavior of cows under HS.

Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium.

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D3 and Ca (HS+D3/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D3/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D3/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D3/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D3/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D3/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D3/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D3/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D3 and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.

Effects of direct-fed Bacillus subtilis and Bacillus licheniformis on production performance and milk fatty acid profile in dairy cows.

The aim of the study was to determine the effect of a Bacillus-based direct-fed microbial on performance of mid-lactating Holstein dairy cows and on their milk fatty acid composition. Six multiparous cows fitted with a rumen cannula were used in a randomized replicated crossover design. Cows received 200 g/d of either whey powder as a control or BioPlus 2B (Chr. Hansen), a commercial direct-fed microbial providing Bacillus subtilis and Bacillus licheniformis, representing a daily dose of 6.4 × 1011 cfu, and using whey powder as a carrier. The 2 experimental periods lasted 14 d and were separated by a 7-d washout interval. Samples were collected on d 0, 13, and 14 of each period. Data from d 0 were used as covariate. Significance was declared at P ≤ 0.05 and tendency at 0.05

The impact of environmental and nutritional stresses on milk fat synthesis in dairy cows.

Stress reduces milk and milk components synthesis and increases maintenance requirements of cows. The major stress-related alterations involve enhanced secretion of glucocorticoids and increased sympathetic nervous system activity, which results in biochemical and physiologic changes. In dairy cows exposed to social (ie housing conditions, overstocking, regrouping, feed delivery), physiological (ie initiation of lactation and parturition), or physical (ie heat or cold stress) stressors, responses involve alterations in energy balance and nutrient partitioning. The capacity of the animal to synthesize milk fat largely depends on the availability of substrates for lipid synthesis from the diet, ruminal fermentation or adipose tissue stores, all of which can be altered under stress conditions. Indeed, milk fat concentration is particularly responsive to diet and environment modifications, where a wide range of nutritional and non-nutritional factors influence milk fat output. Milk fat synthesis is an energy demanding process, and extremely sensitive to stress factors during lactation and the involvement of multiple organs. Recent studies examining social, physical, and physiological stressors have provided important insights into how differences in milk yield and milk components may be associated with biological responses to stress factors in dairy cows. This review focuses primarily on the role of stress sources and indicators to which the dairy cow is exposed in regulating milk fat synthesis. We will review the role of nutritional and non-nutritional factors on milk fat synthesis in dairy cows under stress conditions.

Effect of dietary supplementation or cessation of magnesium-based alkalizers on milk fat output in dairy cows under milk fat depression conditions.

We aimed to evaluate the effects of dietary supplementation with magnesium oxide and calcium-magnesium dolomite on milk fat synthesis and milk fatty acid profile or persistency in milk fat synthesis after their cessation in dairy cows under milk fat depression conditions. Twenty-four multiparous dairy cows in early lactation (mean ± standard deviation; 112 ± 14 d in milk) were used in a randomized complete block design. Milk fat depression was induced in all cows for 10 d by feeding a diet containing 35.2% starch, 28.7% neutral detergent fiber, and 4.8% total fatty acid (dry matter). The experiment was conducted in 2 periods. During the Mg-supplementation period (d 1-20), cows were randomly assigned to (1) the milk fat depression diet used during the induction phase (control; n = 8), (2) the control diet plus 0.4% magnesium oxide (MG; n = 8), or (3) the control diet plus 0.8% calcium-magnesium dolomite (CMC; n = 8). Compared with the control group, feeding the magnesium-supplemented diets increased milk fat concentration and yield by 12% within 4 d. During the 20-d Mg-supplementation period, both the MG and CMC diets increased milk fat concentration and yield, as well as 3.5% fat-corrected milk and energy-corrected milk yield, without affecting dry matter intake, milk yield, and milk protein and lactose concentrations. In the Mg-cessation period (d 21-30), all cows received the control diet, which resulted in a greater milk fat concentration and yield in the cows that had already received the MG and CMC diets in the Mg-supplementation period. Whereas, milk fat concentration and yield remained high after discontinuation of the magnesium-containing alkalizer until d 27. The difference in milk fat synthesis was associated with lower trans-10 C18:1 (-22%) and higher trans-11 C18:1 (+12.5%) concentrations in milk during the Mg-supplementation period. Furthermore, it was evident that within 2 d of supplementation, the trans-10:trans-11 ratio was lower in MG and CMC cows compared with cows receiving the control. This suggested that the effect of magnesium-based alkalizers on milk fat synthesis was mediated via a shift in ruminal biohydrogenation of cis-9,cis-12 C18:2 in the rumen. In conclusion, abrupt addition of magnesium oxide and calcium-magnesium dolomite increased milk fat synthesis, which persisted for 7 d after cessation of magnesium-based alkalizers. A similar ability to recover milk fat synthesis and normal fatty acid biohydrogenation pathways was observed for magnesium oxide and calcium-magnesium dolomite.

Potassium carbonate as a supplement to improve milk fat concentration and yield in early-lactating dairy goats fed a high-starch, low-fiber diet.

This study investigated the use of K2CO3 as dietary buffer to prevent or to recover from low milk fat production when early-lactating dairy goats are fed a high-starch, low-fiber (HSLF) diet. At kidding, 30 Alpine goats housed in pens with Calan gate feeders received a total mixed ration with a forage-to-concentrate ratio of 55:45 on a dry matter (DM) basis for a baseline period of 27 ± 4 d. Goats (milk yield, 4.14 ± 0.88 kg/d; milk fat, 4.28 ± 0.52%; mean ± SD) were then assigned to 1 of 10 blocks according to parity (first vs. second or more) and milk fat concentration, and fed a HSLF diet containing 45% forages and 55% concentrates for 2 experimental periods of 28 d. Treatments were identified as (1) control, in which the HSLF diet was fed throughout both periods; (2) preventive, in which the HSLF diet supplemented with K2CO3 (1.6% of DM) was fed during both periods; and (3) recovery, in which the HSLF diet was fed during the first period (P1) and the HSLF diet supplemented with K2CO3 was fed during the second period (P2). Data from P1 and P2 were analyzed separately. In P1, preplanned contrasts were used to evaluate the preventive effect of K2CO3 (control and recovery, both groups receiving the same diet during this period, vs. preventive), and in P2, to assess the potential of K2CO3 to alleviate an already existing state of low milk fat (control vs. recovery and preventive vs. recovery). Feeding the HSLF diet in P1 moderately decreased milk fat concentration (-16%) and yield (-13%) as compared with baseline. Dietary addition of K2CO3 decreased DM intake by 12 and 14% in P1 and P2, respectively. Ruminal pH was not different among treatments. There was also no significant difference in milk yield (4.13 and 3.71 kg/d on average in P1 and P2, respectively) for any tested contrasts. In P1, milk fat concentration and yield did not differ among goats fed control (3.58% and 151 g/d, respectively) and preventive (3.67% and 148 g/d, respectively) diets. In P2, milk fat concentration and yield did not differ among goats fed the control diet (3.38% and 137 g/d, respectively), and diets where K2CO3 was used as preventive (3.44% and 126 g/d, respectively) or recovery treatment (3.25% and 113 g/d, respectively). Supplementing a high-concentrate diet with 1.6% K2CO3 was therefore not effective in either preventing or suppressing already existing conditions of low milk fat production in dairy goats.

Transient reductions in milk fat synthesis and their association with the ruminal and metabolic profile in dairy cows fed high-starch, low-fat diets.

Sub-acute ruminal acidosis (SARA) is sometimes observed along with reduced milk fat synthesis. Inconsistent responses may be explained by dietary fat levels. Twelve ruminally cannulated cows were used in a Latin square design investigating the timing of metabolic and milk fat changes during Induction and Recovery from SARA by altering starch levels in low-fat diets. Treatments were (1) SARA Induction, (2) Recovery and (3) Control. Sub-acute ruminal acidosis was induced by feeding a diet containing 29.4% starch, 24.0% NDF and 2.8% fatty acids (FAs), whereas the Recovery and Control diets contained 19.9% starch, 31.0% NDF and 2.6% FA. Relative to Control, DM intake (DMI) and milk yield were higher in SARA from days 14 to 21 and from days 10 to 21, respectively (P < 0.05). Milk fat content was reduced from days 3 to 14 in SARA (P < 0.05) compared with Control, while greater protein and lactose contents were observed from days 14 to 21 and 3 to 21, respectively (P < 0.05). Milk fat yield was reduced by SARA on day 3 (P < 0.05), whereas both protein and lactose yields were higher on days 14 and 21 (P < 0.05). The ruminal acetate-to-propionate ratio was lower, and the concentrations of propionate and lactate were higher in the SARA treatment compared with Control on day 21 (P < 0.05). Plasma insulin increased during SARA, whereas plasma non-esterified fatty acids and milk ß-hydroxybutyrate decreased (P < 0.05). Similarly to fat yield, the yield of milk preformed FA (>16C) was lower on day 3 (P < 0.05) and tended to be lower on day 7 in SARA cows (P < 0.10), whereas yield of de novo FA (<16C) was higher on day 21 (P < 0.01) in the SARA group relative to Control. The t10- to t11-18:1 ratio increased during the SARA Induction period (P < 0.05), but the concentration of t10-18:1 remained below 0.5% of milk fat, and t10,c12 conjugated linoleic acid remained below detection levels. Odd-chain FA increased, whereas branched-chain FA was reduced during SARA Induction from days 3 to 21 (P < 0.05). Sub-acute ruminal acidosis reduced milk fat synthesis transiently. Such reduction was not associated with ruminal biohydrogenation intermediates but rather with a transient reduction in supply of preformed FA. Subsequent rescue of milk fat synthesis may be associated with higher availability of substrates due to increased DMI during SARA.

Abomasally infused SFA with varying chain length differently affect milk production and composition and alter hepatic and mammary gene expression in lactating cows.

The aim of the present study was to compare the effects of post-ruminally infused fat supplements, varying in fatty acid (FA) chain length, on animal performance, metabolism and milk FA. Eleven multiparous Holstein dairy cows were used in a replicated incomplete 3 × 3 Latin square design with 7-d periods, separated by 7-d washouts. Treatments were administered as abomasal infusions of enrichments providing 280 g/d of FA: (1) palmitic acid (98·4 % 16 : 0; PA), (2) caprylic and capric acids (56·2 % 8 : 0, 43·8 % 10 : 0; medium-chain TAG (MCT)) and (3) stearic acid (99·0 % 18 : 0; SA). Relative to PA, SA decreased the efficiency of fat-corrected milk production, which was associated with a tendency for higher DM intake and lower FA absorption with SA, whereas MCT was not different from PA for these variables. Milk fat concentration and yield were increased by PA relative to SA, but only fat yield tended to be greater relative to MCT. Relative to PA, MCT increased milk fat concentration of FA < 16 C, whereas SA increased FA > 16 C. Expression of mammary stearoyl-coA desaturase 1 was lower with SA than with PA. Relative to PA, liver expression of adenosine monophosphate-activated protein kinase-1 and pyruvate kinase was increased with MCT, whereas expression of these genes tended to be increased by SA. The mechanism of increased fat secretion with PA does not seem to be related to a modulation of the expression of lipogenesis-related genes, but rather to increased substrate availability as reflected by milk FA profile.

Technical note: An in vivo method to determine kinetics of unsaturated fatty acid biohydrogenation in the rumen.

Rumen microbial biohydrogenation (BH) of unsaturated fatty acids (UFA) has been extensively studied in vitro; however, in vitro BH pathways, rates, and extents may not parallel those in vivo. The objective was to develop an assay to assess in vivo rates, pathways, and extent of BH of oleic (OA), linoleic (LA), and α-linolenic (ALA) acids. Each UFA was characterized in a separate experiment, each using 4 ruminally cannulated lactating Holstein cows. A single bolus consisting of 200 g of a UFA-oil [experiment 1 (EXP1): 87% OA sunflower, experiment 2 (EXP2): 70% LA safflower, and experiment 3 (EXP3): 54% ALA flaxseed] and 12 g of heptadecanoic acid (C17:0) was mixed into the rumen through the fistula. Rumen digesta was collected at -1, -0.25, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h relative to the bolus. Overall, the triglyceride boluses increased total fatty acids (FA) in the rumen from 3.9 (standard deviation = ±1.4) to 7.3% (±1.4) of rumen dry matter and enriched C17:0 from 0.4 (±0.1) to 2.5% (±0.5) of FA. The bolus enriched OA from 8.9 (±1.0) to 30.1% (±4.6) of FA in EXP1, LA from 11.1 (±1.8) to 35.9% (±5.0) of FA in EXP2, and ALA from 2.1 (±0.1) to 19.8% (±4.3) of FA in EXP3. The disappearances of C17:0, OA, LA, and ALA were fit to a single exponential decay model. The first-order rate of C17:0 rumen disappearance (turnover) was 9.1, 6.9, and 5.2%/h in EXP1, EXP2, and EXP3, respectively, and was used as a marker of FA passage. The rate of total rumen turnover of OA was 54.1%/h, LA was 60.5%/h, and ALA was 93.0%/h in EXP1, EXP2, and EXP3, respectively. Rumen concentration of all 3 UFA reached prebolus concentrations within 4 h. The calculated extent of lipolysis and initial isomerization was 85.6% for OA, 89.8% for LA, and 94.7% for ALA in EXP1, EXP2, and EXP3, respectively. Assuming that BH equals total disappearance minus passage, the rates of lipolysis and initial isomerization were 45.0, 53.6, and 87.8%/h for OA, LA, and ALA in EXP1, EXP2, and EXP3, respectively. Analysis of the data using compartmental modeling showed that the normal BH pathways proposed in the literature explained 46.0, 37.3, and 49.8% of the BH of OA, LA, and ALA in EXP1, EXP2, and EXP3, respectively. Based on the model, BH of trans C18:1 FA was the rate-limiting step to complete BH. Importantly, oils were provided as triglycerides and the reported rates represent the rate of lipolysis and BH. In conclusion, the rate of ruminal BH of OA, LA, and ALA was higher than that commonly observed in vitro, but the extent of BH was near expected values. The method developed provides a potential in vivo assay of ruminal BH for use in future experiments and modeling efforts.

Alterations in ruminal bacterial populations at induction and recovery from diet-induced milk fat depression in dairy cows.

Ten ruminally cannulated Holstein cows were used in a crossover design that investigated changes in ruminal bacterial populations in response to induction and recovery from diet-induced milk fat depression (MFD). Further, the effect on the ruminal microbiota of the cows with diet-induced milk fat depression inoculated with rumen contents from non-milk fat-depressed donor cows was evaluated. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber, high-starch, and high-polyunsaturated fatty acid diet (26.1% neutral detergent fiber, 28.1% starch, 5.8% total fatty acids, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. Induction was followed by a recovery phase, where all cows were switched to a high-fiber, low-starch, and low-polyunsaturated fatty acid diet (31.8% neutral detergent fiber, 23% starch, 4.2% total fatty acids, and 1.2% C18:2) and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Ruminal samples were collected at the end of induction (d 10) and during recovery (d 13, 16, and 28), separated to solid and liquid fractions, extracted for DNA, PCR- amplified for the V1-V2 region of the 16S rRNA gene, and analyzed for bacterial diversity. Results indicated that bacterial communities were different between fractions. In each fraction, differences were significant between the induction (d 10) and recovery (d 13, 16, and 28) periods; however, differences were less apparent with time during the recovery period. The MFD (d 10) was typified by a reduction in the relative sequence abundance of Bacteroidetes and an increase in the relative sequence abundance of Firmicutes and Actinobacteria across both fractions. At the genus level, relative sequence abundance of unclassified Lachnospiraceae, Butyrivibrio, Bulleidia, and Coriobacteriaceae were higher on d 10 and were positively correlated with trans-10,cis-12 CLA and the trans-10 isomer, suggesting their potential role in altered biohydrogenation reactions. A switch to the recovery diet resulted in a sharp increase in the Bacteroidetes lineages and a decrease in Firmicutes members on d 13; however, this shift appears to stabilize by d 28, indicating the restoration process for ruminal bacteria from an altered state is gradual and complex. Inoculation of 10% of rumen contents from non-MFD donor cows to MFD cows revealed this procedure had transient effects on only a few bacterial populations, and such effects disappeared after d 16 following cessation of inoculation. It can be concluded that alterations in milk FA profiles at induction are preceded by microbial alterations in the rumen driven by dietary changes.

Short communication: Effects of lysolecithin on milk fat synthesis and milk fatty acid profile of cows fed diets differing in fiber and unsaturated fatty acid concentration.

Thirteen multiparous Holstein cows were used in a crossover design that tested the effect of lysolecithin in diets differing in neutral detergent fiber (NDF) and unsaturated fatty acid (FA) concentrations. Experimental periods were 20 d in length and included two 10-d phases. A standard fiber and lower fat diet was fed the first 10 d (30.5% NDF, no added oil, lower-risk phase) and a lower NDF and higher oil diet was fed during the second 10 d (29.0% NDF and 2% oil from whole soybeans and soybean oil, high-risk phase). Treatments were control and 10 g/d of lysolecithin (LYSO) extended in a ground corn carrier. Milk was sampled on d 0, 5, and 10 of each phase for determination of fat and protein concentration and FA profile. We found no effect of treatment or treatment by time interaction for dry matter intake, milk yield, or milk protein concentration. A treatment by time interaction was observed for milk fat concentration and yield. Milk fat concentration was higher in LYSO on d 5 of the lower-risk phase, but decreased progressively in both treatments during the high-risk phase. Milk fat yield was not different among treatments during the lower-risk phase, but was lower in LYSO on d 15 and tended to be lower on d 20 during the high-risk phase. Concentrations of milk de novo FA decreased and preformed FA increased during the high-risk phase, but we found no effect of treatment or treatment by time interactions. We noted an effect of time, but no treatment or treatment by time interactions for milk trans FA isomers. Briefly, trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid progressively decreased as trans-10 C18:1 and trans-10,cis-12 conjugated linoleic acid progressively increased during the high-risk phase. The LYSO increased milk fat concentration when feeding a higher fiber and lower unsaturated FA diet, but decreased milk fat yield when feeding a lower fiber and higher unsaturated FA diet, although biohydrogenation pathways and capacity did not appear to be modified. The effect of lysolecithin on rumen fermentation warrants further investigation, but is not recommended when feeding lower fiber and higher unsaturated fat diets.

Effect of lipid supplementation on milk odd- and branched-chain fatty acids in dairy cows.

Eight ruminally fistulated, multiparous Holstein cows were arranged in a double 4×4 Latin square with 14-d periods to investigate the effects of lipid supplementation on performance, rumen parameters, the milk odd- and branched-chain fatty acid (OBCFA) profile, and the relationships between milk OBCFA and rumen parameters. Lipid supplementation is known to inhibit microbial growth in the rumen, decrease de novo microbial fatty acid synthesis, and increase the uptake of circulating fatty acids by the mammary gland; treatments were selected to isolate these effects on the milk OBCFA profile. The 4 treatments were (1) a lipid-free emulsion medium infused in the rumen (CTL), (2) soybean oil as a source of polyunsaturated fatty acids infused in the rumen (RSO), (3) saturated fatty acids (38% 16:0, 40% 18:0) infused in the rumen (RSF), and (4) saturated fatty acids infused in the abomasum (ASF). Fat supplements were provided continuously as emulsions at a rate of 450g/d. Preplanned contrasts compared CTL to RSO, RSO to RSF, and RSF to ASF. Infusing RSO slightly decreased ruminal pH, but did not affect volatile fatty acids profile and milk fat concentration as compared with CTL. The yields of energy-corrected milk, fat, and protein were greater with RSF compared with RSO. The concentration of odd-chain fatty acids was decreased by RSO, whereas even-chain iso fatty acids were not affected. Milk fat concentration of 17:0 + cis-9 17:1 was higher for RSF than for RSO, due to the saturated fatty acids supplement containing 2% 17:0 + cis-9 17:1. Limited differences were observed in the milk OBCFA profile between RSF and ASF. A multiple regression analysis yielded the following equation for predicting rumen pH based on milk fatty acids: pH=6.24 - (0.56×4:0) + (1.67 × iso 14:0) + (4.22 × iso 15:0) + (9.41×22:0). Rumen propionate concentration was negatively correlated with milk fat concentration of iso 14:0 and positively correlated with milk 15:0, whereas the acetate-to-propionate ratio gave the opposite correlations with milk iso 14:0 and 15:0. Milk fat concentration of 17:0 + cis-9 17:1 was not related to rumen propionate or to acetate-to-propionate ratio, due to the presence of 17:0 and cis-9 17:1 in the saturated fatty acids supplement. The results suggest that although lipid supplementation can affect the profile of milk OBCFA, the promise remains of using these milk fatty acids to evaluate rumen function.

Production, composition, and oxidative stability of milk highly enriched in polyunsaturated fatty acids from dairy cows fed alfalfa protein concentrate or supplemental vitamin E.

Given its elevated content of carotenoids, alfalfa protein concentrates (APC) have the potential to prevent oxidation of milk enriched in polyunsaturated fatty acids. The effects of feeding APC or supplemental vitamin E on production, composition, and oxidative stability of milk enriched in polyunsaturated fatty acids were evaluated using 6 lactating Holstein cows (224±18d in milk) in a replicated 3×3 Latin square (21-d periods, 14d for adaptation). Treatment diets contained (dry matter basis) (1) 9% soybean meal (control, CTL); (2) 9% soybean meal + 300 IU of vitamin E/kg (VitE treatment); or (3) 9% APC (APC treatment). Cows received a continuous abomasal infusion of 450g/d of linseed oil. As a result, milk fat content of cis-9,cis-12 18:2 increased from 1.08±0.13 to 3.9±0.40% (mean ± SD), whereas cis-9,cis-12,cis-15 18:3 increased from 0.40±0.04 to 14.27±1.81% during the experimental period compared with the pretrial period. Milk yield tended to be higher for APC (14.7kg/d) compared with CTL (13.4kg/d), and was greater than that for VitE (13.0kg/d). Protein yield was higher in cows fed APC (518g/d) compared with VitE (445g/d) but was not different from that in cows fed CTL (483g/d). These effects resulted in improved milk N efficiency in cows fed APC (26.1% of N intake secreted in milk) compared with CTL (23.0%) and VitE (22.9%). Feeding APC increased milk fat content of lutein (252µg/g) compared with CTL (204µg/g) and VitE (190µg/g). Milk fat content of vitamin E was higher for APC (34.5µg/g) compared with CTL (19.0µg/g) and tended to be lower than that with VitE (44.9µg/g). Redox potential of fresh milk from cows fed APC (152mV) was similar to that of VitE (144mV), but lower than that of CTL (189mV). Treatments had no effect on fresh milk contents of dissolved oxygen (8.1±1.5mg/L), and conjugated diene hydroperoxides (2.7±0.5mmol/L). The concentrations of volatile lipid oxidation products (propanal, hexanal, hept-cis-4-enal, 1-octen-3-one) tended to be decreased by APC relative to CTL, whereas similar values were observed for VitE, except for hexanal, which was reduced by 40% in VitE. In conclusion, feeding APC to lactating dairy cows could serve as a source of dietary protein that improves dietary N utilization efficiency, and also as a preharvest technology to increase natural antioxidant levels in milk to limit oxidation.

Prediction of enteric methane emissions from Holstein dairy cows fed various forage sources.

Milk fatty acid (FA) profile has been previously used as a predictor of enteric CH4 output in dairy cows fed diets supplemented with plant oils, which can potentially impact ruminal fermentation. The objective of this study was to investigate the relationships between milk FA and enteric CH4 emissions in lactating dairy cows fed different types of forages in the context of commonly fed diets. A total of 81 observations from three separate 3×3 Latin square design (32-day periods) experiments including a total of 27 lactating cows (96±27 days in milk; mean±SD) were used. Dietary forages were included at 60% of ration dry matter and were as follows: (1) 100% corn silage, (2) 100% alfalfa silage, (3) 100% barley silage, (4) 100% timothy silage, (5) 50:50 mix of corn and alfalfa silages, (6) 50:50 mix of barley and corn silages and (7) 50:50 mix of timothy and alfalfa silages. Enteric CH4 output was measured using respiration chambers during 3 consecutive days. Milk was sampled during the last 7 days of each period and analyzed for components and FA profile. Test variables included dry matter intake (DMI; kg/day), NDF (%), ether extract (%), milk yield (kg/day), milk components (%) and individual milk FA (% of total FA). Candidate multivariate models were obtained using the Least Absolute Shrinkage and Selection Operator and Least-Angle Regression methods based on the Schwarz Bayesian Criterion. Data were then fitted into a random regression using the MIXED procedure including the random effects of cow, period and study. A positive correlation was observed between CH4 and DMI (r=0.59, P0.19). Milk FA profile and DMI can be used to predict CH4 emissions in dairy cows across a wide range of dietary forage sources.

Effect of diet fermentability and unsaturated fatty acid concentration on recovery from diet-induced milk fat depression.

Diet-induced milk fat depression is caused by highly fermentable and high-unsaturated fatty acid (FA) diets, and results in reduced milk fat concentration and yield, reduced de novo FA, and increased trans isomers of the alternate biohydrogenation pathways. The hypothesis of the current experiment was that a diet higher in fermentability and lower in unsaturated FA (UFA) would accelerate recovery compared with a high-UFA and lower-fermentability diet. Eight ruminally cannulated and 9 noncannulated multiparous Holstein cows were randomly assigned to treatment sequences in a replicated Latin square design. During each period milk fat depression was induced for 10 d by feeding a low-fiber, high-UFA diet [25.9% neutral detergent fiber (NDF) and 3.3% C18:2]. Following the induction phase, cows were switched to recovery treatments for 18 d designed to correct dietary fermentability, UFA, or both fermentability and UFA concentration. Treatments during recovery were (1) correction of fiber and UFA diet [control; 31.8% NDF and 1.65% C18:2], (2) a diet predominantly correcting fiber, but not UFA [high oil (HO); 31.3% NDF and 2.99% C18:2], and (3) a diet predominantly correcting UFA, but not fiber concentration [low fiber (LF); 28.4% NDF and 1.71% C18:2]. Milk and milk component yield, milk FA profile, ruminal pH, and 11 rumen microbial taxa were measured every third day during recovery. Milk yield decreased progressively in HO and control, whereas it was maintained in the LF diet. Milk fat concentration increased progressively during recovery in all treatments, but was on average 9% lower in LF than control from d 12 to 18. Milk fat yield increased progressively in all treatments and was not different between control and LF at any time point, but was lower in HO than control on d 15. Milk trans-10 C18:1 and trans-10,cis-12 conjugated linoleic acid decreased progressively in all treatments, but was higher in HO than control from d 3 to 18 [136 ± 50 and 188 ± 57% (mean ± SD)], whereas LF caused a smaller increase in these FA compared with control (67 ± 25 and 90 ± 22%). Additionally, milk trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid was decreased in control and LF and increased in HO during recovery. Selected microbial species observed changed during recovery, but major treatment differences were only observed for Streptococcus bovis. The LF diet that was similar in UFA but 3.4% units lower in NDF compared with to the control had a similar decrease in alternate trans biohydrogenation intermediates in milk. The HO diet that was similar in NDF but 2.0% units higher in UFA compared with the control had higher alternate trans biohydrogenation intermediates in milk compared with control. However, recovery of milk fat yield was similar between treatments at most time points.

Effect of forage level and replacing canola meal with dry distillers grains with solubles in precision-fed heifer diets: Digestibility and rumen fermentation.

Objectives of this study were to determine the effects of feeding differing forage-to-concentrate ratios (F:C) and inclusion rates of corn dry distillers grain with solubles (DDGS) on digestion and rumen fermentation in precision-fed dairy heifer rations. A split-plot design with F:C as whole plot and DDGS inclusion level as sub-plot was administered in a 4-period (19 d) 4 × 4 Latin square. Eight rumen-cannulated Holstein heifers (12.5 ± 0.5 mo of age and 344 ± 15 kg of body weight) housed in individual stalls were allocated to 2 F:C [50:50, low forage, or 75:25 high forage; dry matter (DM) basis] and to a sequence of DDGS inclusion (0, 7, 14, and 21%; DM basis). Forage was a mix of 50% corn silage and 50% grass hay (DM basis). Diets were fed to allow for 800 g/d of body weight gain and fed 1×/d. Rumen contents were sampled at -2, 0, 2, 4, 6, 8, 10, 12, and 20 h after feeding for rumen fermentation measures. Low-forage rations had greater DM and organic matter apparent digestibility. We detected a quadratic effect for DM, organic matter, acid detergent fiber, and neutral detergent fiber apparent digestibility, with the 14% DDGS inclusion level having the highest values. Nitrogen retention decreased with increasing levels of DDGS. Molar proportions of acetate tended to be greater for HF and decreased as DDGS increased; propionate increased as DDGS increased, resulting in the opposite effect on acetate to propionate ratio. Rumen protozoa count decreased as DDGS increased. Moderate levels (14% of DM) of DDGS appear to enhance nutrient utilization and fermentation in precision-fed dairy heifers fed different F:C diets.

Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.

The ruminant provides a powerful model for understanding the temporal dynamics of gastrointestinal microbial communities. Diet-induced milk fat depression (MFD) in the dairy cow is caused by rumen-derived bioactive fatty acids, and is commonly attributed to the changes in the microbial population. The aim of the present study was to determine the changes occurring in nine ruminal bacterial taxa with well-characterised functions, and abundance of total fungi, ciliate protozoa and bacteria during the induction of and recovery from MFD. Interactions between treatment and time were observed for ten of the twelve populations. The total number of both fungi and ciliate protozoa decreased rapidly (days 4 and 8, respectively) by more than 90% during the induction period and increased during the recovery period. The abundance of Streptococcus bovis (amylolytic) peaked at 350% of control levels on day 4 of induction and rapidly decreased during the recovery period. The abundance of Prevotella bryantii (amylolytic) decreased by 66% from day 8 to 20 of the induction period and increased to the control levels on day 12 of the recovery period. The abundance of Megasphaera elsdenii and Selenomonas ruminantium (lactate-utilising bacteria) increased progressively until day 12 of induction (>170%) and decreased during the recovery period. The abundance of Fibrobacter succinogenes (fibrolytic) decreased by 97% on day 4 of induction and increased progressively to an equal extent during the recovery period, although smaller changes were observed for other fibrolytic bacteria. The abundance of the Butyrivibrio fibrisolvens/Pseudobutyrivibrio group decreased progressively during the induction period and increased during the recovery period, whereas the abundance of Butyrivibrio hungatei was not affected by treatment. Responsive taxa were modified rapidly, with the majority of changes occurring within 8 d and their time course was similar to the time course of the induction of MFD, demonstrating a strong correlation between changes in ruminal microbial populations and MFD.

Comparison of enriched palmitic acid and calcium salts of palm fatty acids distillate fat supplements on milk production and metabolic profiles of high-producing dairy cows.

A variable response to fat supplementation has been reported in dairy cows, which may be due to cow production level, environmental conditions, or diet characteristics. In the present experiment, the effect of a high palmitic acid supplement was investigated relative to a conventional Ca salts of palm fatty acids (Ca-FA) supplement in 16 high-producing Holstein cows (46.6±12.4kg of milk/d) arranged in a crossover design with 14-d periods. The experiment was conducted in a non-heat-stress season with 29.5% neutral detergent fiber diets. Treatments were (1) high palmitic acid (PA) supplement fed as free FA [1.9% of dry matter (DM); 84.8% C16:0] and (2) Ca-FA supplement (2.3% of DM; 47.7% C16:0, 35.9% C18:1, and 8.4% C18:2). The PA supplement tended to increase DM intake, and increased the yields of milk and energy-corrected milk. Additionally, PA increased the yields of milk fat, protein, and lactose, whereas milk concentrations of these components were not affected. The yields of milk de novo and 16-C FA were increased by PA compared with Ca-FA (7 and 20%, respectively), whereas the yield of preformed FA was higher in Ca-FA. A reduction in milk fat concentration of de novo and 16-C FA and a marginal elevation in trans-10 C18:1 in Ca-FA is indicative of altered ruminal biohydrogenation and increased risk of milk fat depression. No effect of treatment on plasma insulin was observed. A treatment by time interaction was detected for plasma nonesterified fatty acids (NEFA), which tended to be higher in Ca-FA than in PA before feeding. Overall, the palmitic acid supplement improved production performance in high-producing cows while posing a lower risk for milk fat depression compared with a supplement higher in unsaturated FA.