A high-concentrate diet induces mitochondrial dysfunction by activating the MAPK signaling pathway in the mammary gland of dairy cows

Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca²⁺ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1β (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis–related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.     

Changes in fermentation profile of the reticulorumen and hindgut,and nutrient digestion in dry cows fed concentrate-rich diets supplemented with a phytogenic feed additive

This study evaluated the effects of duration of high-concentrate feeding on ruminal and fecal fermentation profile, as well as selected systemic health biomarkers in nonlactating cows supplemented with or without a phytogenic feed additive (PHY). In addition, ruminal degradation kinetics and total-tract nutrient digestibility were evaluated when feeding either only forage or a high-concentrate diet. Nine nonlactating, cannulated Holstein cows were used in a crossover design. Each period included 1 wk of forage feeding (wk 0), diet transition, and 4 wk on the high-concentrate diet (1, 2, 3 and wk 4; 65% dry matter basis). Cows received PHY or not (control). Compared with wk 0, from wk 1 onward, cows on high concentrate showed greater reticular, ruminal, and fecal total volatile fatty acids (VFA), with a greater level of VFA in the rumen than in the hindgut. However, ruminal fermentation was modulated differently by PHY, which showed increased total VFA in wk 1 and increased butyrate in wk 2 in the particle-associated fluid of rumen. In the hindgut, PHY increased propionate in wk 3. Cows fed a high-concentrate diet from wk 1 and onward also showed greater ruminal lactate, as well as lower ruminal and fecal pH, independent of PHY. In addition, compared with cows in wk 1 on a high-concentrate diet, cows in wk 4 had a greater total VFA in free fluid of the rumen and lower fecal pH. Compared with cows at wk 0, cows at wk 1 on high concentrate onward showed greater serum amyloid A and greater activity of glutamate dehydrogenase. In contrast, the high-concentrate diet decreased in situ ruminal degradability of grass silage but increased degradability of corn grain as well as total-tract nutrient digestibility, with total-tract neutral detergent fiber digestibility being greater for cows on the PHY treatment. Overall, from the start of high-concentrate feeding, gut fermentation increased, but differently according to location or PHY, with a stronger build-up of VFA in the rumen compared with the hindgut. In addition, a longer duration on high concentrate exacerbated gut acidification. The enhancing effects of PHY on total VFA and butyrate in particle-associated fluid of the rumen suggest beneficial effects of PHY on particle-associated bacteria, likely contributing to the increased neutral detergent fiber digestibility. The greater production of ruminal butyrate with PHY may be beneficial for the host, given the health benefits of this acid, but more research is needed to elucidate the effects on gut microbiota and the effects of increased butyrate in nonlactating dairy cows.     

Subacute ruminal acidosis downregulates FOXA2, changes oxidative status,and induces autophagy in the livers of dairy cows fed a high-concentrate diet

The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3β (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.     

Effect of fibrolytic enzyme application to low- and high-concentrate diets on the performance of lactating dairy cattle

The objective of this study was to examine the effect of applying a fibrolytic enzyme preparation to diets with high (48% of diet dry matter, DM) or low (33% of diet DM) proportions of concentrate on production performance of lactating dairy cows. Sixty lactating Holstein cows (589 kg ± 20; 22 ± 3 d in milk) were stratified according to milk production and parity and randomly assigned to 4 treatments with a 2 × 2 factorial arrangement. Dietary treatments included the following: 1) low-concentrate diet (LC); 2) LC plus enzyme (LCE); 3) high-concentrate diet (HC); and 4) HC plus enzyme (HCE). The enzyme was sprayed at a rate of 3.4 mg of enzyme/g of DM on the total mixed ration daily and the trial lasted for 63 d. A second experiment with a 4 × 4 Latin square design used 4 ruminally fistulated cows to measure treatment effects on ruminal fermentation and in situ ruminal dry matter degradation during four 18-d periods. Enzyme application did not affect dry matter intake (DMI; 23.9 vs. 22.3 kg/d) or milk production (32.8 vs. 34.2 kg/d) but decreased estimated CH₄ production, increased total volatile fatty acid concentration (114.5 vs. 125.7 mM), apparent total tract digestibility of DM (69.8 vs. 72.6%), crude protein (CP; 69.2 vs. 73.3%), acid detergent fiber (50.4 vs. 54.8%), neutral detergent fiber (53.7 vs. 55.4%), and the efficiency of milk production (1.44 vs. 1.60 kg of milk/kg of DMI). Feeding more concentrates increased DMI (21.5 vs. 24.8 kg/d), milk yield (32.2 vs. 34.7 kg/d), milk protein yield (0.89 vs. 0.99 kg/d), and DM (69.9 vs. 72.6%), but decreased ruminal pH (6.31 vs. 6.06). Compared with cows fed HC, those fed LCE had lower DMI (20.8 vs. 25.7 kg/d) and CP intake (3.9 vs. 4.8 kg/d), greater ruminal pH (6.36 vs. 6.10), and similar milk yield (33.2 ± 1.1 kg/d). Consequently, the efficiency of milk production was greater in cows fed LCE than those fed HC (1.69 vs. 1.42 kg of milk/kg of DMI). This fibrolytic enzyme increased the digestibility of DM, CP, neutral detergent fiber, and acid detergent fiber and the efficiency of milk production by dairy cows. Enzyme application to the low-concentrate diet resulted in as much milk production as that from cows fed the untreated high-concentrate diet.     

Effects of body condition,monensin, and essential oils on ruminal lipopolysaccharide concentration,inflammatory markers,and endoplasmatic reticulum stress of transition dairy cows

Evidence exists that dairy cows experience inflammatory-like phenomena in the transition period. Rumen health and alterations in metabolic processes and gene networks in the liver as the central metabolic organ might be key factors for cows’ health and productivity in early lactation. This study made use of an animal model to generate experimental groups with different manifestations of postpartal fat mobilization and ketogenesis. In total, 60 German Holstein cows were allocated 6 wk antepartum to 3 high-body condition score (BCS) groups (BCS 3.95) and 1 low-BCS group (LC; BCS 2.77). High-BCS cows were fed an antepartal forage-to-concentrate ratio of 40:60 on dry matter basis, in contrast to 80:20 in the LC group, and received a monensin controlled-release capsule (HC/MO), a blend of essential oils (HC/EO), or formed a control group (HC). We evaluated serum haptoglobin, kynurenine, tryptophan, ruminal lipopolysaccharide concentration and mRNA abundance of nuclear factor kappa B (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and endoplasmatic reticulum stress-induced unfolded protein response (UPR) target genes in liver biopsy samples from d ?42 until +56 relative to calving. Nearly all parameters were highly dependent on time, with greatest variation near calving. The ruminal lipopolysaccharide concentration and evaluated target genes were not generally influenced by antepartal BCS and feeding management. The kynurenine-to-tryptophan ratio was higher in LC than in HC/MO treatment on d 7. Ruminal lipopolysaccharide concentration was higher in HC/MO than in the HC group, but not increased in HC/EO group. Abundance of UPR target gene X-box binding protein 1 was higher in HC/MO than in HC/EO group on d 7. Hepatic mRNA abundance of Nrf2 target gene glutathione peroxidase 3 was higher, whereas expression of NF-κB target gene haptoglobin tended to be higher in LC than in HC/EO cows. The HC/MO cows showed the most prominent increase in the abundance of glutathione peroxidase 3 and haptoglobin after calving in comparison to antepartal values. Results indicate the presence of inflammatory-like phenomena near calving. Simultaneously, alterations in UPR and Nrf2 target genes with antioxidative properties and haptoglobin occurred, being most prominent in LC and HC/MO group.     

Thiamine ameliorates metabolic disorders induced by a long-term high-concentrate diet and promotes rumen epithelial development in goats

Data indicate that dietary thiamine supplementation can partly alleviate rumen epithelium inflammation and barrier function in goats fed a high-concentrate diet. The current work aimed to explore whether thiamine promotes rumen epithelium development by regulating carbohydrate metabolism during a long period of feeding high levels of concentrate. For the experiment, 24 female Boer goats (35.62 ± 2.4 kg of body weight) in parity 1 or 2 were allocated to 3 groups (8 goats per replicate) receiving a low-concentrate diet (concentrate:forage 30:70), a high-concentrate diet (HC; concentrate:forage 70:30), or a high-concentrate diet (concentrate:forage 70:30) supplemented with 200 mg of thiamine/kg of dry matter intake (HCT; concentrate:forage 70:30). On the last day of 12 wk, rumen fluid and blood samples were collected to measure ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine differential metabolites, enzyme activities, and gene expression. Liquid chromatography–tandem mass spectrometry analysis revealed that the HCT group had significantly increased concentrations of d-glucose 6-phosphate, d-fructose 6-phosphate, glyceraldehyde 3-phosphate, thiamine pyrophosphate, oxaloacetate, acetyl-CoA, succinyl-CoA, sedoheptulose 7-phosphate, ribose 5-phosphate, and NADPH compared with the HC group. The pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase enzyme activities in the rumen epithelium of the HCT group were higher than those in the HC group. The plasma total antioxidant capacity values for the HCT group were greater than those for the HC group. The rumen epithelium ATP content in the HCT group was higher than that in the HC group. Compared with the HCT group, the HC group had a lower mRNA abundance of CCND1, CCNA2, CDK2, CDK4, CDK6, BCL2, PI3K, and AKT1. Taken together, the results suggest that dietary thiamine supplementation could ameliorate disorders in the tricarboxylic acid cycle and the pentose phosphate pathway induced by a long-term high-concentrate diet and could promote rumen epithelial growth.     

Effects of varying dietary forage particle size in two concentrate levels on chewing activity, ruminal mat characteristics, and passage in dairy cows

The objective of this study was to investigate the effects of varying dietary forage particle size on chewing activity, ruminal mat characteristics, passage, and in situ ruminal and total tract digestion in dairy cows at a low- and high-concentrate inclusion. The experiment was designed as a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments. Four ruminally cannulated late-lactating dairy cows were restrictively fed (17 kg of dry matter/d), in four 23-d periods, 1 of 4 different diets varying in the theoretical particle size (6 and 30 mm) of hay (56.6% NDF of dry matter) and in the levels (approximately 20 and 60%, dry matter basis) of a cereal-based concentrate. Ingredients of the ration were offered separately to the cows; dietary hay and low-level concentrate were offered twice daily at 0800 and 1600 h, whereas concentrate of the high-level treatment was offered in 4 meals a day at 0800, 1200, 1600, and 1900 h. This study showed that altering the forage particle size from 6 to 30 mm in a low-concentrate diet significantly increased the rumination time and ruminal mat consistency without affecting ruminal fermentation and passage. Further, particle breakdown and proportion of mat in the rumen increased, and in situ hay dry matter degradability improved, which in turn indicated a higher capacity of ruminal digesta to degrade fiber. On the other hand, increasing the forage particle size in a diet containing a high amount of concentrate increased the proportion of dry matter retained on a 1.18-mm screen from 37.5 to 42.0% and extended the rumination time by 100 min/d, as well as increasing the ruminal mat consistency. However, ruminal particle breakdown, short-term ruminal pH, fibrolytic capacity of the digesta, and proportion of mat in the rumen decreased. This was also reflected in a higher bailable liquid pool, increased fractional passage rate of solid digesta from the reticulorumen, and increased retention time in the hindgut, which in turn indicated a shift of fiber digestion from the rumen to the lower digestive tract. This study showed that the response of chewing or ruminating activity alone seemed to be insufficient to assess the dietary physical effectiveness or fiber adequacy in limit-fed dairy cows when high-concentrate diets were fed separately. Based on the results of this study, we concluded that inclusion of coarsely chopped hay in the high-concentrate diet did not appear to further improve rumen conditions and digestion when the rations were formulated to exceed the fiber requirements in limit-fed dairy cows.     

Effect of linseed oil supplementation on ruminal digestion in dairy cows fed diets with different forage:concentrate ratios

The effect of linseed oil (LSO) supplementation on total-tract and ruminal nutrient digestibility, N metabolism, and ruminal fluid characteristics was investigated in dairy cows fed diets containing different forage to concentrate ratios (F:C). The experimental design was a 4 x 4 Latin square with 2 x 2 factorial arrangement of treatments. Four lactating Holstein cows were fed a forage-rich diet without LSO (F; F:C = 65:35, dry matter basis), a forage-rich diet with LSO (FO; F:C = 65:32, 3% LSO), a concentrate-rich diet without LSO (C; F:C = 35:65), or a concentrate-rich diet with LSO (CO; F:C = 35:62, 3% LSO). Total-tract digestibility of DM and OM was greater with supplemental LSO. A tendency for greater total-tract digestibility of NDF and ADF also was observed in cows fed LSO. Ruminal digestibility of NDF or ADF decreased when CO was fed compared with C. In contrast, feeding FO increased NDF or ADF digestibility compared with F. Although ruminal starch digestion was nearly complete with all diets, digestibility was greater when cows were fed C or CO compared with F or FO. Bacterial N flow to the duodenum decreased when FO was fed compared with F. In contrast, feeding CO increased bacterial-N flow compared with C. Neither F:C nor LSO supplementation affected ruminal pH or total VFA concentration in ruminal fluid. However, molar proportion of propionate was greater with C or CO compared with F or FO and increased with LSO supplementation regardless of F:C. Molar proportion of n-butyrate decreased with LSO supplementation. Total protozoal numbers in ruminal fluid decreased markedly only when CO was fed. Overall, data show that feeding LSO had no negative effects on total-tract digestion in dairy cows but may decrease ruminal fiber digestibility when fed with high-concentrate diets. The widely spread idea that LSO decreases digestibility, arising from studies with sheep, did not seem to apply to lactating cows fed 3% LSO.     

Short communication: Blood metabolites,body reserves,and feed efficiency of high-producing dairy cows that varied in ruminal pH when fed a high-concentrate diet

Recent studies report considerable variation in ruminal pH for lactating dairy cows even when fed the same diet. We hypothesized that blood metabolites would be indicators of low ruminal pH, and hence could be used as predictors to help manage this variability. The objective of the study was to determine whether blood metabolite concentrations, body reserves, and feed efficiency were associated with ruminal pH in high-producing dairy cows fed a high-concentrate diet. Seventy-eight individually fed lactating dairy cows (days in milk = 103 ± 27; body weight = 638 ± 77 kg at the start; mean ± SD) were fed a diet consisting of 35% forage and 65% concentrate (dry matter basis). Cows were adapted for 14 d and then were sampled for 10 d. Ruminal pH was measured by rumenocentesis for all cows at the end of the study 4 h after feeding, and reticular pH was measured on a subsample of 14 cows via indwelling sensors for 5 consecutive days. Cows were classified according to rumenocentesis pH as high (pH ≥ 6.0; n = 26), medium (5.8 ≤ pH < 6; n = 21), and low (pH < 5.8; n = 31). Cows were also classified according to reticular pH as high if pH <5.8 persisted <330 min/d (an average of 78 min/d; n = 5) or low if duration of pH <5.8 was ≥330 min/d (an average of 920 min/d; n = 9). The classification based on rumenocentesis pH revealed that serum activity of aspartate aminotransferase (AST) was greater in cows with low ruminal pH (70.7 U/L) than cows with high (56.6 U/L) and medium (59.9 U/L) ruminal pH. Also, the blood urea nitrogen concentration was greater in cows with low ruminal pH (13.6 mg/dL) than cows with medium (12.2 mg/dL) and high (12.5 mg/dL) ruminal pH. Blood albumin concentration was greater for cows with low ruminal pH than for cows with medium and high ruminal pH. The classification based on reticular pH also resulted in a trend of greater AST activity and greater blood urea nitrogen concentration in the blood of cows with low pH. Regression analysis showed high serum concentration of AST was associated with high valerate concentration in ruminal fluid (R² = 0.14), low rumenocentesis pH (R² = 0.10), and low milk fat percentage (R² = 0.06). Glucose, triglyceride, cholesterol, globulin, alkaline phosphates, and serum amyloid A did not differ among the different ruminal pH classes. Low pH cows (reticular and ruminal) had less backfat thickness measured via ultrasound, and cows with low ruminal pH tended to have greater milk:feed ratio. Results indicated that cows that differ in ruminal pH also had different concentrations of blood metabolites and backfat thickness, and AST activity in blood may be a plausible indicator of ruminal pH in dairy cows. Further studies on the applicability of AST in blood as a biomarker for detecting low ruminal pH in dairy cows are warranted.     

Effects of calcium carbonate on ruminal fermentation, nutrient digestibility, and cow performance

Sixteen Holstein cows were used in a 4 x 4 Latin square design (four replicates) to investigate the effect of feeding calcium carbonate on feed intake, ruminal fermentation, apparent total tract nutrient digestibility, milk yield, and milk composition. Supplementation of calcium carbonate to diets that contained 60% concentrate and 40% corn silage (DM basis) decreased DM intake and milk production and was not effective in altering ruminal fluid pH, ruminal fluid dilution rate or outflow, molar proportions of ruminal fluid VFA, or synthesis of milk fat and milk protein by dairy cows. Calcium carbonate supplementation to the diet tended to improve efficiency of feed utilization (4% FCM/DM intake). The exact site of action of calcium carbonate, if any, is not known. However, these data suggest that calcium carbonate exerts little or no buffering effect in the rumen when the pH is 6 or above regardless of its reactivity rate in strong acid or its mean particle size. This lack of effect is probably because of its low solubility in ruminal fluid at pH above 5.5.     

Role of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil extracellular trap formation in dairy cows with subclinical hypocalcemia

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 Ca²⁺ <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 Ca²⁺ 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 Ca²⁺, 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.     

Influence of carbohydrate source and buffer on rumen fermentation characteristics, milk yield, and milk composition in early-lactation Holstein cows

The effects of concentrate to forage ratio and sodium bicarbonate (buffer) supplementation on intake, ruminal fermentation characteristics, digestibility coefficients, milk yield, and milk composition were examined in 4 cannulated Holstein cows (100 +/- 20 d in milk). A 4 x 4 Latin square design with 2 x 2 factorial arrangement of treatments was implemented for 3-wk experimental periods. The 4 treatments were a 50:50 concentrate to forage ratio with 1.2% of dry matter (DM) and without added buffer and a 75:25 concentrate to forage ratio with (1.2% of DM) and without (0% of DM) buffer. The forage component of the ration was a 50:50 mixture of alfalfa and barley and triticale silage, and diets were fed ad libitum as a total mixed ration. Although feed intake was not influenced by treatments, substantial treatment differences were observed for milk yield and milk composition. Cows fed high-concentrate diet had lower ruminal pH, ruminal acetate, and butyrate concentrations, whereas propionate concentrations were significantly elevated. The addition of buffer, at both levels of concentrate inclusion, resulted in elevated total volatile fatty acids and acetate concentrations. We concluded that altering the forage concentrate ratio in the diet of lactation cows influenced milk yield and milk composition, but the addition of buffer to the diet prevented the elevation in trans-C18:1 fatty acids in milk fat, and related milk fat depression, associated with feeding high-concentrate diets.     

Effects of feeding whole cottonseed to lactating dairy cows on glucose and palmitate metabolism

Eight lactating Holstein dairy cows were fed corn silage-based diets with or without whole cottonseed at 18.5% of the dietary dry matter. At 42 days postpartum, a pulse injection of 100 mg glucose/kg body weight was given intravenously and plasma glucose concentration was monitored for 45 min. At 50 d postpartum, biopsies of adipose tissue and mammary tissue were taken and tissue slices were incubated in vitro with either uniform carbon-14 glucose or 1-carbon-14 palmitate. Basal concentration of plasma glucose was not affected by diet, although apparent distribution volume of glucose did seem to decrease due to feeding whole cottonseed. Feeding whole cottonseed decreased uptake of glucose and palmitate in both adipose tissue and mammary tissue and also decreased oxidation of glucose to carbon dioxide in both tissues. Palmitate oxidation was not affected by diet. Incorporation of carbon-14 from glucose into adipose tissue lipids was decreased in cows fed whole cottonseed. Results indicate that fat supplementation in the form of whole cottonseed may decrease palmitate incorporation and glucose utilization for glycerol and reducing equivalent synthesis in both adipose tissue and mammary tissue of lactating dairy cows.     

Effects of ground,steam-flaked,and super-conditioned corn grain on production performance and total-tract digestibility in dairy cows

This study aimed to evaluate the effects of feeding ground, steam-flaked, or super-conditioned corn on production performance, rumen fermentation, nutrient digestibility, and milk fatty acid (FA) profile of lactating dairy cows. Twenty-four lactating Holstein cows (130 ± 12 d in milk) in a completely randomized block design experiment were assigned to 1 of 3 treatments that contained 31% of one of the following corn types: (1) ground corn; (2) steam-flaked corn; and (3) super-conditioned corn. Actual milk yield was greater in the super-conditioned corn diet than in the steam-flaked and ground corn diets. Dry matter intake, 3.5% fat-corrected milk and energy-corrected milk remained unaffected by treatments; however, milk fat concentration decreased in the super-conditioned corn diet compared with the ground and steam-flaked corn diets. The molar proportion of ruminal acetate decreased in the super-conditioned corn diet compared with the ground and steam-flaked corn diets, whereas the molar proportion of propionate spiked in the super-conditioned corn diet. Ruminal pH dropped in cows fed super-conditioned corn compared with the other 2 diets. A similar pattern was observed for ruminal NH₃-N and acetate-to-propionate ratio. Total-tract starch digestibility increased the most in the super-conditioned corn diet followed by the steam-flaked and ground corn diets (96.8, 95.1, and 92.5%, respectively). The neutral detergent fiber digestibility declined in cows fed the super-conditioned corn diet as opposed to other diets (~3.9%). The concentrations of 16:0 and mixed-FA in milk fat dropped in the super-conditioned corn-based diet compared with the ground corn diet. Milk trans-10 18:1 FA increased, whereas trans-11 18:1 FA decreased in cows fed the super-conditioned diet. We concluded that super-conditioned corn has the potential to increase milk yield and starch digestibility in lactating dairy cows; however, reduced milk fat output caused by altering ruminal pH and ruminal FA biohydrogenation pathways may not be desirable in certain markets. Future research is warranted to investigate how super-conditioned corn affects feed efficiency.     

Supplementing phytogenic compounds or autolyzed yeast modulates ruminal biogenic amines and plasma metabolome in dry cows experiencing subacute ruminal acidosis

Subacute ruminal acidosis (SARA) causes ruminal dysbiosis, thereby increasing the risk of systemic metabolic disorders in cattle. We recently showed that supplementation with phytogenic compounds (PHY) or autolyzed yeast (AY) counteracted negative effects of SARA by improving ruminal pH and microbiome. This study investigated the effects of an intermittent SARA challenge on the ruminal concentration of biogenic amines (BA) and lipopolysaccharides (LPS), as well as on the blood metabolome. We also evaluated effects of PHY and AY on the latter variables. Eight rumen-cannulated nonlactating Holstein cows were arranged in an incomplete 4 × 3 Latin square design with 4 experimental runs and 3 treatment groups. During each run, cows were switched from an all-forage diet (baseline) to an intermittent concentrate-challenge diet with a forage:concentrate ratio of 35:65 (dry matter basis) to induce SARA for 1 (SARA1) or 2 (SARA2) wk, separated by 1 wk of forage-only feeding. The 3 treatment groups were no additive as control, PHY, or AY. During baseline, SARA1 and SARA2 rumen fluid samples were collected for analysis of BA and LPS. Blood samples were taken during baseline and SARA1 for a targeted metabolomics approach. High-concentrate feeding caused a 9-fold increase in ruminal LPS during SARA1 and an 11-fold increase in SARA2 compared with the baseline. Elevated concentrations of ruminal BA were found during both SARA periods, with histamine showing the strongest increase during SARA1. Moreover, a decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelines, and several AA in the blood during SARA1 were detected. Supplementation of PHY decreased concentrations of LPS (?43%), histamine (?66%), pyrrolidine (?38%), and spermine (?54%) in SARA1 and cadaverine in SARA2 (?50%). Moreover, cows that received PHY had higher concentrations of cholesterol (+26%), several AA, and phosphatidylcholines in SARA1 compared with control cows. For AY, decreases in ruminal ethanolamine (?21%), methylamine (?52%), histamine (?54%), spermidine (?44%), and spermine (?80%) in SARA1 were observed, whereas in the blood an increase in tryptophan was noticed. In conclusion, the SARA was associated with markedly increased concentrations of LPS and BA in the rumen fluid and undesirable shifts in the plasma metabolome. Supplementation of PHY and AY counteracted some of these changes and therefore may help in attenuating negative effects of high-concentrate feeding in dairy cattle.     

Severity of ruminal acidosis in primiparous holstein cows during the periparturient period

The objectives of this study were: 1) to determine the effect of providing additional prepartum concentrate on the occurrence and severity of ruminal acidosis (RA) and lactational performance during the periparturient period in primiparous cows; and 2) to characterize the occurrence and severity of RA during the periparturient period. We hypothesized that providing additional concentrate prepartum would reduce postpartum RA. Fourteen ruminally cannulated Holstein heifers were paired by expected calving date and body condition score. The heifers were assigned to 1 of 2 prepartum feeding regimens: 1) a control treatment consisting of a far-off diet (forage:concentrate, F:C = 80:20) fed from d −60 to d −25 and a close-up diet (F:C = 54:46) fed from d −24 until parturition; or 2) a high-concentrate (HC) feeding program consisting of 4 prepartum diets, HC-1 (F:C = 68:32) fed from d −60 to d −43, HC-2 (F:C = 60:40) fed from d −42 to d −25, HC-3 (F:C = 52:48) fed from d −24 to d −13, and HC-4 (F:C = 46:54) fed from d −12 until parturition. All cows received the same lactation diet postpartum. Ruminal pH was measured continuously from d −5 to d +5, and for 3 consecutive days starting on d +17 ± 1.2, d +37 ± 1.4, and d +58 ± 1.5 relative to parturition using an indwelling ruminal pH system. Ruminal acidosis was considered to occur when ruminal pH was <5.8 (total RA). Ruminal acidosis was further partitioned into: 1) mild RA (5.8 > ruminal pH > 5.5), 2) moderate RA (5.5 > ruminal pH > 5.2), and 3) acute RA (ruminal pH < 5.2). Feeding additional concentrate prepartum did not reduce postpartum RA. In fact, cows fed the HC treatment had more daily episodes of acute RA than cows fed the control treatment. Day relative to parturition affected the occurrence and severity of RA; RA increased following parturition and was sustained thereafter. The DM intake during the last 5 d of gestation was lower for cows fed the HC treatment compared with cows fed the control treatment, but lactational performance was not affected. We conclude that, under the conditions imposed, feeding additional concentrate prepartum does not reduce postpartum RA. Furthermore, the incidence and severity of RA increases immediately postpartum, emphasizing the need to develop and implement feeding strategies that reduce this risk.     

Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production

The objective of this study was to investigate the potential of reducing enteric methane production from dairy cows by incorporating into the diet various sources of long-chain FA varying in their degree of saturation and ruminal availability. The experiment was conducted as a crossover design with 16 lactating dairy cows maintained in 2 groups and fed 4 dietary treatments in four 28-d periods. Eight ruminally cannulated primiparous cows (96 ± 18 d in milk) were assigned to group 1 and 8 multiparous cows (130 ± 31 d in milk) were assigned to group 2. The dietary treatments were: 1) a commercial source of calcium salts of long-chain fatty acids (CTL), 2) crushed sunflower seeds (SS), 3) crushed flaxseed (FS), and 4) crushed canola seed (CS). The oilseeds added 3.1 to 4.2% fat to the diet (DM basis). All 3 oilseed treatments decreased methane production (g/d) by an average of 13%. When corrected for differences in dry matter intake (DMI), compared with CTL, methane production (g/kg of DM intake) was decreased by feeding FS (−18%) or CS (−16%) and was only numerically decreased (−10%) by feeding SS. However, compared with the CTL, feeding SS or FS lowered digestible DMI by 16 and 9%, respectively, because of lowered digestibility. Thus, only CS lowered methane per unit of digestible DM intake. Feeding SS and CS decreased rumen protozoal counts, but there were no treatment effects on mean ruminal pH or total volatile fatty acid concentration. Milk efficiency (3.5% fat corrected milk/DMI), milk yield, and component yield and concentrations were not affected by oilseed treatments. The study shows that adding sources of long-chain fatty acids to the diet in the form of processed oilseeds can be an effective means of reducing methane emissions. However, for some oilseeds such as SS or FS, the reduction in methane can be at the expense of diet digestibility. The use of crushed CS offers a means of mitigating methane without negatively affecting diet digestibility, and hence, milk production.