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We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at -7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and =5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 +/- 2.1%; least squares means +/- standard error) and a higher proportion of apoptosis (14.4 +/- 2.0%) than healthy (92.4 +/- 1.3 and 6.7 +/- 1.3%, respectively) or SCE (95.3 +/- 2.4 and 3.8 +/- 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 +/- 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 +/- 0.08 MFI) or CE (6.8 +/- 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 +/- 5.1%) than SCE (27.4 +/- 7.3%) and CE (27.7 +/- 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 +/- 8.6%) than in healthy (18.4 +/- 7.6%) cows, but did not differ from SCE cows (25.9 +/- 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows. Key words: transition period, inflammation, immune function, uterine disease

The aim of the present study was to assess the counts, viability, and functionality of circulating and endometrial polymorphonuclear leukocytes (PMN) isolated from fourteen clinically and metabolically healthy multiparous dairy cows in the peripartum period. For this, blood samples were collected at −5, +9, +21 and + 37 days (d) relative to calving. Cytology samples were collected from the vagina, cervix, and uterus at +9, +21 and + 37 d, using the cytobrush technique. Additional vaginal samples were collected at −5 d. Cytology smears were prepared and the PMN-to-all nucleated cell proportions (PMN%) were calculated. The endometrial cytobrush samples were also used for flow cytometric assessment of endometrial PMN (ePMN) viability and functionality. Functionality tests for circulating PMN (cPMN) included phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation. For ePMN, we evaluated PC only. The effect of day relative to calving on PMN viability and functionality were fitted in linear regression models, accounting for repeated measures. The endometrial PMN% were higher at +9 d (23.5 ± 0.4%; least-squares means ± standard error) and +21 d (8.5 ± 0.3%) than at +37 d (1.4 ± 0.3%). No changes in PMN% were found on either vaginal or cervical cytology along the peripartum period. The cPMN counts were higher pre- (6.2 ± 0.4 x 106/mL) than postpartum (4.9 ± 0.4 x 106/mL). Upon viability analysis, only the percentage of viable cPMN tended to be lower at −5 d (90.1 ± 1.5%) than at +37 d (94.1 ± 1.4%), and no other changes in the percentage of apoptotic and necrotic cPMN, nor in their functionality were found during the peripartum period. Analysis of ePMN viability showed that the percentage of viable ePMN did not change over time. In marked contrast, the percentage of apoptotic ePMN was higher at +9 d (37.8 ± 5.1%) than at +21 d (20.9 ± 5.1%) and +37 d (11.9 ± 5.3%), while the percentage of necrotic ePMN was lower at +9 d (27.0 ± 6.3%) than at +37 d (54.9 ± 6.6%). The percentage of ePMN PC was higher at +9 d (27.5 ± 3.4%) than at +37 d (13.3 ± 4.9%). In conclusion, during the peripartum period ePMN in the healthy postpartum uterus are highly dynamic in terms of counts, viability, and functionality compared to their circulating counterparts.

In a temperate climate, the mycotoxins deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROC) and zearalenone (ZEN) are often found in maize silage. Although rumen microbiota are able to degrade some mycotoxins (e.g. DON), others are known to stay mainly intact (e.g. ROC). In addition, mycotoxin degradation can be hampered by a low ruminal pH or decrease in rumen microbial activity. Hence, these mycotoxins can reach the small intestine and exert a cytotoxic effect on intestinal epithelial cells. In this study, a real-time kinetic analysis of the cytotoxicity of these six mycotoxins and some of their metabolites (α- and β-zearalenol, α- and β-ZOL) was performed in a calf small intestinal epithelial cell line (CIEB). Confluency as well as the cell death parameters apoptosis and necrosis were determined to evaluate the mycotoxin-induced cytotoxicity. A combination of Annexin-V green and Cytotox red staining was used to determine early and late apoptosis as well as necrosis. Six different concentrations were tested ranging from 0.78 to 12.5 μM. Compared to cells not exposed to mycotoxins, DON and NIV exert a fast toxic effect with DON being more toxic than NIV within the first hours of incubation, whereas the inverse was observed at 16 h of incubation. On the other hand, MPA and ZEN induced increased Annexin V green positive cells within several hours of incubation with higher toxicity over time. Increased Annexin V green and Cytotox red positive cells were seen for ROC only at the highest concentration tested. For ENN B, increased Annexin V green positive cells were observed only after 12 h and α- and β-ZOL did not show cytotoxic effects. Hence, mycotoxin exposure causes either severe (DON and NIV) or more limited (ZEN, ROC, MPA, and ENN B) risk of bovine intestinal epithelial damage.

The transition period is a challenging period for high-producing dairy cattle. Cows in early lactation are considered as a group at risk of subacute ruminal acidosis (SARA). Variability in SARA susceptibility in early lactation is hypothesized to be reflected in fecal characteristics such as fecal pH, dry matter content, volatile and odd- and branched-chain fatty acids (VFA and OBCFA, respectively), as well as fecal microbiota. This was investigated with 38 periparturient dairy cows, which were classified into four groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6. Furthermore, we investigated whether fecal differences were already obvious during a period prior to the SARA risk (prepartum).

In high-yielding dairy cows, some fertility traits can be influenced by the fatty acid (FA) composition of the follicular fluid during early lactation. The first objective of the current study was to evaluate the potential of dietary supplements enriched in specific FA to influence the FA composition of follicular fluid lipid classes in early lactation dairy cows. The second objective was to determine the influence of the resulting follicular fluid FA composition on the folliculogenesis, lipid and energy metabolism of granulosa cells, as well as oocyte quality and embryo development. Twenty Holstein multiparous cows in late gestation were randomly assigned to 200 g/d of FA supplements enriched in (1) palmitic acid (control treatment; 82% 16:0; PA) in the rumen or (2) palmitoleic acid (sea buckthorn oil; 27% cis-9 16:1, 28% 16:0, 22% cis-9 18:1, and 11% cis-9,cis-12 18:2; SBT) in the abomasum. The treatment period ranged from 20 ± 5 d precalving to 67 ± 2 d postcalving. Cumulus-oocyte complexes, granulosa cells, and follicular fluid were recovered from 2 sequential sessions of ovum pick-up (OPU-1 and OPU-2) at 46 and 67 ± 2 d postcalving (mean ± standard deviation). On the same days, blood samples were collected. Milk performance was recorded, and feed and milk samples were collected from d 8 to 10 ± 3 (onset of lactation), d 35 to 37 ± 2 (before OPU-1), and d 63 to 65 ± 2 (before OPU-2). Treatments did not affect milk yield or fat concentration throughout the experimental trial. Compared with PA, SBT increased the cis-9 16:1 concentration in milk fat, in plasma esterified lipid classes (phospholipids, cholesterol esters, and triacylglycerols), and in follicular fluid phospholipids and cholesterol esters at OPU-1. Abundance of mRNA for stearoyl-CoA desaturase 1 and 5, and perilipin 2 in granulosa cells was not different between treatments, but an increase in the level of stearoyl-CoA desaturase 5 was observed between the 2 OPU periods. Treatments did not affect oocyte quality and developmental capacity or embryo lipid metabolism when cultivated in vitro. These results suggest that limited modifications in the FA composition of the oocyte microenvironment via dietary lipid supplements enriched in specific FA had no major effects on granulosa cell metabolism and oocyte developmental capacity in early lactation cows.

PURPOSE: To examine the intra- and inter-individual variability in fatty acid composition of follicular fluid (FF) of 23 patients undergoing assisted reproductive treatment. METHODS: The average coefficient of variation within each patient (CV(w)) and intra-class correlation coefficient (ICC) values of FF fatty acid composition as well as correlation between the fatty acid composition of individual, pooled or first-punctured follicles, were assessed. RESULTS: The proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 20:5n-3, total MUFA and n-3 PUFA showed good reproducibility (CV(w)

In this study, the in vitro apparent rumen degradability of organic matter (ARDOM) and plant secondary metabolites (ARDPSM) of three tropical legumes (Mucuna pruriens, Canavalia ensiformis and Leucaena leucocephala) were assessed. For this, single end-point incubations (24 h) as well as kinetic assessments (0, 2, 4, 6, 8, 10, 12 and 24 h) were set up with different inocula sources, i.e. ruminal inocula from Belgian and Cuban sheep. L-mimosine, L-canavanine, Concanavaline A (Con A) and trypsin inhibitor (TI) were the plant secondary metabolites (PSM) targeted in this study. Bean and forage/bean meals of M. pruriens and C. ensiformis as well as their PSM were extensively degraded during 24 h incubation (0.70 and 0.78 g ARDOM/g of organic matter (OM), respectively and > 0.80 g/g for L-canavanine, > 0.76 TIU/TIU for TI and > 0.95 g/g for Con A, for both legumes). Forage meal of L. leucocephala was considerably less degraded, with apparent ruminal degradabilities of 0.20 and 0.35 g ARDOM/g OM after 24 h incubation with Belgian or Cuban sheep inoculum, respectively. This could - at least partially - be related to L-mimosine, present in L. leucocephala, which was hardly degraded in the Belgian incubation, while a more extensive ruminal breakdown was observed under the Cuban conditions (0.05 to 0.78 g ARDPSM/g PSM, respectively). The negative effect of L-mimosine on OM degradability was supported by in vitro incubations with straw and inoculum from Belgian sheep, as ruminal degradation of straw was 31% lower when L-mimosine was supplemented.

To investigate the metabolism of 18:2n-6 and 18:3n-3 by pure cultures of Sharpea azabuensis, two different strains (RL 1 and ST18) were each incubated in the presence of 40 µg ml-1 18:2n-6 or 18:3n-3. Pure cultures of Butyrivibriofibrisolvens D1 and Butyrivibrio proteoclasticus P18 were included as control treatments. Similar to the metabolism of B. fibrisolvens, both S. azabuensis strains converted 18:2n-6 or 18:3n-3 to cis-9, trans-11 CLA or cis-9, trans-11, cis-15 CLnA, after which it was further reduced to trans-11 18:1 or trans-11, cis-15 18:2, respectively. B. proteoclasticus additionally reduced trans-11 18:1 to 18:0. Trans-11, cis-15 18:2 was also further metabolized by B. proteoclasticus, although trans-11 18:1 did not accumulate, and only minor amounts of 18:0 were formed. The time frame of 18:2n-6 and 18:3n-3 biohydrogenation by S. azabuensis was comparable with B. fibrisolvens, indicating that S. azabuensis and B. fibrisolvens might be alternative biohydrogenators of 18:2n-6 and 18:3n-3 in the rumen.

The aim of this work was to determine the effect of 3-nitrooxypropanol (3-NOP) on the enteric methane (CH4) emissions and performance of lactating dairy cows when mixed in with roughage or incorporated into a concentrate pellet. After 2 pretreatment weeks without 3-NOP supplementation, 30 Holstein Friesian cows were divided into 3 homogeneous treatment groups: no additive, 3-NOP mixed in with the basal diet (roughage; NOPbas), and 3-NOP incorporated into a concentrate pellet (NOPconc). The pretreatment period was followed by a 10-wk treatment period in which the NOPbas and NOPconc cows were fed 1.6 g of 3-NOP/cow per day. After the treatment period, a 2-wk washout period followed without 3-NOP supplementation. The CH4 emissions were measured using a GreenFeed unit (C-Lock Inc., Rapid City, SD) installed in a freestall with cubicles during the entire experimental period. On average for the total treatment period and compared with the no-additive group, CH4 production (g/d) was 28 and 23% lower for NOPbas and NOPconc, respectively. Methane yield (g/kg of dry matter intake) and methane intensity (g/kg of milk) were 23 and 24% lower for NOPbas, respectively, and 21 and 22% lower for NOPconc, respectively. No differences were found between NOPbas and NOPconc. Moreover, supplying 3-NOP did not affect total dry matter intake, milk production, or milk composition. The results of this experiment show that 3-NOP can reduce enteric CH4 emissions of dairy cattle when incorporated into a concentrate pellet and that this reduction is not different from the effect of mixing in 3-NOP with the basal diet (roughage). This broadens the possibilities for using 3-NOP in the dairy sector worldwide, as it is not always feasible to provide an additive mixed in with the basal diet.

The study, conducted on 70 smallholder dairy farms in Northern Ethiopia, aimed to evaluate whether variation in milk yield (in early and mid-lactation) of multiparous Holstein-Friesian crossbred cows is related to diet composition and quality. At early stage (1-120 days in milk (DIM)), a total of 70 dairy farms were used, while at mid-lactation (121-240 DIM), 54 dairy farms continued to be part of the study. K-means clustering was applied to group the cows based on energy-corrected milk yield (ECMY) into three milk production farm clusters (MPFC): Low MPFC (5.7-9.3 L/day), medium MPFC (9.4-12.8 L/day), and high MPFC (12.9-17.6 L/day). The dry matter intake (DMI) of cows during early lactation for high MPFC and low MPFC was 14.1 and 11.2 kg/day, respectively. The dietary proportion of crop residues in diets offered to crossbred cows tended to be lower in the high MPFC during early as well as in mid-lactation. Cows from the high MPFC consumed diets with higher (rumen degradable) protein levels both in early and in mid-lactation, while dietary fiber fractions and in vitro dry matter digestibility (IVDMD) only differed in early lactation. Multiple regression models indicated that DMI (kg/day) in combination with either neutral detergent fiber, crude protein, or IVDMD (g/kg DM) explained about 25% of the variation in daily ECMY expressed relative to body weight (mL/kg). Hence, higher milk production is linked to both increased DMI and better quality of diets.

The accuracy of the milk fatty acid profile as a diagnostic tool for the diagnosis of sub-acute ruminal acidosis (SARA) has been determined when SARA was experimentally induced. This had not yet been done not on commercial dairy farms, where SARA can occur naturally. The objective of this study was to determine this accuracy in individual cows on commercial dairy farms. A total of 336 cows from 24 commercial dairy farms in Quebec were included. Farms were blocked based on geographical location and management, with each block having one high risk SARA farm and one low risk SARA farm. Farm Risk of SARA was determined based on the milk fat content and the proportions of de novo fatty acids and long chain unsaturated fatty acids in the bulk tank. On each farm, 7 early/mid-lactation (< 150 days in milk DIM) and 7 mid/late lactation (< 150 DIM) cows were randomly selected. The fatty acid profile of pooled milk samples from these cows were determined by gas chromatography. Farm risk of SARA did not affect the milk fat proportion of fatty acids, with the exception of trans 10 cis 12 C18:2, which was higher in At Risk Farms. Later lactation cows had a higher milk fat content and higher milk fat proportions of de novo, C16 fatty, and odd and branch chain fatty acids. The prevalence of SARA was likely higher in earlier lactation cows than in later lactation cows, but non-SARA related animal and dietary factors also affect the milk fatty acid profile. Hence, the milk fatty acid profile alone may not be accurate enough to diagnose SARA on farm. This profile can, however, contribute to this diagnosis, the identification of causes of milk fat depression, and the development of strategies to optimize the milk fatty acid profile.

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 +/- 0.29 cm; least squares means +/- standard error) and serum NEFA (0.16 +/- 0.04 mmol/L) in comparison to overconditioned cows (3.14 +/- 0.43 cm and 0.38 +/- 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 +/- 0.40% and 6.06 +/- 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

Rumen lipid metabolism largely determines the fatty acid composition of dairy products. As such, milk fatty acids can be used as indicators of ruminal and hence animal health. Control of rumen metabolism might also facilitate the production of dairy products enriched in unsaturated fatty acids for an enhanced human health. These include encapsulation technologies, either in a formaldehyde-protein, gelled protein or solid fat matrix. To better understand the origin of fatty acids in dairy products, the first sections address ruminal metabolism, intestinal digestion, transfer to the mammary gland and fatty acid metabolism in the mammary gland. The chapter then addresses the potential to improve the fatty acid composition of dairy products for enhanced human health, with a particular focus on technologies to protect unsaturated fatty acids from rumen biohydrogenation. Finally, the chapter discusses the use of use variation in the milk fatty acid profile, induced by changes in the ruminal lipid metabolism, for monitoring animal health.

The nutritionally imbalanced poor-quality diet feeding is the major constraint of dairy production in tropical regions. Hence, alternative high-quality roughage-based diets are required to improve milk yield and reduce methane emission (CH4). Thus, we tested the effects of feeding natural pasture hay, improved forage grass hays (Napier and Brachiaria Hybrid), and treated crop residues (Eragrostis tef straw) on nutrient digestibility, milk yield, nitrogen balance, and methane emission. The eight lactating Fogera cows selected for the experiment were assigned randomly to a 4 &times, 4 Latin square design. Cows were housed in well-ventilated individual pens and fed a total mixed ration (TMR) comprising 70% roughage and 30% concentrate. The four roughage-based basal dietary treatments supplemented with formulated concentrate were: Control (natural pasture hay (NPH)), treated teff straw silage (TTS), Napier grass hay (NGH), and Brachiaria hybrid grass hay (BhH). Compared with the control diet, the daily milk yield increased (p &lt, 0.01) by 31.9%, 52.9%, and 71.6% with TTS, NGH, and BhH diets, respectively. Cows fed BhH had the highest dry matter intake (8.84 kg/d), followed by NGH (8.10 kg/d) and TTS (7.71 kg/d), all of these intakes were greater (p = 0.01) than that of NPH (6.21 kg/d). Nitrogen digestibility increased (p &lt, 0.01) from the NPH diet to TTS (by 27.7%), NGH (21.7%), and BhH (39.5%). The concentration of ruminal ammonia nitrogen was higher for cows fed NGH than other diets (p = 0.01) and positively correlated with plasma urea nitrogen concentration (R&sup2, = 0.45). Feeding TTS, NGH, and BhH hay as a basal diet changed the nitrogen excretion pathway from urine to feces, which can help protect against environmental pollution. Estimated methane yields per dry matter intake and milk yield were decreased in dairy cows fed BhH, NGH, and TTS diets when compared to cows fed an NPH diet (p &lt, 0.05). In conclusion, feeding of TTS, NGH, and BhH roughages as a basal diet to lactating dairy cows in tropical regions improved nutrient intake and digestibility, milk yield, nitrogen utilization efficiency, and reduced enteric methane emission.

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to &alpha, ZEL and &beta, ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen.

Background: Oil palm fronds (OPF) when pretreated with white rot fungi (WRF) shows increased rumen degradability but with significant biomass loss. Thus, effects of pre-treated OPF with enzyme extracts from WRF on rumen degradability were studied in vitro. The enzyme extracts were prepared by inoculating OPF with three WRF, i.e Ceriporiopsis subvermispora, Lentinula edodes and Ganoderma lucidum, for 15, 30 and 45 days with either ammonium sulphate, (NH4)2SO4 and sodium nitrate, NaNO3 added to the culture media for each inoculation period. After preparation of enzyme extracts, the enzyme activities were determined. OPF was then pre-treated with enzyme extracts in a citrate buffer (pH 5.0) in a forced air oven at 40 oC during 5 days. Further, the in vitro rumen degradation of OPF pre-treated with enzyme extracts, with respect to the short chain fatty acid (SCFA) production, was determined after 24 h incubation. Activity of lignolytic (laccase and MnP), cellulolytic (CMCase and avicelase) and hemicellulolytic (xylanase) enzymes were measured in all of the extracts irrespective on the inoculation period. Results: Treatment of OPF with enzyme extracts from G. lucidum after 45 days of inoculation showed a numerical increase (13%) in total SCFA and apparently rumen degradable carbohydrates (ARDC) after 24 h in vitro incubation, without any loss of biomass. However, this increase was not clearly correlated to results of the enzyme assays. Conclusion: This study indicates pre-treatment of OPF with enzyme extracts from specific WRF to be promising to enhance the ruminal degradability of OPF without simultaneous loss of biomass.

To reduce competition with human-edible feed resources, it is of interest to incorporate by-products from the food industry in animal feeds. The current research investigated the effect of including increasing amounts of tofu by-product (TF) in practical pig diets on animal performance, nitrogen balance and ammonia emissions from manure. Two experiments were conducted including a control diet without TF, containing 160 g/kg dietary non-starch polysaccharides (NSPs) and three diets including 122, 246 and 360 g TF/kg DM (TF122, TF246 and TF360, respectively) to reach 220, 280 and 360 g/kg NSP. All diets had the same level of CP and protein digestible in the small intestine which particularly was realized by replacing rice bran with TF. Animal performance was assessed in a first experiment with 40 growing barrows with initial BW of 26.6 ± 1.80 kg (M ± SD) being allocated to the 4 treatments, during 2 growth phases (i.e. until 50 kg BW and from 50 to 80 kg BW). In the growth phase until 50 kg, feed intake and average daily gain (ADG) were linearly reduced by dietary TF inclusion, while this negative impact disappeared during the second growth phase (50 to 80 kg BW). Tofu by-product inclusion even positively affected the feed conversion ratio during this second growth phase (3.4 to 2.7 kg feed/kg ADG for 0 to 360 g/kg dietary TF). Over the entire growth period, performance and feed intake were negatively affected at the highest dietary TF level. Experiment 2 was conducted to assess digestibility, nitrogen balance and ammonia emission from manure. For this purpose, 16 pigs with BW of 62.8 ± 3.6 kg (M ± SD) were assigned to either 1 of the 4 treatments. There was no difference in total tract apparent digestibility of dietary organic matter or CP, while NDF digestibility increased with increasing TF level, suggesting increasing importance of the hindgut fermentation when digesting diets with increasing TF levels. Nevertheless, this was not reflected in increasing levels of faecal volatile fatty acids or purines, nor in reduced manure pH. As a result, ammonia emission from slurry was not reduced through dietary TF inclusion, despite the linear decrease in urinary nitrogen. In conclusion, TF can be included in pigs’ diets up to an inclusion rate of 25% without risk of impaired animal performance; however, this dietary strategy fails to mitigate ammonia emission from slurry.

Background Bacteria involved in ruminal formation of trans-10 intermediates are unclear. Therefore, this study aimed at identifying rumen bacteria that produce trans-10 intermediates from 18-carbon unsaturated fatty acids. Results Pure cultures of 28 rumen bacterial species were incubated individually in the presence of 40 μg/mL 18:3n-3, 18:2n-6 or trans-11 18:1 under control or lactate-enriched (200 mM Na lactate) conditions for 24 h. Of the 28 strains, Cutibacterium acnes (formerly Propionibacterium acnes) was the only bacterium found to produce trans-10 intermediates from 18:3n-3 and 18:2n-6, irrespective of the growth condition. To further assess the potential importance of this species in the trans-11 to trans-10 shift, different biomass ratios of Butyrivibrio fibrisolvens (as a trans-11 producer) and C. acnes were incubated in different growth media (control, low pH and 22:6n-3 enriched media) containing 40 μg/mL 18:2n-6. Under control conditions, a trans-10 shift, defined in the current study as trans-10/trans-11 ≥ 0.9, occurred when the biomass of C. acnes represented between 90 and 98% of the inoculum. A low pH or addition of 22:6n-3 inhibited cis-9, trans-11 CLA and trans-10, cis-12 CLA formation by B. fibrisolvens and C. acnes, respectively, whereby C. acnes seemed to be more tolerant. This resulted in a decreased biomass of C. acnes required at inoculation to induce a trans-10 shift to 50% (low pH) and 90% (22:6n-3 addition). Conclusions Among the bacterial species studied, C. acnes was the only bacterium that have the metabolic ability to produce trans-10 intermediates from 18:3n-3 and 18:2n-6. Nevertheless, this experiment revealed that it is unlikely that C. acnes is the only or predominant species involved in the trans-11 to trans-10 shift in vivo.

The nutritional intake of Tibetan sheep on the harsh Qinghai–Tibetan Plateau is often under maintenance requirements, especially during the long, cold winter. However, they have adapted well and even thrive under these conditions. The aim of the present study was to gain insight into how the rumen epithelium of Tibetan sheep has adapted to the consumption of low-energy-level diets. For this purpose, we compared Tibetan and small-tailed Han sheep (n 24 of each breed, all wethers and 1·5 years of age), which were divided randomly into one of four groups and offered ad libitum diets of different digestible energy (DE) densities: 8·21, 9·33, 10·45 and 11·57 MJ DE/kg DM. The Tibetan sheep had higher rumen concentrations of total SCFA, acetate, butyrate and iso-acids but lower concentrations of propionate than small-tailed Han sheep. The Tibetan sheep had higher absorption capability of SCFA due to the greater absorption surface area and higher mRNA expression of the SCFA absorption relative genes than small-tailed Han sheep. For the metabolism of SCFA in the rumen epithelium, the small-tailed Han sheep showed higher utilisation of the ketogenesis pathway than Tibetan sheep; however, Tibetan sheep had greater regulation capacity in SCFA metabolism pathways. These differences between breeds allowed the Tibetan sheep to have greater capability of absorbing SCFA and better capacity to regulate the metabolism of SCFA, which would allow them to cope with low energy intake better than small-tailed Han sheep.

In the current study, linseed oil emulsions were cross-linked by polyphenol oxidase to study the effect of emulsion characteristics on the protection of polyunsaturated fatty acids against biohydrogenation by rumen bacteria in vitro. Polyphenol oxidase (PPO) catalyzes the oxidation of diphenolic substrate to o-quinones which are reactive towards amino- and thiol- groups on proteins, forming a complex of covalently bound protein cross-linked by protein-bound phenols (PBP). Such cross-linking of protein-stabilized emulsions was shown to yield high in vitro ruminal protection of polyunsaturated fatty acids before, by addition of PPO extract and diphenolic substrate to protein-stabilized emulsions. The current study examined the relationship between characteristics of cross-linked emulsions and in vitro ruminal protection by evaluation of two hypotheses: protection efficiency would be impaired when 1) the amount of PPO in the emulsion decreased or 2) the amount of non-adsorbed protein in the emulsion increased. Hence, in a first experiment, emulsions containing the same protein concentration and varying amounts of PPO extract (0,2-0,8 g/g) were analyzed, while in experiment 2, emulsions containing varying amounts of non-adsorbed protein and a similar amount of PPO extract (0,8 g/g) were investigated. In the latter experiment, variation in non-adsorbed protein was obtained by altering the emulsifier concentration (10 or 0 mg/g whey protein isolate) and/or removal of the majority (>90%) of the non-adsorbed protein after emulsion preparation, i.e. washing of the emulsion, before addition of PPO extract. In vitro ruminal biohydrogenation was evaluated after 24 h in a rumen-simulated environment containing rumen inoculum. Results of experiment 1 showed protection efficiency decreased for decreasing amounts of PPO extract. Additionally, in vitro ruminal protection was found to be positively correlated to mean droplet size, protein load and proportion of PBP in the free protein fraction of the emulsions after cross-linking, while proportion of PBP in the adsorbed protein layer did not differ between treatments. In experiment 2, an increase in emulsifier concentration from 10 to 50 g/g WPI lowered the amount of PBP in both protein fractions. Washing of 50 mg/g WPI emulsions, enhanced in vitro ruminal protection of 18:3n-3 and 18:2n-6 and increased the proportion of PBP in both free and adsorbed protein. Moreover, protein load and the amount of total adsorbed PBP were positively affected by washing. Comparison of emulsions with similar protein load between experiments showed in vitro ruminal protection efficiency increased for higher proportions of PBP in the adsorbed protein layer. Moreover, an excess of PBP on the possible binding positions on the adsorbed protein seemed to promote in vitro ruminal protection efficiency.

The current study was carried out to assess 2 hypotheses: (1) cows differ in susceptibility to a subacute ruminal acidosis (SARA) challenge, and (2) the milk fatty acid (FA) pattern can be used to differentiate susceptible from nonsusceptible cows. For this, 2 consecutive experiments were performed. During experiment 1, the milk FA pattern was determined on 125 cows fed an increasing amount of concentrate during the first 4 wk in milk (WIM). The coefficient of variation of several SARA indicative milk FA (i.e., C15:0, C18:1 trans-10, C18:2 cis-9,trans-11, and C18:1 trans-10 to C18:1 trans-11 ratio) increased, indicating that cows reacted differently upon the concentrate build-up. A first grouping was based on the milk fat C18:1 trans-10 proportion in the third WIM. Fifteen cows with the highest proportion of the latter FA (HT10) and their counterparts with low C18:1 trans-10 and equal parity distribution (LT10) were compared, which revealed that milk fat content and milk fat to protein ratio were lower for the HT10 group. From each of the HT10 and LT10 groups, 5 animals were selected for experiment 2. The subselection of the HT10 group, referred to as HT10s, showed a high proportion of C18:1 trans-10 at 3 WIM (>0.31 g/100 g of FA), a high level of C15:0 (on average ≥1.18 g/100 g of FA over the 4 WIM), and a sharp decrease of C18:1 trans-11 (Δ ≥ 0.25 g/100 g of FA during the 4 WIM). Their counterparts (LT10s) had a low milk fat C18:1 trans-10 proportion at 3 WIM (

Monitoring systems (MS) provide additional information that many developers and researchers expect will reduce the uncertainty surrounding decision-making in livestock production and therefore enhance management decisions. However, the actual economic value of the information (VoI) yielded by MS has hardly been investigated. The aim of this study was to fill that void based on two objectives. The first is to estimate the VoI of MS prior to implementation using decision analysis based on scarce data from different sources. The second objective is to identify which factors most influence the VoI of MS and to develop recommendations about the focus of future MS development. To illustrate our objectives, we used a case study of two milk biomarkers used to monitor subacute ruminal acidosis (SARA) in dairy cows: fat-to-protein ratio (FPR) and the fatty acid profile (FAP). FPR is presently used to monitor SARA, while FAP is a newly developed test, currently in the pre-commercial phase, with reports of better accuracy than FPR. A stochastic decision tree model was used to estimate the expected monetary value of three levels of information with regards to SARA: (i) no monitoring, monitoring (ii) with FPR or (iii) with FAP. The VoI of FPR and FAP were calculated as the difference in expected monetary value of monitoring with FPR and FAP as compared with no monitoring, respectively. Several scenarios were modeled using sensitivity and elasticity analyses. The aim was not only to compensate for the scarcity of data for some variables, but also to identify under which conditions decisions based on FAP monitoring were indeed the best. In all the scenarios, monitoring SARA with FPR had the lowest expected monetary value. No monitoring was a better decision in 70% of the iterations in the scenario that described the most probable situation. The VoI of FAP was positive when SARA prevalence was between 0.21 and 0.79 with its maximum value at 0.61, when the treatment costs were lower than €116/case/year and when the disease costs were higher than €260/case/year. Moreover, an increase of specificity of the FAP to 0.95 yielded a positive VoI, whereas an increase of its sensitivity to 1.0 still yielded a negative VoI, suggesting that developers of the FAP should focus on improving its specificity rather than its sensitivity. To avoid suboptimal use of finite resources while developing MS, we recommend ex-ante investigation of the VoI of the MS under development.

The aim of this study was to investigate the methane (CH(4)) reducing potential of a combination of prenatal and/or postnatal treatment with coconut oil medium chain fatty acids (CO MCFA) in goat kids. The hypothesis is that influencing rumen function during early life has more chances for success than in the adult life, related to the resilience of the mature rumen microbiota. Forty-eight pregnant does were split into two experimental groups: treated does (D+) received 40 g/d of CO MCFA in a test compound feed, while control does (D−) received a control compound feed, during the last 3 wk of gestation. Twin kids from 10 does of each group were split up into a treated (K+) and nontreated (K−) group, resulting in four experimental groups: D+K+, D+K−, D−K+, and D−K−. The K+ kids received 1.8 mL/d of CO MCFA from birth until 2-wk postweaning (11 wk). Irrespective of treatment, the experimental rearing conditions resulted in absence of rumen protozoa at all sampling times, assessed by quantitative PCR (qPCR). In vitro incubations with rumen fluid at 4 wk old showed 82% lower CH(4) production of inoculum from D+K+ kids compared to D−K− kids (P = 0.01). However, this was accompanied by lower total volatile fatty acids (tVFA) production (P = 0.006) and higher hydrogen accumulation (P = 0.008). QPCR targeting the mcrA and rrs genes confirmed a lower abundance of total methanogens (P < 0.02) and total eubacteria (P = 0.02) in D+K+ kids at 4 wk old. Methanogenic activity, as assessed by mcrA expression by RT-qPCR, was also lower in these kids. However, activity did not always reflect methanogen abundance. At 11 and 28 wk old, prenatal and postnatal effects on in vitro fermentation and rumen microbiota disappeared. Nevertheless, lower milk replacer intake in the first 4 wk resulted in reduced BW in K+ kids, persisting until 28 wk of age. Additionally, differences assigned to postnatal treatment were found in papillae density, width, and length in different areas of the rumen, recorded at 28 wk old. Conclusion: prenatal and postnatal supplementation with CO MCFA reduced in vitro CH(4) emissions until 4 wk old by depressing methanogen abundance and activity but at the expense of rumen fermentation and eubacterial abundance. Unfortunately, daily gain of K+ kids was suppressed. Some rumen papillae characteristics differed at 28 wk old due to postnatal treatment which ended at 11 wk old, indicating rumen papillary development can be affected by the early-life nutritional circumstances.

The production of trans-10, cis-12 C18:2 isomer in the rumen is associated with milk fat depression. Two experiments were conducted to determine the effects of some additives on rumen fermentation and apparent biohydrogenation of linoleic (LA) and linolenic (LNA) acids, and the production of the trans-10, cis-12 C18:2 isomer associated with milk fat depression. In experiment 1, a 1:1 forage:concentrate diet containing linseed oil (84 g/kg of DM) was incubated in a 2 h or 6 h batch culture with rumen fluid in 2 replicated periods. Treatments in the 2 h incubations were: control; lipase 1 and 2 (4 and 40 μl/l) and a lipase inhibitor (4 and 20 mg/l); and in the 6 h incubations were: Oxy-propyl-thiosulfate (PTSO; 60 and 120 mg/l); Eugenol (EUG; 150 and 500 mg/l) and Cinnamaldehyde (CIN; 150 and 500 mg/l). After incubation, samples were collected to analyze ammonia-N, volatile fatty acids (VFA) and the fatty acid (FA) profile. In experiment 2, 8 continuous culture fermenters (1320 ml) were used in 3 replicated periods (5 d of adaptation and 3 d of sampling). Fermenters were fed 95 g/d of DM of a 60:40 forage:concentrate diet containing 50 g/kg DM of linseed oil. Treatments were control, lipase 1 (4 μl/l), PTSO (90 mg/l) and CIN (250 mg/l), and were tested at 2 pH levels (6.4 and 5.6). During the last 3 d of each period, samples were taken to analyze VFA, ammonia-N and the FA profile. In experiment 1, Lipase 1 increased the apparent biohydrogenation of LNA, but these results were not observed in experiment 2. Both CIN and PTSO seem to inhibit the apparent biohydrogenation of LNA, but only the PTSO inhibited the biohydrogenation of LA in a 6 h incubation compared with control. Cinnamaldehyde in Experiment 1, and PTSO in Experiment 1 and 2 decreased total VFA concentrations. Although the short-term batch incubation suggested some potential benefits of use of some additives for modifying rumen biohydrogenation pathways, effects observed in the long-term fermentation were not relevant for the purpose of decreasing the production of trans-10, cis-12 C18:2 isomer involved in milk fat depression.

The aim of this study was to evaluate the dose response on in vitro methane (CH) production of PUFA to which the inoculum donor animals had been exposed early in life. Sixteen Holstein calves (160 ± 3 and 365 ± 2 kg BW) at 6 and 12 mo of age were used as inoculum donors. Half of the calves were given increasing amounts of extruded linseed from birth (22 g/d) until 4 mo of age (578 g/d) first mixed with milk and then included in their concentrate. Linseed oil (LSO) was supplemented in vitro at 5 different doses (0, 0.6, 1.2, 2.4, and 4.8 mg/mL). Supplementation of LSO in the rumen inocula at both ages linearly decreased ( < 0.05) the in vitro CH production. Total in vitro VFA production was not affected by LSO supplementation. Inhibition of CH was smaller when using the rumen inoculum from calves that had received a similar treatment early in life ( < 0.05). Differences in response to in vitro supplementation of a type of fatty acids similar to those applied during early life suggest some "changes" in the functioning of the rumen microbial community.

Effects of grain type and dietary oil supplement on milk fat depression and milk fatty acid (FA) composition of dairy cows were evaluated using eight multiparous Holstein cows (77±22.1 days in milk; mean±SD) in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of diets. Experimental diets contained either ground barley or ground corn supplemented with either fish oil or soybean oil at 2% of dietary dry matter (DM). Experimental periods were 25 d, with the final 7 d used for sample and data collection. Dry matter intake tended ( P =0.09) to be greater for barley- vs. corn-based diets (23.2 vs. 22.3 kg/d), but was reduced for the fish oil compared to soybean oil supplemented diets (21.1 vs. 24.3 kg/d; P P cis- 9 C16:1) but decreased the concentration of preformed FA ( P trans -11 C18:1 ( P =0.03) and cis -9, trans -11 C18:2 ( P =0.01) which was a reflection of greater intake of cis -9, cis -12 C18:2 as substrate for rumen biohydrogenation. Severity of MFD was greater for fish oil than for soybean oil which was evidenced by the increased concentration and yield of biohydrogenation intermediates (especially trans- 10 C18:1) associated with MFD ( r =–0.61; n =32) in milk fat. However, fish oil increased concentration of both trans -11 C18:1 ( P =0.001) and cis- 9, trans- 11 C18:2 ( P P P de novo synthesized- and preformed-FA as compared with soybean oil supplemented diets.

Polyunsaturated fatty acids (PUFAs) may affect colon microbiome homeostasis by exerting (specific) antimicrobial effects and/or interfering with mucosal biofilm formation at the gut mucosal interface. We used standardized batch incubations and the Mucosal-Simulator of the Human Microbial Intestinal Ecosystem (M-SHIME) to show the in vitro luminal and mucosal effects of the main PUFA in the Western diet, linoleic acid (LA). High concentrations of LA were found to decrease butyrate production and Faecalibacterium prausnitzii numbers dependent on LA biohydrogenation to vaccenic acid (VA) and stearic acid (SA). In faecal batch incubations, LA biohydrogenation and butyrate production were positively correlated and SA did not inhibit butyrate production. In the M-SHIME, addition of a mucosal environment stimulated biohydrogenation to SA and protected F. prausnitzii from inhibition by LA. This was probably due to the preference of two biohydrogenating genera Roseburia and Pseudobutyrivibrio for the mucosal niche. Co-culture batch incubations using Roseburia hominis and F. prausnitzii validated these observations. Correlations networks further uncovered the central role of Roseburia and Pseudobutyrivibrio in protecting luminal and mucosal SHIME microbiota from LA-induced stress. Our results confirm how cross-shielding interactions provide resilience to the microbiome and demonstrate the importance of biohydrogenating, mucosal bacteria for recovery from LA stress.

27 páginas, 6 figuras, 2 tablas., To meet the energy requirements of high-yielding dairy cows, grains and fats have increasingly been incorporated in ruminant diets. Moreover, lipid supplements have been included in ruminant diets under experimental or practical conditions to increase the concentrations of bioactive n-3 fatty acids and conjugated linoleic acids in milk and meat. Nevertheless, those feeding practices have dramatically increased the incidence of milk fat depression in dairy cattle. Although induction of milk fat depression may be a management tool, most often, diet-induced milk fat depression is unintended and associated with a direct economic loss. In this review, we give an update on the role of fatty acids, particularly originating from rumen biohydrogenation, as well as of rumen microbes in diet-induced milk fat depression. Although this syndrome seems to be multi-etiological, the best-known causal factor remains the shift in rumen biohydrogenation pathway from the formation of mainly trans-11 intermediates toward greater accumulation of trans-10 intermediates, referred to as the trans-11 to trans-10 shift. The microbial etiology of this trans-11 to trans-10 shift is not well understood yet and it seems that unraveling the microbial mechanisms of diet-induced milk fat depression is challenging. Potential strategies to avoid diet-induced milk fat depression are supplementation with rumen stabilizers, selection toward more tolerant animals, tailored management of cows at risk, selection toward more efficient fiberdigesting cows, or feeding less concentrates and grains, L. Dewanckele received a PhD grant from the Special Research Fund of Ghent University (BOF-Belgium, grant number BOF15/DOC/246). P. G. Toral benefits from a Ramón y Cajal research contract from the MINECO (RYC-2015-17230), co-funded by the European Social Fund. The authors have not stated any conflicts of interest.

With the existence of clearly demarcated fields that are cropped every year, the agricultural system of the Tembien highlands can be characterised as a permanent upland farming system, practised by a sedentary population. Livestock are predominantly cattle (especially oxen) and sheep, goats, donkeys and mules.

The low transfer in ruminants of dietary polyunsaturated fatty acids to the milk or peripheral tissues is largely due to ruminal biohydrogenation. Lipids emulsified by a polyphenol oxidase (PPO) rich protein extract of red clover were shown before to be protected against this breakdown after cross-linking with 4-methylcatechol. Protein extracts of 13 other vegetal resources were tested. Surprisingly, the effectiveness to protect emulsified lipids against in vitro ruminal biohydrogenation largely depended on the origin of the extract and its protein concentration but was not related to PPO activity. Moreover, PPO isoforms in vegetal sources, effectively protecting emulsified lipids, were diverse and their presence at the emulsion interface did not seem essential. Potato tuber peels were identified as an interesting biological source of emulsifying proteins and PPO, particularly since protein extracts of industrial potato sidestreams proved to be suitable for the current application.

The aim of the present research was to describe characteristics of adipose tissue lipolysis in dairy cows with a variable body condition score (BCS). Ten clinically healthy Holstein Friesian cows were selected based on BCS and euthanized 10 to 13 d before the expected parturition date. Immediately after euthanasia, adipose tissue samples were collected from subcutaneous and omental fat depots. In both depots, we observed an increase in adipocyte size with increasing BCS. Using an in vitro explant culture of subcutaneous and omental adipose tissue, we aimed to determine the influence of adipocyte size and localization of adipose depot on the lipolytic activity in basal conditions and after addition of isoproterenol (nonselective β-agonist) and insulin in different concentrations. Glycerol release in the medium was used as a measure for lipolytic activity. We observed that the basal lipolytic activity of subcutaneous and omental adipose tissue increased with adipocyte volume, meaning that larger fat cells have higher basal lipolytic activity independent of the location of the adipose depot. Dose-response curves were created between the concentration of isoproterenol or insulin and the amount of glycerol released. The shape of the dose-response curves is determined by the concentration of isoproterenol and insulin needed to elicit the half-maximal effect and the maximal amount of stimulated glycerol release or the maximal inhibitory effect of insulin. We observed that larger fat cells released more glycerol upon maximal stimulation with isoproterenol and this was more pronounced in subcutaneous adipose tissue. Additionally, larger fat cells had a higher sensitivity toward lipolytic signals. We observed a trend for larger adipocytes to be more resistant to the maximal antilipolytic effect of insulin. The insulin concentration needed to elicit the half-maximal inhibitory effect of insulin was within the physiological range of insulin and was not influenced by adipocyte size or adipose depot. We conclude that overconditioned cows have larger adipocytes and are predisposed to excessive mobilization of body fat due to a higher basal and stimulated lipolytic activity of large adipocytes while the antilipolytic effect of insulin is preserved.

SUMMARYRelationships between milk fatty acids (MFA) and methane (CH4) emissions from dairy cattle were explored. Data from a total of 12 studies including 39 treatments were gathered in the database. Methane was expressed as daily emissions (g/d), relative to dry matter intake (g/kg), milk production (g/kg milk) and body weight (g/kg). The univariate correlations between MFA and CH4 were based on absolute means and on relative changes of each treatment compared with its corresponding control. Saturated fatty acids, odd- and branched-chain FA and long-chain poly-unsaturated FA were positively related to CH4, while cis- and trans-isomers of mono-unsaturated FA were negatively related to CH4. However, most of the coefficients of determination (R2) of these univariate regressions ranged from 0·2 to 0·7, indicating that individual MFA only explain a limited part of the variation in CH4. Significant relationships between MFA and CH4 varied depending on the unit in which emissions were expressed. Similarly, some MFA seemed more suited to predict relative changes in CH4 emissions rather than absolute amounts. The present findings contribute to the exploration of the potential of MFA as biomarkers for CH4 emissions from dairy cattle, although differences between studies in the detail of MFA analysis and hence the number of MFA reported in the current study, complicates this kind of literature survey.