Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation and metabolism in mammary and liver tissue during an intramammary lipopolysaccharide challenge.

The objective of this experiment was to examine the effects of supplementation and dose of rumen-protected choline (RPC) on markers of inflammation and metabolism in liver and mammary tissue during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC (20.4 g/d of choline ions; CHOL45), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30), or no RPC (CON) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM (CHOL45, n = 9; CHOL45-LPS, n = 9; CHOL30, n = 11; CHOL30-LPS, n = 10; CON, n = 10; CON-LPS, n = 9). Hepatic and mammary tissues were collected from all cows on d 17 postpartum. Hepatic and mammary tissues were collected at ∼7.5 and 8 h, respectively, after the LPS challenge. An additional mammary biopsy was conducted on LPS-challenged cows (CHOL45-LPS, CHOL30-LPS, and CON-LPS) at 48 h postchallenge. Hepatic and mammary RNA copy numbers were quantified for genes involved in apoptosis, methylation, inflammation, oxidative stress, and mitochondrial function using NanoString technology. Targeted metabolomics was conducted only on mammary tissue samples (both 8 and 48 h biopsies) to quantify 143 metabolites including choline metabolites, amino acids, biogenic amines and derivatives, organic acids, carnitines, and glucose. Hepatic IFNG was greater in CHOL45 as compared with CON in unchallenged cows, suggesting an improvement in type 1 immune responses. Hepatic CASP3 was greater in CHOL45-LPS as compared with CON-LPS, suggesting greater apoptosis. Mammary IL6 was reduced in CHOL30-LPS cows as compared with CHOL45-LPS and CON-LPS (8 and 48 h). Mammary GPX4 and COX5A were reduced in CHOL30-LPS as compared with CON-LPS (8 h), and SDHA was reduced in CHOL30-LPS as compared with CON-LPS (8 and 48 h). Both CHOL30-LPS and CHOL45-LPS cows had lesser mammary ATP5J than CON-LPS, suggesting that dietary RPC supplementation altered mitochondrial function following LPS challenge. Treatment did not affect mammary concentrations of any metabolite in unchallenged cows, and only 4 metabolites were affected by dietary RPC supplementation in LPS-challenged cows. Mammary concentrations of isobutyric acid and 2 acyl-carnitines (C4:1 and C10:2) were reduced in CHOL45-LPS as compared with CHOL30-LPS and CON-LPS. Taken together, reductions in medium- and short-chain carnitines along with an increase in long-chain carnitines in mammary tissue from CHOL45-LPS cows suggests less fatty acid entry into the ß oxidation pathway. Although the intramammary LPS challenge profoundly affected markers for inflammation and metabolism in liver and mammary tissue, dietary RPC supplementation had minimal effects on inflammatory markers and the mammary metabolome.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation, metabolism, and performance during an intramammary lipopolysaccharide challenge.

Recent studies have suggested that dietary rumen-protected choline (RPC) supplementation can modulate immune function, attenuate inflammation, and improve performance in periparturient dairy cattle; however, this has yet to be evaluated during a mastitis challenge. Therefore, the objective of this study was to examine the effects of supplementation and dose of RPC on metabolism, inflammation, and performance during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows (parity, mean ± SD, 1.9 ± 1.1 at enrollment) were blocked by calving month and randomly assigned within block to receive either 45 g/d of RPC (20.4 g/d of choline ions; CHOL45, n = 18), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30, n = 21), or no RPC (CON, n = 19) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM. Before the challenge, CHOL45 and CHOL30 cows produced 3.4 and 3.8 (±1.2 SED) kg/d more milk than CON, respectively. Dietary RPC supplementation did not mitigate the milk loss associated with the intramammary LPS challenge; however, CHOL45 and CHOL30 cows produced 3.1 and 3.5 (±1.4 SED) kg/d more milk than CON, respectively in the carryover period (22 to 84 DIM). Dietary RPC supplementation enhanced plasma ß-hydroxybutyrate (BHB) concentrations before the LPS challenge, and increased plasma nonesterified fatty acids (NEFA) and acetylcarnitine concentrations during the LPS challenge, potentially reflecting greater adipose tissue mobilization, fatty acid transport and oxidation. Aside from trimethylamine N-oxide and sarcosine, which were increased in CHOL45-LPS as compared with CON-LPS, most other choline metabolite concentrations in plasma were unaffected by treatment, likely because more choline was being secreted in milk. Plasma lactic acid concentrations were decreased in CHOL45-LPS and CHOL30-LPS as compared with CON-LPS, suggesting a reduction in glycolysis or an enhancement in the flux through the lactic acid cycle to support gluconeogenesis. Plasma concentrations of fumaric acid, a byproduct of AA catabolism and the urea cycle, were increased in both choline groups as compared with CON-LPS during the LPS challenge. Cows in the CHOL45 group had greater plasma antioxidant potential before the LPS challenge and reduced plasma methionine sulfoxide concentrations during the LPS challenge compared with CON-LPS, suggesting an improvement in oxidant status. Nevertheless, concentrations of inflammatory markers such as haptoglobin and tumor necrosis factor α (TNFα) were not affected by treatment. Taken together, our data suggest that the effects of dietary RPC supplementation on milk yield could be mediated through metabolic pathways and are unlikely to be related to the resolution of inflammation in periparturient dairy cattle. Lastly, dose responses to dietary RPC supplementation were not found for various economically important outcomes including milk yield, limiting the justification for feeding a greater dietary RPC dose in industry.

Effects of dietary rumen-protected choline supplementation on colostrum yields, quality, and choline metabolites from dairy cattle.

Colostrum is a critical nutrient source that provides passive immunity to dairy calves. Choline is a trimethylated molecule that is frequently supplemented in the diet to periparturient dairy cows to support postpartum health and performance. Whereas choline and its metabolites have been characterized in milk, the effects of dietary rumen-protected choline (RPC) supplementation on choline metabolites in colostrum from dairy cattle have yet to be explored. Therefore, the objective of the present study was to assess the effects of dietary supplementation and dose of RPC on colostrum yields, quality, and choline metabolites. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d (20.4 g/d of choline ions) of RPC (CHOL45, n = 22), 30 g/d (13.6 g/d of choline ions) of RPC (CHOL30, n = 20), or no RPC (control, n = 19) starting 24 d before expected calving. The effects of dietary supplementation and dose of RPC were assessed on colostrum yields, component yields, somatic cell score (SCS), quality (as assessed by Brix), and choline metabolites. Data were analyzed using a linear mixed model with the fixed effects of treatment, parity, and the 2-way interaction and the random effect of block. Regardless of dose, dietary RPC supplementation increased colostrum yields and protein yields. No effects of dietary RPC supplementation were found on colostrum component percentages, SCS, or colostrum quality. For choline metabolites, treatment interacted with parity for phosphocholine where colostrum from second-parity CHOL45 and CHOL30 cows had greater concentrations of phosphocholine than colostrum from second-parity control cows, but no treatment effect was seen in the colostrum from 3+ parity cows. Dietary choline supplementation, regardless of dose, increased trimethylamine N-oxide concentrations. Dietary choline supplementation did not affect the concentrations of choline, betaine, glycerophosphocholine, sphingomyelin, phosphatidylcholine, or total choline in colostrum. In conclusion, dietary choline supplementation increased phosphocholine concentrations in colostrum from second-parity cows, enhanced trimethylamine N-oxide concentrations, and increased colostrum yields without affecting colostrum quality.

Effects of prenatal dietary rumen-protected choline supplementation during late gestation on calf growth, metabolism, and vaccine response.

The objective of this study was to examine the effects of prenatal supplementation and dose of rumen-protected choline (RPC) on neonatal calf growth, metabolism, and vaccine response. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC [20.4 g/d of choline ions (CHOL45), n = 19], 30 g/d of RPC [13.6 g/d of choline ions (CHOL30), n = 22], or no RPC (CON, n = 19) as a top-dress, starting 24 d before expected calving. Calf body weights were recorded for the first 3 wk of life. All calves were fed colostrum replacer (300 g of IgG) at birth, and apparent efficiency of IgG absorption was calculated. On d 1, 7, 14, and 21, blood samples were taken to quantify plasma reactive oxygen and nitrogen species, antioxidant potential, haptoglobin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, and glucose. Calves received an intranasal vaccine at birth, and nasal secretions were collected on d 0, 7, 10, 14, and 21 to quantify bovine respiratory syncytial virus-specific IgA. Data were analyzed using linear mixed models including the fixed effects of treatment, time (when applicable), calf sex, and prepartum dam data (-24 d) along with interactions. Treatment did not affect calf body weight, ß-hydroxybutyrate, or glucose concentrations. For apparent efficiency of IgG absorption, treatment interacted with the dam's prepartum body condition score. Where the dam's body condition score was ≤3.25, IgG absorption was reduced in calves born from CHOL45 dams as compared with calves from either CHOL30 or CON dams. Calves from CHOL30 dams had a lesser oxidative stress index (OSi; reactive oxygen and nitrogen species/antioxidant potential) than calves from CON dams. Haptoglobin concentrations were less in heifer calves from CHOL45 dams as compared with heifers from CON dams. The dam's prepartum NEFA concentration interacted with treatment. When dam NEFA was minimal, calves from CHOL45 and CHOL30 dams had greater or tended to have greater NEFA, respectively. Conversely, when dam NEFA was greater, calves from CHOL30 and CHOL45 dams had lesser or tended to have lesser NEFA than calves from CON dams, respectively. For vaccine response, treatment interacted with the dam's prepartum OSi. Among calves born from dams with a greater OSi, calves from CHOL45 and CHOL30 dams had lesser bovine respiratory syncytial virus-specific IgA concentrations in nasal secretions as compared with CON. Prenatal RPC supplementation during late gestation affected IgG absorption, neonatal calf metabolism, and vaccine response with some effects dependent on the dam's prepartum parameters.

Assessing availability of amino acids from various feedstuffs in dairy cattle using a stable isotope-based approach.

Nitrogen efficiency in dairy cows can be improved by more precisely supplying essential amino acids (EAA) relative to animal needs, which requires accurate estimates of the availability of individual EAA from feedstuffs. The objective of this study was to determine EAA availability for 7 feed ingredients. Seven heifers (258 ± 28 kg BW) were randomly chosen and assigned to 8 treatment sequences in a 7 × 8 incomplete Latin square design. Treatments were a basal diet (BD), and 10% (on a dry matter basis) of BD replaced by corn silage (CS), grass hay (GH), alfalfa hay (AH), dried distillers grain (DDGS), soybean hulls (SH), wet brewers grain (BG), or corn grain (CG). Total plasma AA entry rates were estimated for each EAA within each diet by fitting a 4-pool dynamic model to observed plasma, 13C AA enrichment resulting from a 2-h constant infusion of a 13C algal AA mixture. Individual EAA availability from each test ingredient was determined by regression of entry rates for that AA on crude protein intake for each ingredient. The derived plasma total EAA entry rates for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain, and corn grain were 30.6 ± 3.4, 27.4 ± 3.2, 31.3 ± 3.4, 37.2 ± 3.2, 26.4 ± 3.2, 37.8 ± 3.2, and 33.5 ± 3.2% (±standard error) of EAA from each ingredient, respectively. Using the previous estimate of 8.27% EAA utilization by splanchnic tissues during first pass, total rumen-undegradable protein EAA absorbed from the gut lumen was 33.4, 29.9, 34.1, 40.6, 28.8, 41.2, and 36.5% of the EAA in each ingredient respectively.

Assessing bioavailability of ruminally protected methionine and lysine prototypes.

Met and Lys are essential AA that can limit lactational performance in dairy cattle fed protein-sufficient diets. Thus, there is industry demand for ruminally protected (RP) sources of Met and Lys. One method of providing ruminal protection for Met and Lys is lipid encapsulation. The objective of this work was to assess 3 lipid-encapsulated Met prototypes (P1, P2, and P3) and 1 Lys prototype (P4) to determine ruminal protection, small intestine absorption (experiment 1), and animal production responses (experiment 2). Ruminal protection was estimated from 8-h in situ retention during ruminal incubation and intestinal absorption from plasma appearance after an abomasal bolus of the in situ retentate. Blood samples were collected over time to determine plasma Met and Lys concentration responses compared with unprotected Lys and Met infused abomasally. The prototypes were not exposed to the total diet or subjected to typical feed handling methods before evaluation. The bioavailability of P1, P2, and P3 Met prototypes was found to be 14, 21, and 18% of the initial AA material, respectively. The RP-Lys prototype had a bioavailability of 45%. To evaluate production responses, 20 Holstein cows were randomly assigned to 2 trials (n = 10 each) in a replicated Latin square design with 14-d periods. The base diet was predicted to be deficient in metabolizable Met (-14.8 g/d) and Lys (-16.1 g/d) per the Cornell Net Carbohydrate and Protein System (version 6.55). In the Met trial, the base diet was supplemented with RP-Lys to meet Lys requirements, and treatments were as follows: no added RP-Met (NCM), NCM plus Smartamine M (SM; Adisseo, Alpharetta, GA), and NCM plus P1, P2, or P3 at 148% of the Met content of SM. In the Lys trial, the base diet was supplemented with RP-Met to meet the Met requirement, and treatments were as follows: no added Lys (NCL), NCL plus AjiProL (AL; Ajinomoto Heartland Inc., Chicago, IL), and NCL plus P4 at 55, 78, or 102% of the reported absorbed Lys in AL. All products were top dressed on the diet without prior mixing or extended exposure to the rest of the diet. Milk protein concentration significantly increased when diets were supplemented with P2, P3, or SM (3.12, 3.12, and 3.11%, respectively) compared with NCM (3.02%). Only P1 (3.04%) was significantly lower than SM. Prototype P2 had the greatest numerical milk protein output response among the 3 RP-Met prototypes, suggesting that it may have had the greatest efficacy when supplemented into these rations. There was a numerical milk protein concentration response to AL and a linear increase in milk protein concentration for P4. The P4 and AL treatments resulted in comparable milk protein production regardless of P4 dose.

An in vivo stable isotope-based approach for assessment of absorbed amino acids from individual feed ingredients within complete diets.

Accurate assessment of the nutritional content of feed ingredients is required for precise diet formulation. Characterizing ingredients in terms of absorption and digestibility of individual AA is challenging, and this information often relies on indirect methods. The purpose of this research was to evaluate an in vivo stable isotope-based method of determining plasma entry rates of individual AA from feather meal (FM), blood meal (BM), and a rumen-protected AA (RPMet). Abomasal infusions of unprotected Ile, Leu, Met, and sodium caseinate were used as control treatments to assess technique reliability and accuracy. Isotopic enrichment of plasma AA in response to a 2-h constant jugular infusion of a mixture of 13C labeled AA was measured and modeled using a dynamic 4-pool model, which was fitted to each AA by infusion to derive diet entry rates. The resulting entry rate matrix was used to derive plasma entry rates of individual AA from each ingredient by regression. The mean of plasma AA entry for abomasally infused Ile, Leu, and Met was 93.4 ± 7.35% of that infused, indicating that 6.6% was used by splanchnic tissues during first pass. The mean of the plasma essential AA entry for abomasally infused casein was 86.7 ± 4.81% of that present in the source protein, which represents a mean of 8.7% first-pass use assuming 95% digestibility. Individual AA appearances ranged from 86 to 93% of the source content except Ile, which was 73%. These fractional appearance percentages were similar to those previously reported when using a dietary regression approach. The mean plasma essential AA entry rate for FM was 52.7% of the AA in the source ingredient, with a range across AA of 48 to 58%. The mean plasma essential AA entry rate for BM was 47.5%, with a range of 30 to 61%. However, estimated Met availability from the RPMet was lower (9.9%) than expected (42%). This may be due to the relatively larger errors of measurement for Met entry rates and a small change in RPMet inclusion. Assuming that rumen-undegraded protein absorption is reflective of aggregated essential AA entry rates after correction for first-pass use, 52.6 and 61.2% of dietary FM and BM CP was absorbed from the intestine, respectively, which yielded an estimated intestinal digestibility of 70 and 66%, respectively. This method appears to provide an accurate and precise in vivo assessment of individual AA plasma entry rates that can be used to better characterize individual feed ingredients in ruminants. Such information will result in more robust economic assessments of feeds and increased precision of diet formulation.