Between-cow variation in milk fatty acids associated with methane production.

We evaluated the between-cow (b-cow) variation and repeatability in omasal and milk fatty acids (FA) related to methane (CH4) emission. The dataset was originated from 9 studies with rumen-cannulated dairy cows conducted using either a switch-back or a Latin square design. Production of CH4 per mole of VFA (Y_CH4VFA) was calculated based on VFA stoichiometry. Experiment, diet within experiment, period within experiment, and cow within experiment were considered as random factors. Empirical models were developed between the variables of interest by univariate and bivariate mixed model regression analysis. The variation associated with diet was higher than the b-cow variation with low repeatability (< 0.25) for milk odd- and branch-chain FA (OBCFA). Similarly, for de novo synthesized milk FA, diet variation was ~ 3-fold greater than the b-cow variation; repeatability for these FA was moderate to high (0.34-0.58). Also, for both cis-9 C18:1 and cis-9 cis-12 cis-15 C18:3 diet variation was more than double the b-cow variation, but repeatability was moderate. Among the de novo milk FA, C4:0 was positively related with stoichiometric Y_CH4VFA, while for OBCFA, anteiso C15:0 and C15:0 were negatively related with it. Notably, when analyzing the relationship between omasal FA and milk FA we observed positive intercept estimates for all the OBCFA, which may indicate endogenous post-ruminal synthesis of these FA, most likely in the mammary gland. For milk iso C13:0, iso C15:0, anteiso C15:0, and C15:0 were positively influenced by omasal proportion of their respective FA and by energy balance. In contrast, the concentration of milk C17:0, iso C18:0, C18:0, cis-11 C18:1, and cis-9 cis-12 cis-15 C18:3 were positively influenced by omasal proportion of their respective FA but negatively related to calculated energy balance. Our findings demonstrate that for most milk FA examined, a larger variation is attributed to diet than b-cow differences with low to moderate repeatability. While some milk FA were positively or negatively related with Y_CH4VFA, there was a pronounced effect of calculated energy balance on these estimates. Additionally, even though OBCFA have been indicated as markers of rumen function, our results suggest that endogenous synthesis of these FA may occur, which therefore, may limit the utilization of milk FA as a proxy for CH4 predictions for cows fed the same diet.

Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques.

Nitrogen is a component of essential nutrients critical for the productivity of ruminants. If excreted in excess, N is also an important environmental pollutant contributing to acid deposition, eutrophication, human respiratory problems, and climate change. The complex microbial metabolic activity in the rumen and the effect on subsequent processes in the intestines and body tissues make the study of N metabolism in ruminants challenging compared with nonruminants. Therefore, using accurate and precise measurement techniques is imperative for obtaining reliable experimental results on N utilization by ruminants and evaluating the environmental impacts of N emission mitigation techniques. Changeover design experiments are as suitable as continuous ones for studying protein metabolism in ruminant animals, except when changes in body weight or carryover effects due to treatment are expected. Adaptation following a dietary change should be allowed for at least 2 (preferably 3) wk, and extended adaptation periods may be required if body pools can temporarily supply the nutrients studied. Dietary protein degradability in the rumen and intestines are feed characteristics determining the primary AA available to the host animal. They can be estimated using in situ, in vitro, or in vivo techniques with each having inherent advantages and disadvantages. Accurate, precise, and inexpensive laboratory assays for feed protein availability are still needed. Techniques used for direct determination of rumen microbial protein synthesis are laborious and expensive, and data variability can be unacceptably large; indirect approaches have not shown the level of accuracy required for widespread adoption. Techniques for studying postruminal digestion and absorption of nitrogenous compounds, urea recycling, and mammary AA metabolism are also laborious, expensive (especially the methods that use isotopes), and results can be variable, especially the methods based on measurements of digesta or blood flow. Volatile loss of N from feces and particularly urine can be substantial during collection, processing, and analysis of excreta, compromising the accuracy of measurements of total-tract N digestion and body N balance. In studying ruminant N metabolism, nutritionists should consider the longer term fate of manure N as well. Various techniques used to determine the effects of animal nutrition on total N, ammonia- or nitrous oxide-emitting potentials, as well as plant fertilizer value, of manure are available. Overall, methods to study ruminant N metabolism have been developed over 150 yr of animal nutrition research, but many of them are laborious and impractical for application on a large number of animals. The increasing environmental concerns associated with livestock production systems necessitate more accurate and reliable methods to determine manure N emissions in the context of feed composition and ruminant N metabolism.

Temporal changes in milk fatty acid composition during diet-induced milk fat depression in lactating cows.

Diet-induced milk fat depression (MFD) in lactating cows has been attributed to alterations in ruminal lipid metabolism leading to the formation of specific fatty acid (FA) biohydrogenation intermediates that directly inhibit milk fat synthesis. However, the mechanisms responsible for decreased lipid synthesis in the mammary gland over time are not well defined. The aim of this study was to evaluate the effect of diet on milk FA composition and milk fat production over time, especially during MFD, and explore the associations between MFD and FA biohydrogenation intermediates in omasal digesta and milk. Four lactating Finnish Ayrshire cows used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d experimental periods were fed diets formulated to cause differences in ruminal and mammary lipid metabolism. Treatments consisted of an iso-nitrogenous total mixed ration based on grass silage with a forage to concentrate ratio of 65:35 or 35:65 without added oil, or with sunflower oil at 50 g/kg of diet dry matter. The high-concentrate diet with sunflower oil (HSO) induced a 2-stage drop in milk fat synthesis that was accompanied by specific temporal changes in the milk FA composition. The MFD on HSO was associated especially with trans-10 18:1 and also with trans-9,cis-11 conjugated linoleic acid (CLA) in milk and omasal digesta across all diets and was accompanied by the appearance of trans-10,cis-15 18:2. Trans-10,cis-12 CLA was increased in HSO, but milk fat secretion was not associated with omasal or milk trans-10,cis-12 CLA. The temporal changes in milk fat content and yield and milk FA composition reflect the shift from the predominant ruminal biohydrogenation pathway to an alternative pathway. The ambiguous role of trans-10,cis-12 CLA suggests that trans-10 18:1, trans-9,cis-11 CLA and trans-10,cis-15 18:2 or additional mechanisms contributed to the diet-induced MFD in lactating cows.

Symposium review: Uncertainties in enteric methane inventories, measurement techniques, and prediction models.

Ruminant production systems are important contributors to anthropogenic methane (CH4) emissions, but there are large uncertainties in national and global livestock CH4 inventories. Sources of uncertainty in enteric CH4 emissions include animal inventories, feed dry matter intake (DMI), ingredient and chemical composition of the diets, and CH4 emission factors. There is also significant uncertainty associated with enteric CH4 measurements. The most widely used techniques are respiration chambers, the sulfur hexafluoride (SF6) tracer technique, and the automated head-chamber system (GreenFeed; C-Lock Inc., Rapid City, SD). All 3 methods have been successfully used in a large number of experiments with dairy or beef cattle in various environmental conditions, although studies that compare techniques have reported inconsistent results. Although different types of models have been developed to predict enteric CH4 emissions, relatively simple empirical (statistical) models have been commonly used for inventory purposes because of their broad applicability and ease of use compared with more detailed empirical and process-based mechanistic models. However, extant empirical models used to predict enteric CH4 emissions suffer from narrow spatial focus, limited observations, and limitations of the statistical technique used. Therefore, prediction models must be developed from robust data sets that can only be generated through collaboration of scientists across the world. To achieve high prediction accuracy, these data sets should encompass a wide range of diets and production systems within regions and globally. Overall, enteric CH4 prediction models are based on various animal or feed characteristic inputs but are dominated by DMI in one form or another. As a result, accurate prediction of DMI is essential for accurate prediction of livestock CH4 emissions. Analysis of a large data set of individual dairy cattle data showed that simplified enteric CH4 prediction models based on DMI alone or DMI and limited feed- or animal-related inputs can predict average CH4 emission with a similar accuracy to more complex empirical models. These simplified models can be reliably used for emission inventory purposes.

In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.

A great deal of uncertainty still exists about intermediate metabolites and pathways explaining the biohydrogenation (BH) of 20- and 22-carbon polyunsaturated fatty acids (PUFA). Therefore, this study was conducted to provide further insight into the ruminal metabolism of 20:5 n-3 (EPA), 22:5 n-3 (DPA), and 22:6 n-3 (DHA), the main n-3 PUFA present in the marine lipids used in dairy ruminant feeding, and to examine potential differences between bovine and ovine. To meet this aim, we investigated the 20- and 22-carbon metabolites accumulated during in vitro incubation of EPA, DPA, and DHA with rumen inocula from cows and ewes. The PUFA were added at a dose of 2% incubated dry matter and digesta samples were analyzed after 24 h of incubation using complementary gas-liquid chromatography of fatty acid methyl esters and gas chromatography-mass spectrometry of 4,4-dimethyloxazoline derivatives. Results suggested that the main BH pathway of EPA and DPA would proceed via the reduction of the double bond closest to the carboxyl group (cis-5 in EPA and cis-7 in DPA); curiously, this mechanism seemed of much lower importance for DHA. Thus, DPA would not be a major intermediate product of DHA and their BH might actually follow separate pathways, with the accumulation of numerous unique metabolites in each case. A principal component analysis supported this hypothesis, with a clear separation between PUFA treatments in the score and loading plots. Within EPA and DPA groups, cow and ewe samples loaded separately from each other but not distant. No conjugated 20:5, 22:5, or 22:6 isomer compatible with the initial product of EPA, DPA, or DHA metabolism, respectively, was identified in the ruminal digesta, although this would not unequivocally exclude their transient formation. In this regard, results from DPA incubations provided the first indication that the metabolism of this very long chain PUFA may involve the formation of conjugated double bond structures. The BH of EPA, DPA, and DHA resulted in the appearance of several tentative trans-10-containing metabolites, showing a general trend to be more abundant in the digesta of ewes than in that of cows. This finding was speculated to have some relationship with the susceptibility of dairy sheep to marine lipid-induced milk fat depression. Differences in the relative proportion of intermediate products would also suggest an influence of ruminant species on BH kinetics, with a process that would likely be slower and less complete in cows than in ewes.

Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows.

Fish oil (FO) alters ruminal biohydrogenation causing trans fatty acid (FA) intermediates to accumulate, but the effects of 18-carbon polyunsaturated FA supply on ruminal long-chain FA metabolism and microbial communities in cattle fed FO are not well established. Four cows fitted with rumen cannula were used in a 4 × 4 Latin square with 21-d experimental periods to evaluate the effects of FO alone or in combination with plant oils high in 18:2n-6 or 18:3n-3 on rumen microbial ecology and flow of FA at the omasum. Treatments comprised a basal grass silage-based diet containing no additional oil (control) or supplements of FO (200 g/d) or FO (200 g/d) plus 500 g/d of sunflower oil (SFO) or linseed oil (LFO). Flow of FA was determined using the omasal sampling technique. The relative abundance of key biohydrogenating bacteria was assessed by quantitative PCR on 16S rRNA genes in omasal digesta. Fish oil-supplemented treatments increased the amounts of trans-18:1, trans-18:2, and 20- to 22-carbon polyunsaturated FA escaping the rumen. Relative to the control, oil supplements had no effect on the amount of 18:0 leaving the rumen, but LFO decreased the flow of 18:0 at the omasum compared with SFO. Both SFO and LFO increased trans-18:1 relative to FO, whereas LFO resulted in the highest trans-18:2 and 20- to 22-carbon FA flow. Supplements of FO plus plant oils shifted biohydrogenation toward trans-10 18:1 formation. Compared with FO alone, the ruminal metabolism of 22:6n-3 in the rumen of lactating cows is more extensive on diets containing higher amounts of 18-carbon polyunsaturated FA. However, the biohydrogenation of 22:5n-3 was less extensive in LFO than SFO, but showed no difference between FO and diets containing plant oils. Ruminal outflow of 20:5n-3 was not altered when plant oils were added to FO. Alterations in the amount of intermediates at the omasum or ruminal biohydrogenation pathways were not accompanied by major changes in analyzed bacterial populations. In conclusion, dietary supplements of FO alone or in combination with plant oils increase the amount of biohydrogenation intermediates containing 1 or more trans double bonds escaping the rumen, which may have implications for host metabolism and the nutritional quality of ruminant foods.

Plant oil supplements reduce methane emissions and improve milk fatty acid composition in dairy cows fed grass silage-based diets without affecting milk yield.

Four lipid supplements varying in chain length or degree of unsaturation were examined for their effects on milk yield and composition, ruminal CH4 emissions, rumen fermentation, nutrient utilization, and microbial ecology in lactating dairy cows. Five Nordic Red cows fitted with rumen cannulas were used in a 5 × 5 Latin square with five 28-d periods. Treatments comprised total mixed rations based on grass silage with a forage-to-concentrate ratio of 60:40 supplemented with no lipid (CO) or 50 g/kg of diet dry matter (DM) of myristic acid (MA), rapeseed oil (RO), safflower oil (SO), or linseed oil (LO). Feeding MA resulted in the lowest DM intake, and feeding RO reduced DM intake compared with CO. Feeding MA reduced the yields of milk, milk constituents, and energy-corrected milk. Plant oils did not influence yields of milk and milk constituents, but reduced milk protein content compared with CO. Treatments had no effect on rumen fermentation characteristics, other than an increase in ammonia-N concentration due to feeding MA, RO, and SO compared with CO. Lipid supplements reduced daily ruminal CH4 emission; however, the response was to some extent a result of lower feed intake. Lipids modified microbial community structure without affecting total counts of bacteria, archaea, and ciliate protozoa. Dietary treatments had no effect on the apparent total tract digestibility of organic matter, fiber, and gross energy. Treatments did not affect either energy secreted in milk as a proportion of energy intake or efficiency of dietary N utilization. All lipids lowered de novo fatty acid synthesis in the mammary gland. Plant oils increased proportions of milk fat 18:0, cis 18:1, trans and monounsaturated fatty acids, and decreased saturated fatty acids compared with CO and MA. Both SO and LO increased the proportion of total polyunsaturated fatty acids, total conjugated linolenic acid, and cis-9,trans-11 conjugated linoleic acid. Feeding MA clearly increased the Δ9 desaturation of fatty acids. Our results provide compelling evidence that plant oils supplemented to a grass silage-based diet reduce ruminal CH4 emission and milk saturated fatty acids, and increase the proportion of unsaturated fatty acids and total conjugated linoleic acid while not interfering with digestibility, rumen fermentation, rumen microbial quantities, or milk production.

Effects of replacement of late-harvested grass silage and barley with early-harvested silage on ruminal digestion efficiency in lactating dairy cows.

The objective of this experiment was to quantify the effects of graded replacement of late-harvested grass silage and barley with early-harvested silage on nutrient digestion and rumen fermentation. Four experimental diets were fed to 4 multiparous rumen-cannulated Nordic Red cows in 4 × 4 Latin square design with 21-d periods. Dietary treatments consisted of late-cut grass silage (LS) and rolled barley, which was gradually replaced with early-cut grass silage [ES; 0, 33, 67, and 100% of the forage component (ES + LS) of the diet]. With increased proportion of ES in the diet, the proportion of barley decreased from 47.2 to 26.6% on a dry matter basis. Early- and late-cut silages were harvested at 2-wk intervals (predicted concentrations of metabolizable energy 11.0 and 9.7 MJ/kg of dry matter). The 4 diets were formulated to support the same milk production. Nutrient flows were quantified using omasal sampling technique applying the triple-marker method (Cr, Yb, and indigestible neutral detergent fiber) and 15N as a microbial marker. Feed intake decreased with graded replacement of LS and barley with ES, but milk production was not influenced by diet. Digestibility of nutrients improved with graded addition of ES in the diet with the greatest difference observed in digestibility of neutral detergent fiber (NDF) and potentially digestible NDF (pdNDF). The results suggested that improved cell wall digestibility with graded level of ES in the diet was partly related to higher intrinsic digestibility of ES than LS, and partly due to negative associative effects with an increased proportion of LS and barley in the diet. Efficiency of microbial N synthesis was not influenced by the diet, but ruminal protein degradability increased with ES in the diet. Rumen fermentation pattern was not affected by the diet despite large difference in the profile of dietary carbohydrates. Rumen pool size of NDF and pdNDF, and ruminal turnover time of NDF decreased with graded addition of ES in the diet, whereas digestion rate of pdNDF improved. The results of this study indicate that increased CH4 yield in a parallel production study with graded addition of ES in the diet were more related to greater ruminal and total digestibility of organic matter than to the changes in rumen fermentation pattern.

In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.

The modulation of milk fat nutritional quality through fish oil supplementation seems to be largely explained by the action of n-3 very long chain polyunsaturated fatty acids (PUFA) on ruminal biohydrogenation (BH) of C18 fatty acids (FA). However, relationships among this action, disappearance of those PUFA in the rumen, and potential detrimental consequences on ruminal fermentation remain uncertain. This study compared the effect of 20:5n-3 (eicosapentaenoic acid; EPA), 22:5n-3 (docosapentaenoic acid; DPA), and 22:6n-3 (docosahexaenoic acid; DHA) on rumen fermentation and BH of C18 FA and was conducted simultaneously in cows and sheep to provide novel insights into interspecies differences. The trial was performed in vitro using batch cultures of rumen microorganisms with inocula collected from cannulated cows and ewes. The PUFA were added at a dose of 2% incubated dry matter, and treatment effects on ruminal C18 FA concentrations, PUFA disappearances, and fermentation parameters (gas production, ammonia and volatile FA concentrations, and dry matter and neutral detergent fiber disappearances) were examined after 24 h of incubation. A principal component analysis suggested that responses to PUFA treatments explained most of the variability; those of ruminant species were of lower relevance. Overall, EPA and DHA were equally effective for inhibiting the saturation of trans-11 18:1 to 18:0 and had a similar influence on ruminal fermentation in cows and sheep (e.g., reductions in gas production and acetate:propionate ratio). Nevertheless, DHA further promoted alternative BH pathways that lead to trans-10 18:1 accumulation, and EPA seemed to have specific effects on 18:3n-3 metabolism. Only minor variations attributable to DPA were observed in the studied parameters, suggesting a low contribution of this FA to the action of marine lipids. Although most changes due to the added PUFA were comparable in bovine and ovine, there were also relevant specificities, such as a stronger inhibition of 18:0 formation in cows and a greater increase in 18:3n-3 metabolites in sheep. No direct relationship between in vitro disappearance of the incubated PUFA and effect on BH (in particular, inhibition of the last step) was found in either cows or ewes, calling into question a putative link between extent of disappearance and toxicity for microbiota. Conversely, an association between the influence of these PUFA on ruminal lipid metabolism and fermentation may exist in both species. In vivo verification of these findings would be advisable.

Effects of replacement of late-harvested grass silage and barley with early-harvested silage on milk production and methane emissions.

This study evaluated the effects of gradual replacement of a mixture of late-cut grass silage (LS) and barley with early-cut grass silage (ES) on milk production, CH4 emissions, and N utilization in Swedish Red cows. Two grass silages were prepared from the same primary growth of timothy grass sward but harvested 2 wk apart [11.0 and 9.7 MJ of metabolizable energy/kg of dry matter (DM)]. Four diets, fed as a total mixed ration, were formulated to meet the metabolizable energy and protein requirements of 35 kg of energy-corrected milk (ECM) by gradually replacing a mixture of LS and barley with ES (0, 33, 67, and 100% of the forage component of the diet), whereas the proportion of barley decreased from 47.2 to 26.6% of diet DM. Expeller canola meal was used as a protein supplement. Sixteen Swedish Red cows were used in 4 replicated 4 × 4 Latin squares. Cows were offered diets ad libitum and milked twice daily. Each period of 28 d comprised 14 d of diet adaptation followed by 14 d of data collection. Intake and milk yield were recorded daily, and milk samples were collected on d 19 to 21 and d 26 to 28 of each period. Diet digestibility was determined by grab sampling using indigestible neutral detergent fiber as an internal marker. Gas emissions were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD). Dry matter intake (DMI) linearly decreased from 22.6 to 19.3 kg/d as the proportion of ES increased in the diet. The ECM yield did not differ among treatments, but milk protein yield decreased with increasing proportion of ES in the diet. Because of reduced DMI with increasing ES, feed efficiency (ECM/DMI) improved with an increased proportion of ES in the diet. Nitrogen efficiency (milk N/N intake) did not change despite a linear increase in milk urea N concentration from 9.7 (LS alone) to 11.9 mg/dL (ES alone) with graded replacement of LS and barley by ES in the diet. Lower DMI responses in ES diets were partly compensated for by increased organic matter digestibility (656 g/kg of DM for LS alone; 715 g/kg of DM for ES alone) related to improved forage digestibility at early harvesting. Total CH4 emissions and CH4 intensity (CH4/ECM) were not influenced by diet, but CH4 yield (CH4/DMI) increased linearly from 19.5 to 23.0 g/kg of DMI with greater inclusion of ES in the diet. In conclusion, replacing LS and barley with ES improved the conversion of feed to milk without increasing CH4 emissions or compromising N efficiency.

Between-cow variation in digestion and rumen fermentation variables associated with methane production.

A meta-analysis based on an individual-cow data set was conducted to investigate the effects of between-cow variation and related animal variables on predicted CH4 emissions from dairy cows. Data were taken from 40 change-over studies consisting of a total of 637 cow/period observations. Animal production and rumen fermentation characteristics were measured for 154 diets in 40 studies; diet digestibility was measured for 135 diets in 34 studies, and ruminal digestion kinetics was measured for 56 diets in 15 studies. The experimental diets were based on grass silage, with cereal grains or by-products as energy supplements, and soybean or canola meal as protein supplements. Average forage:concentrate ratio across all diets on a dry matter basis was 59:41. Methane production was predicted from apparently fermented substrate using stoichiometric principles. Data were analyzed by mixed-model regression using diet and period within experiment as random effects, thereby allowing the effect of experiment, diet, and period to be excluded. Dry matter intake and milk yield were more repeatable experimental measures than rumen fermentation, nutrient outflow, diet digestibility, or estimated CH4 yield. Between-cow coefficient of variation (CV) was 0.010 for stoichiometric CH4 per mol of volatile fatty acids and 0.067 for predicted CH4 yield (CH4/dry matter intake). Organic matter digestibility (OMD) also displayed little between-cow variation (CV = 0.013), indicating that between-cow variation in diet digestibility and rumen fermentation pattern do not markedly contribute to between cow-variation in CH4 yield. Digesta passage rate was much more variable (CV = 0.08) between cows than OMD or rumen fermentation pattern. Increased digesta passage rate is associated with improved energetic efficiency of microbial N synthesis, which partitions fermented substrate from volatile fatty acids and gases to microbial cells that are more reduced than fermented carbohydrates. Positive relationships were observed between CH4 per mol of volatile fatty acids versus OMD and rumen ammonia N concentration versus OMD; and negative relationships between the efficiency of microbial N synthesis versus OMD and digesta passage rate versus OMD, suggesting that the effects of these variables on CH4 yield were additive. It can be concluded that variations in OMD and efficiency in microbial N synthesis resulting from variations in digesta passage contribute more to between-animal variation in CH4 emissions than rumen fermentation pattern.

Dietary forage to concentrate ratio and sunflower oil supplement alter rumen fermentation, ruminal methane emissions, and nutrient utilization in lactating cows.

The effects of supplementing high- or low-concentrate diets with sunflower oil (SO) on rumen fermentation, nutrient utilization, and ruminal methane (CH4) emissions in lactating cows were examined. Four multiparous Nordic Red dairy cows fitted with rumen cannulae were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d periods. Experimental treatments comprised iso-nitrogenous total mixed rations based on grass silage with forage to concentrate ratio of 65:35 or 35:65 supplemented with 0 or 50 g/kg diet DM of SO. Apparent ruminal OM and starch digestibility was greater (P < 0.05) with high- than low-concentrate diets but was unaffected by SO. Inclusion of SO in high-concentrate diet decreased (P ≤ 0.05) apparent total tract OM, fiber, and GE, and apparent ruminal fiber digestibility. High-concentrate diets and SO shifted (P < 0.05) fiber digestion from rumen to the hindgut. High-concentrate diet resulted in a lower rumen pH and elevated total rumen VFA concentration compared with low-concentrate diet, whereas SO increased rumen pH and decreased rumen VFA concentration when included in high-, but not low-concentrate diet (P < 0.05 for interaction). High-concentrate diet reduced rumen ammonia-N (P < 0.01) and molar proportion of acetate to propionate (P < 0.01), and decreased (P < 0.05) ruminal CH4 emissions when expressed as g/d or g/kg OM digested in the rumen. With both low- and high-concentrate diets, SO reduced (P < 0.05) daily emissions of CH4 as g/d or g/kg OM digested in the rumen, but SO reduced CH4 emissions expressed as g/kg OM intake, OM digested in total digestive tract, energy-corrected milk or % of GE intake only with low-concentrate diet (P ≤ 0.05 for interaction). In conclusion, replacing grass silage with concentrates led to a reduction in daily ruminal CH4 emissions that were accompanied by a shift in rumen fermentation toward the synthesis of propionate, and decreases in rumen pH and fiber digestion. Sunflower oil was effective in reducing daily CH4 emissions in lactating cows which was accompanied by a noticeable lower feed intake with high- but not low-concentrate diet. Overall the effects of SO and greater proportion of concentrates in the diet on daily CH4 emissions were additive but the additivity declined or vanished when different indices of CH4 emission intensity were considered. Consequently, SO was more effective in reducing CH4 emissions when low-concentrate diet was fed.

Effect of incremental amounts of camelina oil on milk fatty acid composition in lactating cows fed diets based on a mixture of grass and red clover silage and concentrates containing camelina expeller.

Camelina is an ancient oilseed crop that produces an oil rich in cis-9,cis-12 18:2 (linoleic acid, LA) and cis-9,cis-12,cis-15 18:3 (α-linolenic acid, ALA); however, reports on the use of camelina oil (CO) for ruminants are limited. The present study investigated the effects of incremental CO supplementation on animal performance, milk fatty acid (FA) composition, and milk sensory quality. Eight Finnish Ayrshire cows (91d in milk) were used in replicated 4×4 Latin squares with 21-d periods. Treatments comprised 4 concentrates (12kg/d on an air-dry basis) based on cereals and camelina expeller containing 0 (control), 2, 4, or 6% CO on an air-dry basis. Cows were offered a mixture of grass and red clover silage (RCS; 1:1 on a dry matter basis) ad libitum. Incremental CO supplementation linearly decreased silage and total dry matter intake, and linearly increased LA, ALA, and total FA intake. Treatments had no effect on whole-tract apparent organic matter or fiber digestibility and did not have a major influence on rumen fermentation. Supplements of CO quadratically decreased daily milk and lactose yields and linearly decreased milk protein yield and milk taste panel score from 4.2 to 3.6 [on a scale of 1 (poor) to 5 (excellent)], without altering milk fat yield. Inclusion of CO linearly decreased the proportions of saturated FA synthesized de novo (4:0 to 16:0), without altering milk fat 18:0, cis-9 18:1, LA, and ALA concentrations. Milk fat 18:0 was low (<5g/100g of FA) across all treatments. Increases in CO linearly decreased the proportions of total saturates from 58 to 45g/100g of FA and linearly enriched trans-11 18:1, cis-9,trans-11 18:2, and trans-11,cis-15 18:2 from 5.2, 2.6, and 1.7 to 11, 4.3, and 5.8g/100g of FA, respectively. Furthermore, CO quadratically decreased milk fat trans-10 18:1 and linearly decreased trans-10,cis-12 18:2 concentration. Overall, milk FA composition on all treatments suggested that one or more components in camelina seeds may inhibit the complete reduction of 18-carbon unsaturates in the rumen. In conclusion, CO decreased the secretion of saturated FA in milk and increased those of the trans-11 biohydrogenation pathway or their desaturation products. Despite increasing the intake of 18-carbon unsaturated FA, CO had no effect on the secretions of 18:0, cis-9 18:1, LA, or ALA in milk. Concentrates containing camelina expeller and 2% CO could be used for the commercial production of low-saturated milk from grass- and RCS-based diets without major adverse effects on animal performance.

Effect of adsorbants on in vitro biohydrogenation of 22:6n-3 by mixed cultures of rumen microorganisms.

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.

Effects of plant species, stage of maturity, and level of formic acid addition on lipolysis, lipid content, and fatty acid composition during ensiling.

Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; L.) silage increases milk fat 18:3-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis and . The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation on lipolysis. A randomized block design with a 2 × 3 × 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy ( L.) and meadow fescue ( Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3-3 and total fatty acids (TFA) were higher ( < 0.001) for RC than for grass. For both forage species, 18:3-3 and TFA content decreased linearly ( < 0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher ( < 0.001) for RC than grass (11 vs. 0.11 µkatal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease ( = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true ( = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis . For both plant species, total PPO activity was not associated with the extent of lipolysis . However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.

Comparison of the nutritional regulation of milk fat secretion and composition in cows and goats.

A study with 2 ruminant species (goats and cows) with inherent differences in lipid metabolism was performed to test the hypothesis that milk fat depression (MFD) due to marine lipid supplements or diets containing high amounts of starch and plant oil is caused by different mechanisms and that each ruminant species responds differently. Cows and goats were allocated to 1 of 3 groups (4 cows and 5 goats per group) and fed diets containing no additional oil (control) or supplemented with fish oil (FO) or sunflower oil and wheat starch (SOS) according to a 3 × 3 Latin square design with 26-d experimental periods. In cows, milk fat content was lowered by FO and SOS (-31%), whereas only FO decreased milk fat content in goats (-21%) compared with the control. Furthermore, FO and SOS decreased milk fat yield in cows, but not in goats. In both species, FO and SOS decreased the secretion of C16 FA output. However, SOS increased milk secretion of >C16 FA in goats. Compared with the control, SOS resulted in similar increases in milk trans-10,cis-12 conjugated linoleic acid (CLA) in both species, but caused a 2-fold larger increase in trans-10 18:1 concentration in cows than for goats. Relative to the control, responses to FO in both species were characterized by a marked decrease in milk concentration of 18:0 (-74%) and cis-9 18:1 (-62%), together with a ~5-fold increase in total trans 18:1, but the proportionate changes in trans-10 18:1 were lower for goats. Direct comparison of animal performance and milk FA responses to FO and SOS treatments demonstrated interspecies differences in mammary lipogenesis, suggesting a lower sensitivity to the inhibitory effects of trans-10,cis-12 CLA in goats and that ruminal biohydrogenation pathways are more stable and less prone to diet-induced shifts toward the formation of trans-10-containing intermediates in goats compared with cows. Even though a direct cause and effect could not be established, results suggest that regulation of milk fat synthesis during FO-induced MFD may be related to a shortage of 18:0 for endogenous mammary cis-9 18:1 synthesis, increase in the incorporation of trans FA in milk triacylglycerols, and limitations in the synthesis of FA de novo to maintain milk fat melting point. However, the possible contribution of biohydrogenation intermediates with putative antilipogenic effects in the mammary gland, including trans-9,cis-11 CLA, trans-10 18:1, or cis-11 18:1 to FO-induced MFD cannot be excluded.

Dietary fish oil supplements depress milk fat yield and alter milk fatty acid composition in lactating cows fed grass silage-based diets.

The potential of dietary fish oil (FO) supplements to increase milk 20:5n-3 and 22:6n-3 concentrations and the associated effects on milk fatty acid (FA) composition, intake, and milk production were examined. Four multiparous lactating cows offered a grass silage-based diet (forage:concentrate ratio 58:42, on a dry matter basis) supplemented with 0, 75, 150, or 300g of FO/d (FO0, FO75, FO150, and FO300, respectively) were used in a 4×4 Latin square with 28-d experimental periods. Milk FA composition was analyzed by complementary silver-ion thin-layer chromatography, gas chromatography-mass spectrometry, and silver-ion HPLC. Supplements of FO decreased linearly dry matter intake, yields of energy-corrected milk, milk fat and protein, and milk fat content. Compared with FO0, milk fat content and yield were decreased by 30.1 and 40.6%, respectively, on the FO300 treatment. Supplements of FO linearly increased milk 20:5n-3 and 22:6n-3 concentrations from 0.07 to 0.18 and 0.03 to 0.10g/100g of FA, respectively. Enrichment of 20:5n-3 and 22:6n-3 was accompanied by decreases in 4- to 18-carbon saturated FA and increases in total conjugated linoleic acid (CLA), trans FA, and polyunsaturated FA concentrations. Fish oil elevated milk fat cis-9,trans-11 CLA content in a quadratic manner, reaching a maximum on FO150 (from 0.61 to 2.15g/100g of FA), whereas further amounts of FO increased trans-10 18:1 with no change in trans-11 18:1 concentration. Supplements of FO also resulted in a dose-dependent appearance of 37 unique 20- and 22-carbon intermediates in milk fat. Concentrations of 16-, 18-, 20-, and 22-carbon trans FA were all increased by FO, with enrichment of trans 18:1 and trans 18:2 being quantitatively the most important. Decreases in milk fat yield to FO were not related to changes in milk trans-10,cis-12 CLA concentration or estimated milk fat melting point. Partial least square regression analysis indicated that FO-induced milk fat depression was associated with changes in the concentrations of multiple FA in milk. Even though a direct cause and effect could not be established, a decrease in 18:0 supply in combination with increased mammary uptake of cis-11 18:1, trans-10 18:1, and trans 20- and 22-carbon FA may contribute. In conclusion, dietary FO supplements enrich 20:5n-3 and 22:6n-3 in milk, but also elevate mono- and polyenoic trans FA concentrations, and in high amounts alter the distribution of individual trans FA isomers.

Effect of camelina oil or live yeasts (Saccharomyces cerevisiae) on ruminal methane production, rumen fermentation, and milk fatty acid composition in lactating cows fed grass silage diets.

The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53±7 d in milk) fitted with rumen cannula were used in a 4×4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 10(10) cfu/d (treatments A and B), or supplements of 60g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.

Evaluation of between-cow variation in milk urea and rumen ammonia nitrogen concentrations and the association with nitrogen utilization and diet digestibility in lactating cows.

Concentrations of milk urea N (MUN) are influenced by dietary crude protein concentration and intake and could therefore be used as a biomarker of the efficiency of N utilization for milk production (milk N/N intake; MNE) in lactating cows. In the present investigation, data from milk-production trials (production data set; n=1,804 cow/period observations from 21 change-over studies) and metabolic studies involving measurements of nutrient flow at the omasum in lactating cows (flow data set; n=450 cow/period observations from 29 studies) were used to evaluate the influence of between-cow variation on the relationship of MUN with MNE, urinary N (UN) output, and diet digestibility. All measurements were made on cows fed diets based on grass silage supplemented with a range of protein supplements. Data were analyzed by mixed-model regression analysis with diet within experiment and period within experiment as random effects, allowing the effect of diet and period to be excluded. Between-cow coefficient of variation in MUN concentration and MNE was 0.13 and 0.07 in the production data set and 0.11 and 0.08 in the flow data set, respectively. Based on residual variance, the best model for predicting MNE developed from the production data set was MNE (g/kg)=238 + 7.0 × milk yield (MY; kg/d) - 0.064 × MY(2) - 2.7 × MUN (mg/dL) - 0.10 body weight (kg). For the flow data set, including both MUN and rumen ammonia N concentration with MY in the model accounted for more variation in MNE than when either term was used with MY alone. The best model for predicting UN excretion developed from the production data set (n=443) was UN (g/d)=-29 + 4.3 × dry matter intake (kg/d) + 4.3 × MUN + 0.14 × body weight. Between-cow variation had a smaller influence on the association of MUN with MNE and UN output than published estimates of these relationships based on treatment means, in which differences in MUN generally arise from variation in dietary crude protein concentration. For the flow data set, between-cow variation in MUN and rumen ammonia N concentrations was positively associated with total-tract organic matter digestibility. In conclusion, evaluation of phenotypic variation in MUN indicated that between-cow variation in MUN had a smaller effect on MNE compared with published responses of MUN to dietary crude protein concentration, suggesting that a closer control over diet composition relative to requirements has greater potential to improve MNE and lower UN on farm than genetic selection.