Effects of type of substrate and dilution rate on fermentation in serial rumen mixed cultures.

Forages and concentrates have consistently distinct patterns of fermentation in the rumen, with forages producing more methane (CH4) per unit of digested organic matter (OM) and higher acetate to propionate ratio than concentrates. A mechanism based on the Monod function of microbial growth has been proposed to explain the distinct fermentation pattern of forages and concentrates, where greater dilution rates and lower pH associated with concentrate feeding increase dihydrogen (H2) concentration through increasing methanogens growth rate and decreasing methanogens theoretically maximal growth rate, respectively. Increased H2 concentration would in turn inhibit H2 production, decreasing methanogenesis, inhibit H2-producing pathways such as acetate production via pyruvate oxidative decarboxylation, and stimulate H2-incorporating pathways such as propionate production. We examined the hypothesis that equalizing dilution rates in serial rumen cultures would result in a similar fermentation profile of a high forage and a high concentrate substrate. Under a 2 × 3 factorial arrangement, a high forage and a high concentrate substrate were incubated at dilution rates of 0.14, 0.28, or 0.56 h-1 in eight transfers of serial rumen cultures. Each treatment was replicated thrice, and the experiment repeated in two different months. The high concentrate substrate accumulated considerably more H2 and formate and produced less CH4 than the high forage substrate. Methanogens were nearly washed-out with high concentrate and increased their initial numbers with high forage. The effect of dilution rate was minor in comparison to the effect of the type of substrate. Accumulation of H2 and formate with high concentrate inhibited acetate and probably H2 and formate production, and stimulated butyrate, rather than propionate, as an electron sink alternative to CH4. All three dilution rates are considered high and selected for rapidly growing bacteria. The archaeal community composition varied widely and inconsistently. Lactate accumulated with both substrates, likely favored by microbial growth kinetics rather than by H2 accumulation thermodynamically stimulating electron disposal from NADH into pyruvate reduction. In this study, the type of substrate had a major effect on rumen fermentation largely independent of dilution rate and pH.

Kinetics of omega-3 fatty acid transfer to milk differs between fatty acids and stage of lactation in dairy cows.

Fatty acids (FA) differ in their transfer efficiencies and metabolic partitioning and lactating cows provide a robust model to investigate kinetics of FA transport. The objective was to compare kinetics of n-3 polyunsaturated FA (PUFA) trafficking through plasma and into milk. In the first experiment, ten ruminally cannulated multiparous Holstein cows were used in a crossover design with 7 d periods. Cows were milked at 6 h intervals and abomasal treatments provided a single dose of 80.1 g of α-linolenic acid as free FA (ALA-FFA) or 45.5 g EPA and 32.9 g DHA (LCn3-FFA). Transfer of n-3 PUFA to milk was nearly 50% higher for ALA-FFA than LCn3-FFA (48.2 and 32.7% of the bolus) and fit a bi-exponential model. Rapid transport of n-3 PUFA, assumed to be directly through chylomicrons, was nearly twice as high in ALA-FFA than LCn3-FFA and the subsequent slow transport, assumed to be indirect transfer through tissue recycling, was over 2.5-fold higher in LCn3-FFA than in ALA-FFA. Plasma analysis revealed LCn3-FFA enriched phospholipids and cholesterol esters, which had a slow clearance. In the second experiment, 4 cows received a bolus of a mixture of ALA, EPA, and DHA prepartum while not lactating and around d 10, 55, and 225 of lactation. Transfer of ALA to milk did not differ between stages of lactation, but DHA was lower in early compared to mid and late lactation. In conclusion, dietary ALA is rapidly and efficiently transferred to milk in cows while EPA and DHA are rapidly incorporated into plasma or tissue fractions not available to the mammary gland. This demonstrates clear differences in trafficking and partitioning of n-3 PUFA that ultimately impact tissue and organelle enrichment with implications for effective doses.

3-Nitrooxypropanol substantially decreased enteric methane emissions of dairy cows fed true protein- or urea-containing diets.

Methane is a potent but short-lived greenhouse gas targeted for short-term amelioration of climate change, with enteric methane emitted by ruminants being the most important anthropogenic source of methane. Ruminant production also releases nitrogen to the environment, resulting in groundwater pollution and emissions of greenhouse gas nitrous oxide. We hypothesized that inhibiting rumen methanogenesis in dairy cows with chemical inhibitor 3-nitrooxypropanol (3-NOP) would redirect metabolic hydrogen towards synthesis of microbial amino acids. Our objective was to investigate the effects of 3-NOP on methane emissions, rumen fermentation and nitrogen metabolism of dairy cows fed true protein or urea as nitrogen sources. Eight ruminally-cannulated cows were fed a plant protein or a urea-containing diet during a Control experimental period followed by a methanogenesis inhibition period with 3-NOP supplementation. All diets were unintentionally deficient in nitrogen, and diets supplemented with 3-NOP had higher fiber than diets fed in the Control period. Higher dietary fiber content in the 3-NOP period would be expected to cause higher methane emissions; however, methane emissions adjusted by dry matter and digested organic matter intake were 54% lower with 3-NOP supplementation. Also, despite of the more fibrous diet, 3-NOP shifted rumen fermentation from acetate to propionate. The post-feeding rumen ammonium peak was substantially lower in the 3-NOP period, although that did not translate into greater rumen microbial protein production nor lesser nitrogen excretion in urine. Presumably, because all diets resulted in low rumen ammonium, and intake of digestible organic matter was lower in the 3-NOP period compared to the Control period, the synthesis of microbial amino acids was limited by nitrogen and energy, precluding the evaluation of our hypothesis. Supplementation with 3-NOP was highly effective at decreasing methane emissions with a lower quality diet, both with true protein and urea as nitrogen sources.

Long-Term and Carryover Effects of Supplementation with Whole Oilseeds on Methane Emission, Milk Production and Milk Fatty Acid Profile of Grazing Dairy Cows.

Research is ongoing to find nutritional methane (CH4) mitigation strategies with persistent effects that can be applied to grazing ruminants. Lipid addition to dairy cow diets has shown potential as means to decrease CH4 emissions. This study evaluated the effects of oilseeds on CH4 emission and production performance of grazing lactating dairy cows. Sixty Holstein Friesian cows grazing pasture were randomly allocated to 1 of 4 treatments (n = 15): supplemented with concentrate without oilseeds (CON), with whole cottonseed (CTS), rapeseed (RPS) or linseed (LNS). Oilseeds were supplemented during weeks 1-16 (spring period) and 17-22 (summer period), and the autumn period (wk 23-27) was used to evaluate treatment carryover effects. Cows fed CTS decreased CH4 yield by 14% compared to CON in spring, but these effects did not persist after 19 weeks of supplementation (summer). Compared to CON, RPS decreased milk yield and CTS increased milk fat concentration in both spring and summer. In summer, CTS also increased milk protein concentration but decreased milk yield, compared to CON. In spring, compared to CON, CTS decreased most milk medium-chain fatty acids (FA; 8:0, 12:0, 14:0 and 15:0) and increased stearic, linoleic and rumenic FA, and LNS increased CLA FA. There were no carry-over effects into the autumn period. In conclusion, supplementation of grazing dairy cows with whole oilseeds resulted in mild effects on methane emissions and animal performance. In particular, supplementing with CTS can decrease CH4 yield without affecting milk production, albeit with a mild and transient CH4 decrease effect. Long term studies conducted under grazing conditions are important to provide a comprehensive overview of how proposed nutritional CH4 mitigation strategies affect productivity, sustainability and consumer health aspects.

Dietary SFAs and ω-6 Fatty Acids Alter Incorporation of ω-3 Fatty Acids into Milk Fat of Lactating CD-1 Mice and Tissues of Offspring.

BACKGROUND: Methods to increase the amount of omega-3 (n-3) PUFAs in milk are desirable for neonatal health. The n-3 PUFA, α-linolenic acid (18:3n-3), can be elongated to EPA (20:5n-3) and DHA (22:6n-3). n-6 PUFAs suppress tissue n-3 PUFA incorporation, but the effect of SFAs is not clear. OBJECTIVES: In this study, we compared the effects of SFAs and n-6 PUFAs on n-3 PUFA incorporation into milk and tissues of lactating mice and tissues of their offspring. METHODS: Female CD-1 mice were bred at 8 wk of age. All experimental diets included 3% flaxseed oil and were begun on day 8 of lactation: low-fat diet (LFD); high-SFA diet (SAT), with an additional 12% saturated oil; or high-linoleic-acid diet (HLA), with 12% high-linoleic-acid oil (% kcal, carbohydrates:fat:protein: LFD, 49:24:27; both SAT and HLA, 35:46:19; n = 5/treatment). After 5 d, pup stomach milk clot FA profiles, tissue FA profiles in dams and pups, and mammary and hepatic expression of lipid metabolism genes in dams were analyzed. Data were analyzed by ANOVA with treatment diet as a fixed effect. RESULTS: Dams in all groups had similar total milk fat concentrations, but both SAT and HLA decreased the concentration of n-3 PUFAs (SAT: -23%; HLA: -31%) compared with LFD, and HLA increased milk n-6 FAs by 347% compared with SAT. SAT pups had n-3 PUFA tissue concentrations similar to LFD, but HLA pups had lower n-3 PUFAs than SAT pups in multiple tissues (liver, -32%; kidney, -29%; heart, -28%; muscle, -18%). Mammary expression of lipid metabolism genes was mostly unchanged, but hepatic expression of elongases and desaturases was decreased with SAT compared with LFD [elongation of very-long-chain fatty acid (Elov)5, -42%; Elov6, -64%; fatty acid desaturase (Fads)1, -33%; Fads2, -44%]. CONCLUSIONS: HLA decreased n-3 PUFA concentrations across multiple pup tissues compared with SAT. This suggests that high dietary n-6 PUFAs suppress n-3 PUFA incorporation in neonates.

Inhibiting Methanogenesis Stimulated de novo Synthesis of Microbial Amino Acids in Mixed Rumen Batch Cultures Growing on Starch but Not on Cellulose.

Ameliorating methane (CH4) emissions from ruminants would have environmental benefits, but it is necessary to redirect metabolic hydrogen ([H]) toward useful sinks to also benefit animal productivity. We hypothesized that inhibiting rumen methanogenesis would increase de novo synthesis of microbial amino acids (AA) as an alternative [H] sink if sufficient energy and carbon are provided. We examined the effects of inhibiting methanogenesis with 9, 10-anthraquione (AQ) on mixed rumen batch cultures growing on cellulose or starch as sources of energy and carbon contrasting in fermentability, with ammonium (NH4+) or trypticase (Try) as nitrogen (N) sources. Inhibiting methanogenesis with AQ inhibited digestion with cellulose but not with starch, and decreased propionate and increased butyrate molar percentages with both substrates. Inhibiting methanogenesis with 9, 10-anthraquinone increased de novo synthesis of microbial AA with starch but not with cellulose. The decrease in the recovery of [H] caused by the inhibition of methanogenesis was more moderate with starch due to an enhancement of butyrate and AA as [H] sinks. There may be an opportunity to simultaneously decrease the emissions of CH4 and N with some ruminant diets and replace plant protein supplements with less expensive non-protein nitrogen sources such as urea.

Kinetics of trans-10, cis-12-conjugated linoleic acid transfer to plasma and milk following an abomasal bolus in lactating dairy cows.

Trans-10, cis-12-conjugated linoleic acid (CLA) is a potent bioactive fatty acids (FA) that causes milk fat depression in lactating animals. FA are transferred to milk directly through chylomicrons and indirectly by recycling through other tissues. The objective of this study was to characterise the kinetics of trans-10, cis-12 CLA transfer to plasma and milk after a single bolus infusion. Five multiparous mid-lactation cows received a single abomasal bolus infusion of an enriched CLA mixture providing 15 g of trans-10, cis-12 CLA and 15 g of cis-9, trans-11 CLA over a 30-min period. Plasma concentration of trans-10, cis-12 and cis-9, trans-11 CLA peaked 2 h post-bolus, reaching 0·29 and 0·38 % of total plasma FA, respectively, and returned to pre-bolus values at 72 h post-infusion. Milk trans-10, cis-12 CLA yield and concentration peaked 14 h post-bolus (0·25 g/h) and was not detectable in milk after 86 h. Total apparent transfer of trans-10, cis-12 CLA to milk was 41 %, with 73 % transferred to milk through the direct pool (chylomicrons) and the remaining 27 % transferred through the indirect pool (tissue recycling). Compartmental modelling revealed the existence of a transient unavailable pool of trans-10, cis-12 CLA in extravascular tissues represented primarily by the mammary gland, which slowly exchanges with an available pool for secretion in milk fat and transfer to milk. In conclusion, trans-10, cis-12 CLA is predominantly transferred to milk through the direct pathway; however, how this CLA isomer is processed within the mammary gland requires further investigation.

Acetate Dose-Dependently Stimulates Milk Fat Synthesis in Lactating Dairy Cows.

Background: Acetate is a short-chain fatty acid (FA) that is especially important to cows because it is the major substrate for de novo FA synthesis. However, the effect of acetate supply on mammary lipid synthesis is not clear.Objective: The objective of this experiment was to determine the effect of increasing acetate supply on milk fat synthesis in lactating dairy cows.Methods: Six multiparous lactating Holstein cows were randomly assigned to treatments in a replicated design to investigate the effect of acetate supply on milk fat synthesis. Treatments were 0 (control), 5, 10, and 15 mol acetate/d continuously infused into the rumen for 4 d. Rumen short-chain FAs, plasma hormones and metabolites, milk fat concentration, and milk FA profile were analyzed on day 4 of each treatment. Polynomial contrasts were used to test the linear and quadratic effects of increasing acetate supply.Results: Acetate increased milk fat yield quadratically (P < 0.01) by 7%, 16%, and 14% and increased milk fat concentration linearly (P < 0.001) by 6%, 9%, and 11% for 5, 10, and 15 mol acetate/d, respectively, compared with the control treatment. Increased milk fat yield predominantly was due to a linear increase in 16-carbon FAs (P < 0.001) and a quadratic increase in de novo synthesized FAs (<16-carbon FAs; P < 0.01), indicating that there was stimulation of de novo synthesis pathways. Apparent transfer of acetate to milk fat was 33.4%, 36.2%, and 20.6% for 5, 10, and 15 mol/d, respectively. Acetate infusion linearly increased the relative concentration of rumen acetate (P < 0.001) before feeding, but not after feeding. Acetate linearly increased plasma ß-hydroxybutyric acid by 29%, 50%, and 78%, respectively, after feeding compared with the control treatment (P < 0.01).Conclusions: Increasing acetate supply to lactating cows increases milk fat synthesis, suggesting that nutritional strategies that increase ruminal acetate absorption would be expected to increase milk fat by increasing de novo FA synthesis.