Effects of a dietary supplement enriched in palmitoleic acid on fatty acid composition of follicular fluid, granulosa cell metabolism, and oocyte developmental capacity in early lactation dairy cows.

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.

Invited review: Role of rumen biohydrogenation intermediates and rumen microbes in diet-induced milk fat depression: An update.

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 fiber-digesting cows, or feeding less concentrates and grains.

Intra- and inter-individual variability of fatty acid composition of the follicular fluid in a cohort of 23 women undergoing assisted reproductive treatment.

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 (CVw) 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 (CVw < 10%). Although CVw values of 18:3n-3 and 20:3n-6 exceeded 10%, variation between patients exceeded intra-individual variation as indicated by elevated ICC values (0.61 and 0.66, respectively). Nevertheless, 20:4n-6 and 22:6n-3 showed non-negligible intra-patient variation. With the exception of some minor fatty acids (< 0.30 g/100 g), strong relationships were demonstrated between the average proportion in individually analysed follicles and the proportion determined in pooled samples and in the first, largest follicle. CONCLUSION: The CVw and ICC values of proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 18:3n-3, 20:5n-3, total MUFA and n-3 PUFA showed limited intra-individual variation and moderate to good reliability. However, this is not the case for some other PUFA, such as 20:4n-6 and 22:6n-3. Nevertheless, for all of these fatty acid(s) (groups), calculated average fatty acid proportions were highly correlated with proportions determined in pooled samples and in the first, largest follicle. This implies that single or pooled follicle aspiration suffices to assess intra-individual variation in the FF of these fatty acids.

Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.

Nine Holstein dairy cows were fed diets with increasing proportions of rapidly fermentable carbohydrates (RFCH) to investigate the effect on reticular pH, milk fat content (MFC), 18-carbon fatty acid proportions in blood plasma and milk, and bacterial community in buccal swab samples. Inter-animal variation was expected in terms of reticular pH response upon higher RFCH proportions, which would be reflected in the occurrence or not of milk fat depression (MFD). Moreover, this variation in occurrence of MFD was hypothesized to be related to differences in blood and milk fatty acid proportions and in the bacterial community in buccal samples. Cows were fed a total mixed ration throughout the experiment, which consisted of 4 periods: adaptation (d 0-4) and low (d 5-18), increasing (d 19-24), and high RFCH (d 25-28). During the increasing RFCH period, the standard concentrate (211 g of starch/kg of dry matter) was gradually and partly replaced by a concentrate high in RFCH (486 g of starch/kg of dry matter). The reticular pH was measured using a bolus and the time below pH 6.00 was calculated on a daily basis. On d 13, 14, 25, 27, and 28, plasma and milk samples were collected and analyzed for 18-carbon fatty acid proportions, and buccal swabs were collected for bacterial community analysis based on 16S rRNA gene amplicon sequencing. Inter-animal variation was observed in terms of reticular pH, which allowed us to divide the cows into 2 groups: tolerant (time below pH 6.00 ≤ 0.1 h/d) and susceptible cows (time below pH 6.00 ≥ 1.26 h/d). The lower reticular pH of susceptible cows was accompanied by lower MFC. Both groups already differed in reticular pH and MFC during the low-RFCH period. Furthermore, higher RFCH amounts did not decrease the reticular pH in either of the 2 groups. Nevertheless, MFD was observed in both groups during the high-RFCH period compared with the low-RFCH period. Lower MFC in animals with lower reticular pH or during the high-RFCH period was associated with a shift in 18-carbon fatty acids toward trans-10 at the expense of trans-11 intermediates, which was observed in plasma as well as in milk samples. Moreover, lower MFC was accompanied by shifts in the relative abundance of specific bacteria in buccal samples. Genera Dialister, Sharpea, Carnobacterium, Acidaminococcus, and uncultured genera belonging to the Betaproteobacteria were more abundant in situations with greater trans-10 proportions.

Susceptibility of dairy cows to subacute ruminal acidosis is reflected in milk fatty acid proportions, with C18:1 trans-10 as primary and C15:0 and C18:1 trans-11 as secondary indicators.

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 (<0.23 g/100 g of FA), an average C15:0 proportion of 0.99 g/100 g of FA or lower, and a rather stable C18:1 trans-11 proportion. The HT10s group was hypothesized to be more susceptible to a SARA challenge, achieved by increasing amounts of rapidly fermentable carbohydrates in experiment 2. The HT10s cows had a lower nadir, mean, and maximum reticulo-ruminal pH; longer period of reticulo-ruminal pH below 6.0; and higher daily reticulo-ruminal pH variation compared with LT10s cows. Throughout experiment 2, HT10s and LT10s cows differed in levels of SARA indicative milk FA. Five animals, including one LT10s and 4 HT10s cows, experienced SARA, defined as reticulo-ruminal pH <6.0 for more than 360 min/d. These results indicate that it is possible to distinguish cows with different susceptibility to a SARA challenge within a herd by monitoring the milk FA composition when cows receive the same diet.

Maternal metabolic stress may affect oviduct gatekeeper function.

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC-albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

Postruminal synthesis modifies the odd- and branched-chain fatty acid profile from the duodenum to milk.

Milk odd- and branched-chain fatty acids (OBCFA) have been suggested as potential biomarkers for rumen function. The potential of milk OBCFA as a biomarker depends on whether their profile reflects the profile observed in the duodenum. The objective of this study was to evaluate whether the OBCFA profile in duodenum samples is reflected in plasma and milk. For this, 2 dairy cattle experiments were used. In experiment 1, 4 Holstein cows fitted with rumen and proximal duodenum cannulas were used in a 4×4 Latin square design. The treatments consisted of 2 nitrogen levels (143 vs. 110g of crude protein/kg of dry matter for high and low N, respectively) combined with either 1 of the 2 energy sources (i.e., starch from barley, corn, and wheat or fiber from soybean hulls and dehydrated beet pulp). In experiment 2, 4 Holstein cows fitted with rumen and proximal duodenum cannulas were used in a 3×3 Latin square design, with the treatments consisting of 3 diets: (1) RNB-, a diet with a crude protein content of 122g/kg of dry matter, predicted to provide protein digested in the small intestine according to the requirement of the animals, but with a shortage of rumen degradable protein; (2) RNB- to which 6g/d of niacin was added through inclusion in the mineral and vitamin premix, and (3) RNB- to which urea was added to balance rumen degradable N supply resulting in a CP content of 156g/kg of dry matter. In both experiments, samples of duodenal digesta, plasma, and milk were collected and analyzed for fatty acids. Additionally, lipids in plasma samples were separated in lipid classes and analyzed for fatty acids. The OBCFA profile in milk was enriched in 15:0, iso-17:0, anteiso-17:0, and cis-9-17:1 as compared with duodenal samples, and milk secretions even exceeded duodenal flows, which suggests occurrence of postruminal synthesis, such as de novo synthesis, desaturation, and elongation. The postruminal modification of the OBCFA profile might hamper the application of OBCFA as diagnostic tools of rumen function.

Grain-based versus alfalfa-based subacute ruminal acidosis induction experiments: Similarities and differences between changes in milk fatty acids.

Subacute ruminal acidosis (SARA) is one of the most important metabolic disorders, traditionally characterized by low rumen pH, which might be induced by an increase in the dietary proportion of grains as well as by a reduction of structural fiber. Both approaches were used in earlier published experiments in which SARA was induced by replacing part of the ration by a grain mixture or alfalfa hay by alfalfa pellets. The main differences between both experiments were the presence of blood lipopolysaccharide and Escherichia coli and associated effects on the rumen microbial population in the rumen of grain-based induced SARA animals as well as a great amount of quickly fermentable carbohydrates in the grain-based SARA induction experiment. Both induction approaches changed rumen pH although the pH decrease was more substantial in the alfalfa-based SARA induction protocol. The goal of the current analysis was to assess whether both acidosis induction approaches provoked similar shifts in the milk fatty acid (FA) profile. Similar changes of the odd- and branched-chain FA and the C18 biohydrogenation intermediates were observed in the alfalfa-based SARA induction experiment and the grain-based SARA induction experiment, although they were more pronounced in the former. The proportion of trans-10 C18:1 in the last week of the alfalfa-based induction experiment was 6 times higher than the proportion measured during the control week. The main difference between both induction experiments under similar rumen pH changes was the decreasing sum of iso FA during the grain-based SARA induction experiment whereas the sum of iso FA remained stable during the alfalfa-based SARA induction experiment. The cellulolytic bacterial community seemed to be negatively affected by either the presence of E. coli and the associated lipopolysaccharide accumulation in the rumen or by the amount of starch and quickly fermentable carbohydrates in the diet. In general, changes in the milk FA profile were related to changes in rumen pH. Nevertheless, feed characteristics (low in structural fiber vs. high in starch) also affected the milk FA profile and, as such, both effects should be taken into account when subacute acidosis occurs.

Effects of feeding different linseed sources on omasal fatty acid flows and fatty acid profiles of plasma and milk fat in lactating dairy cows.

The aim of this experiment was to study the effects of feeding different linseed sources on omasal fatty acid (FA) flows, and plasma and milk FA profiles in dairy cows. Four ruminally cannulated lactating Holstein-Friesian cows were assigned to 4 dietary treatments in a 4×4 Latin square design. Dietary treatments consisted of supplementing crushed linseed (CL), extruded whole linseed (EL), formaldehyde-treated linseed oil (FL) and linseed oil in combination with marine algae rich in docosahexaenoic acid (DL). Each period in the Latin square design lasted 21 d, with the first 16 d for adaptation. Omasal flow was estimated by the omasal sampling technique using Cr-EDTA, Yb-acetate, and acid detergent lignin as digesta flow markers. The average DM intake was 20.6 ± 2.5 kg/d, C18:3n-3 intake was 341 ± 51 g/d, and milk yield was 32.0 ± 4.6 kg/d. Milk fat yield was lower for the DL treatment (0.96 kg/d) compared with the other linseed treatments (CL, 1.36 kg/d; EL, 1.49 kg/d; FL, 1.54 kg/d). Omasal flow of C18:3n-3 was higher and C18:3n-3 biohydrogenation was lower for the EL treatment (33.8 g/d; 90.9%) compared with the CL (21.8 g/d; 94.0%), FL (15.5 g/d; 95.4%), and DL (4.6 g/d; 98.5%) treatments, whereas whole-tract digestibility of crude fat was lower for the EL treatment (64.8%) compared with the CL (71.3%), FL (78.5%), and DL (80.4%) treatments. The proportion of C18:3n-3 (g/100 g of FA) was higher for the FL treatment compared with the other treatments in plasma triacylglycerols (FL, 3.60; CL, 1.22; EL, 1.35; DL, 1.12) and milk fat (FL, 3.19; CL, 0.87; EL, 0.83; DL, 0.46). Omasal flow and proportion of C18:0 in plasma and milk fat were lower, whereas omasal flow and proportions of biohydrogenation intermediates in plasma and milk fat were higher for the DL treatment compared with the other linseed treatments. The results demonstrate that feeding EL did not result in a higher C18:3n-3 proportion in plasma and milk fat despite the higher omasal C18:3n-3 flow. This was related to the decreased total-tract digestibility of crude fat. Feeding FL resulted in a higher C18:3n-3 proportion in plasma and milk fat, although the omasal C18:3n-3 flow was similar or lower than for the CL and EL treatment, respectively. Feeding DL inhibited biohydrogenation of trans-11,cis-15-C18:2 to C18:0, as indicated by the increased omasal flows and proportions of biohydrogenation intermediates in plasma and milk fat.

The fatty acid profile of subcutaneous and abdominal fat in dairy cows with left displacement of the abomasum.

The objective of this study was to determine the fatty acid (FA) profile and assess desaturase indices of nonesterified fatty acids (NEFA) in the blood, as well as in the abdominal (ABD) and subcutaneous (SUBC) fat stores, in dairy cows with left displacement of the abomasum (LDA). Blood, ABD, and SUBC samples were taken from 50 Holstein cows offered for surgery to correct LDA. The FA profile of the 3 compartments was determined by gas chromatography after lipid extraction, methylation, and, in the case of blood plasma, separation of lipid classes. The most abundant FA in all 3 compartments were 16:0, 18:0, and 18:1 cis-9, with a total proportion of 82.5, 68.0, and 74.1g/100 g of FA in ABD, NEFA, and SUBC, respectively. A principal component analysis was performed on the entire FA profile as well as on the Δ(9)-desaturase indices (14:1 cis-9/14:0, 16:1 cis-9/16:0, 18:1 cis-9/18:0). The principal component analysis extracted 2 principal components (PC), representing 51.6% (PC1) and 21.1% (PC2) of the total variance in FA composition of the 3 compartments. The loading plot for the regression factors revealed a strong positive correlation between PC1 with the Δ(9)-desaturase indices and the proportions of 14:1 cis-9 and 16:1 cis-9, and revealed a negative correlation with the proportion of 18:0 and saturated FA. The correlation with PC2 was positive for the proportion of unsaturated FA, 18:2n-6, and 18:3n-3, and negative for the proportion of 14:0, 16:0, and saturated FA. The SUBC could be distinguished from the NEFA and ABD by a positive score for PC1, whereas differentiation among the latter 2 compartments could be made by a positive (NEFA) or negative (ABD) score for PC2. The Δ(9)-desaturase indices for C14 and C16 differed between all compartments but were numerically closer for NEFA and ABD versus NEFA and SUBC. The desaturase indices of the main FA (18:1 cis-9 and 18:0) did not differ between NEFA and ABD. These results support the existence of a different FA composition in ABD compared with SUBC. The greater similarity between the FA profiles of ABD and NEFA compared with SUBC and NEFA and the closer desaturase indices of ABD and NEFA support the hypothesis of a preferential mobilization of ABD fat in dairy cows with LDA.

Prediction of ruminal volatile fatty acid proportions of lactating dairy cows based on milk odd- and branched-chain fatty acid profiles: new models, better predictions.

The volatile fatty acids (VFA) produced in the rumen and the proportions in which they are produced are important determinants of a ruminant's metabolism, but their monitoring requires rumen-fistulated animals, which is not feasible under practical conditions or in experimental setups at herd level. An alternative approach was suggested earlier, consisting of predicting the VFA proportions from measured odd- and branched-chain fatty acid concentrations in the milk with a linear model. Here, we have improved this strategy through the development and application of 2 new model structures: the quadratic model, containing quadratic terms and interactions, and the rational model, consisting of a ratio of linear expressions. Both were found to improve prediction accuracy significantly compared with the linear model. Although the quadratic model achieved the best prediction accuracy, the rational model has the interesting property that it takes the dependence of the 3 predicted VFA into account and guarantees that the 3 proportions add up to 1. Adding a study effect to correct for a possible study bias in the multi-study data improved prediction substantially for all 3 methods. Our results demonstrate the potential of using milk odd- and branched-chain fatty acid concentrations to predict rumen VFA proportions.

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature.

AIMS: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α-linolenic acid (α-LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. METHODS AND RESULTS: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1-60%). CLA conversion, occurring in three strains, was lower (2-5%). The LAI gene sequences of the ten LAI-positive strains shared 75-99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6.2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5.5 (30°C) or 37°C (pH 6.2), LA was not converted and α-LNA only slightly converted. CONCLUSIONS: LAB show strain-dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. SIGNIFICANCE AND IMPACT OF THE STUDY: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.

The effect of marine algae in the ration of high-yielding dairy cows during transition on metabolic parameters in serum and follicular fluid around parturition.

Sixteen Holstein cows were assigned to 2 groups to evaluate the caloric and metabolic effect of feeding marine algae (ALG) from 3 wk prepartum until 12 wk postpartum. Milk production characteristics and the profiles of hormones and metabolites in the serum were monitored from -7 to 46 d in milk (DIM) and in follicular fluid (FF) from 14 to 46 DIM. All cows received a corn- and grass silage-based partially mixed ration supplemented with concentrate and protein supplement. In the diet of the ALG group, 2 kg of the concentrate was replaced by a concentrate containing ALG (44 g/d of docosahexaenoic acid). Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. The ALG diet increased milk yield (41.2 vs. 38.2 kg/d) and decreased milk fat yield (1.181 vs. 1.493 kg/d) and milk fat content (31.6 vs. 40.7 g/kg). Protein yield (1.336 vs. 1.301 kg/d) was not affected but a tendency toward decreased milk protein content (32.8 vs. 34.7 g/kg) was observed. Marine algae supplementation increased the ß-hydroxybutyric acid (BHBA) concentration in FF of the ALG cows compared with that in the controls (0.992 vs. 0.718 mmol/L). The total protein concentration in FF was decreased in ALG (62.9 vs. 67.6 g/L). Plasma and serum metabolites did not significantly differ between treatments except for a tendency toward a lower concentration of urea in the serum of the control compared with ALG (4.69 vs. 5.13 mmol/L). Based on metabolizable energy calculations, a daily energy-sparing effect of 3.48 Mcal was obtained due to milk fat depression (MFD). The concomitant increase in milk yield suggests that at least part of this spared energy is used to stimulate milk production. Theoretically, 3.48 Mcal of ME could lead to an increase in milk yield of 7.43 kg/d, which is higher than the observed 3 kg/d. However, when evaluating nutrient requirements during MFD in early lactation, we calculated that increased milk production is caused by a propionate-saving effect of 2.71 mol in the udder when milk fat is depressed. Concurrent increased BHBA concentrations in FF in the ALG group cannot be attributed to a worsened energy status of the animals because all other indicators contradict any change in energy balance, indicating that BHBA might not be an appropriate metabolic parameter to estimate the energy balance in early lactating dairy cows during MFD.

Effects of chemically or technologically treated linseed products and docosahexaenoic acid addition to linseed oil on biohydrogenation of C18:3n-3 in vitro.

Rumen biohydrogenation kinetics of C18:3n-3 from several chemically or technologically treated linseed products and docosahexaenoic acid (DHA; C22:6n-3) addition to linseed oil were evaluated in vitro. Linseed products evaluated were linseed oil, crushed linseed, formaldehyde treated crushed linseed, sodium hydroxide/formaldehyde treated crushed linseed, extruded whole linseed (2 processing variants), extruded crushed linseed (2 processing variants), micronized crushed linseed, commercially available extruded linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil. Each product was incubated with rumen liquid using equal amounts of supplemented C18:3n-3 and fermentable substrate (freeze-dried total mixed ration) for 0, 0.5, 1, 2, 4, 6, 12, and 24h using a batch culture technique. Disappearance of C18:3n-3 was measured to estimate the fractional biohydrogenation rate and lag time according to an exponential model and to calculate effective biohydrogenation of C18:3n-3, assuming a fractional passage rate of 0.060/h. Treatments showed no differences in rumen fermentation parameters, including gas production rate and volatile fatty acid concentration. Technological pretreatment (crushing) followed by chemical treatment applied as formaldehyde of linseed resulted in effective protection of C18:3n-3 against biohydrogenation. Additional chemical pretreatment (sodium hydroxide) before applying formaldehyde treatment did not further improve the effectiveness of protection. Extrusion of whole linseed compared with extrusion of crushed linseed was effective in reducing C18:3n-3 biohydrogenation, whereas the processing variants were not different in C18:3n-3 biohydrogenation. Crushed linseed, micronized crushed linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil did not reduce C18:3n-3 biohydrogenation. Compared with the other treatments, docosahexaenoic acid addition to linseed oil resulted in a comparable trans11,cis15-C18:2 biohydrogenation but a lesser trans10+11-C18:1 biohydrogenation. This suggests that addition of DHA in combination with linseed oil was effective only in inhibiting the last step of biohydrogenation from trans10+11-C18:1 to C18:0.

Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture.

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C(18:1) and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 x 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C(18:0), trans-11 C(18:1) and cis-9, trans-11 CLA, and increased the flow of trans-10 C(18:1), C(18:2n-6), C(18:3n-3), trans-11,cis-15 C(18:2) and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C(18:1) and trans-10 C(18:1), and increased C(18:2n-6), C(18:3n-3) and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C(18:1) and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.

The effect of silage and concentrate type on intake behavior, rumen function, and milk production in dairy cows in early and late lactation.

The objective of this experiment was to evaluate the effect of feeding total mixed rations (TMR) that differ in structural and nonstructural carbohydrates to dairy cows in early and late lactation on short-term feed intake, dry matter intake (DMI), rumen fermentation variables, and milk yield. A 5 x 5 Latin square experiment with 15 dairy cows was repeated during early and late lactation. The 5 treatments were a TMR with (all on dry matter basis) 55% roughage (a 50:50 mixture of corn silage and grass silage) and 45% concentrate (a 50:50 mixture of concentrate rich in structural carbohydrates and concentrate rich in nonstructural carbohydrates; treatment CON), a TMR with the concentrate mixture and 55% grass silage (RGS) or 55% corn silage (RCS), and a TMR with the roughage mixture and 45% of the concentrate rich in structural carbohydrates (CSC) or the concentrate rich in nonstructural carbohydrates (CNS). Meal criteria, determined using the Gaussian-Gaussian-Weibull method per animal per treatment, showed an interaction between lactation stage and treatment. Feed intake behavior variables were therefore calculated with meal criteria per treatment-lactation stage combination. Differences in feed intake behavior were more pronounced between treatments differing in roughage composition than between treatments differing in concentrate composition, probably related to larger differences in chemical composition and particle size between corn silage and grass silage than between the 2 concentrates. The number of meals was similar between treatments, but eating time was greater in RGS (227 min/d) and lesser in RCS (177 min/d) than the other treatments. Intake rate increased when the amount of grass silage decreased, whereas meal duration decreased simultaneously. These effects were in line with a decreased DMI of the RGS diet vs. the other treatments, probably related to the high neutral detergent fiber (NDF) content. However, this effect was not found in CSC, although NDF content of the TMR, fractional clearance rate of NDF, and fractional degradation rate of NDF was similar between CSC and RGS. Rumen fluid pH was lesser, and molar proportions of acetic acid and of propionic acid were lesser and greater, respectively, in RCS compared with all other diets. Milk production did not differ between treatments. There was no effect of type of concentrate on milk composition, but diet RCS resulted in a lesser milk fat content and greater milk protein content than diet RGS. Lactation stage did affect short-term feed intake behavior and DMI, although different grass silages were fed during early and late lactation. The results indicate that short-term feed intake behavior is related to DMI and therefore may be a helpful tool in optimizing DMI and milk production in high-production dairy cows.

Frequent allocation of rotationally grazed dairy cows changes grazing behavior and improves productivity.

Twenty Holstein cows were blocked in 2 groups according to milk yield to evaluate the effect of frequency of allocation to new grazing plots on pasture intake, grazing behavior, rumen characteristics, and milk yield. The 2 treatments, daily allocation to 0.125-ha plots (1D) or allocation every 4 d to 0.5-ha plots (4D) of Lolium perenne L., were tested in a randomized block design (2 rotations with 3 or 4 measuring periods of 4 d each) with mixed model analysis accounting for repeated measures. There were no differences in the chemical composition of offered pasture and in pasture dry matter intake (DMI) between 1D and 4D. However, an interaction between treatment and rotation indicated a difference in pasture DMI between treatments during the first rotation (4D, 16.5 vs. 1D, 18.3 kg/d) but not during the second rotation (4D, 15.0 vs. 1D, 14.7 kg/d), possibly a result of a greater pasture mass in the first rotation. Grazing time (average 562 min/d) and ruminating time (average 468 min/d), observed using IGER graze recorders, were similar between treatments, but grazing time increased numerically (549 to 568 min/d), and ruminating time decreased linearly (471 to 450 min/d) within periods in the 4D treatment. Mean rumen pH (6.16 vs. 6.05) and rumen NH(3)-N concentration (113.7 vs. 90.1 mg/L) were higher in 4D than in 1D, and total volatile fatty acid (VFA) concentrations did not differ. Molar proportions of VFA, except butyrate, differed between treatments, causing the nonglucogenic to glucogenic VFA ratio to be greater in 4D than in 1D. Within days in the 4D treatment, the molar proportion of acetate increased and those of all other VFA decreased linearly. Rumen NH(3)-N concentration within the 4D treatment declined quadratically from 170.3 mg/L on d 1 to 80.7 mg/L on d 4. In contrast to rumen NH(3)-N concentration, milk urea content did not differ between treatments, but decreased quadratically from d 1 to 4 in the 4D treatment (from 26.7 to 20.7 mg/dL). Mean fat- and protein-corrected milk was greater in 1D than in 4D (23.5 vs. 22.8 kg/d), mainly due to a difference in milk yield (24.5 vs. 23.7 kg/d). Fat and protein content were slightly lower in the 1D than in the 4D treatment (3.66 vs. 3.76% and 3.28 vs. 3.34%, respectively). This study confirmed that increasing pasture allocation frequency from once every 4 d to every day improved milk production in grazing dairy cows, especially when offered pasture was high.

Effect of in vitro docosahexaenoic acid supplementation to marine algae-adapted and unadapted rumen inoculum on the biohydrogenation of unsaturated fatty acids in freeze-dried grass.

The objective of this study was to examine the ruminal biohydrogenation of linoleic (18:2n-6) and linolenic (18:3n-3) acid during in vitro incubations with rumen inoculum from dairy cattle adapted or not to marine algae and with or without additional in vitro docosahexaenoic acid (DHA, 22:6n-3) supplementation. Treatments were incubated in 100-mL flasks containing 400 mg of freeze-dried grass, 5 mL of strained ruminal fluid, and 20 mL of phosphate buffer. Ruminal fluid was collected just before the morning feeding from 3 cows receiving a control diet (49% ryegrass silage, 39% corn silage, 1% straw, and 11% concentrate, fresh-weight basis) supplemented with marine algae for 21 d (adapted rumen fluid, aRF) or from the same cows receiving the control diet only for 14 d after marine algae supplementation was stopped (unadapted rumen fluid, uRF). In half of the incubation flasks, pure DHA (5 mg) was added as an oil-ethanol solution (100 mL). Incubations were carried out during 0, 0.5, 1, 2, 4, 6, and 24 h. After 24 h, in vitro addition of DHA resulted in greater amounts (mg/incubation) of 18:3n-3 (0.23, 0.43, 0.26, and 0.34 for aRF, aRF+DHA, uRF, and uRF+DHA), 18:2n-6 (0.14, 0.22, 0.15, and 0.20 for aRF, aRF+DHA, uRF, and uRF+DHA) and trans-11, cis-15-18:2 (0.27, 2.40, 0.06, and 2.21 for aRF, aRF+DHA, uRF, and uRF+DHA), whereas no effect of inoculum source was observed. Trans-11-18:1 accumulated after 24 h when aRF was incubated irrespective of in vitro DHA supplementation, whereas in incubations with uRF, accumulation of trans-11-18:1 only occurred when DHA was added (6.40, 4.35, 1.06, and 3.91 for aRF, aRF+DHA, uRF, and uRF+DHA). The increased amounts of trans-11-18:1 were due to the strong inhibition of the reduction to 18:0 because no 18:0 was formed when trans-11-18:1 accumulated after 24 h. The results of the current experiment shows hydrogenation of trans-11, cis-15-18:2 occurred in the absence of in vitro DHA only, whereas substantial hydrogenation of trans-11-18:1 to 18:0 only took place in incubations without DHA and with unadapted rumen inoculum, confirming the higher sensitivity of the latter process to DHA.

Effect of dietary starch or micro algae supplementation on rumen fermentation and milk fatty acid composition of dairy cows.

Two experiments with rumen-fistulated dairy cows were conducted to evaluate the effects of feeding docosahexaenoic acid (DHA; C22:6 n-3)-enriched diets or diets provoking a decreased rumen pH on milk fatty acid composition. In the first experiment, dietary treatments were tested during 21-d experimental periods in a 4 x 4 Latin square design. Diets included a control diet, a starch-rich diet, a bicarbonate-buffered starch-rich diet, and a diet supplemented with DHA-enriched micro algae [Schizochytrium sp., 43.0 g/kg of dry matter intake (DMI)]. Algae were supplemented directly through the rumen fistula. The total mixed ration consisted of grass silage, corn silage, soybean meal, and a standard or glucogenic concentrate. The glucogenic and buffered glucogenic diet had no effect on rumen fermentation and milk fatty acid composition because, unexpectedly, no reduced rumen pH was detected. The algae diet had no effect on rumen pH but provoked decreased butyrate and increased isovalerate molar proportions in the rumen. In addition, algae supplementation affected rumen biohydrogenation of linoleic and linolenic acid as reflected in the modified milk fatty acid composition toward increased conjugated linoleic acid (CLA) cis-9 trans-11, CLA trans-9 cis-11, C18:1 trans-10, C18:1 trans-11, and C22:6 n-3 concentrations. Concomitantly, on average, a 45% decrease in DMI and milk yield was observed. Based on these drastic and impractical results, a second animal experiment was performed for 20 d in which 9.35 g/kg of total DMI of algae were incorporated in the concentrate and supplemented to 3 rumen-fistulated cows. Algae concentrate feeding increased rumen pH, which was associated with decreased rumen short-chain fatty acid concentrations. Moreover, a different shift in rumen short-chain fatty acid proportions was observed compared with the first experiment because molar proportions of butyrate, isobutyrate, and isovalerate increased, whereas acetate molar proportion decreased. The milk fatty acid profile changed as in experiment 1. However, the decrease in DMI and milk yield was less pronounced (on average 10%) at this algae supplementation level, whereas milk fat percentage decreased from 47.9 to 22.0 g/kg of milk after algae treatment. In conclusion, an algae supplementation level of about 10 g/kg of DMI proved effective to reduce the milk fat content and to modify the milk fatty acid composition toward increased CLA cis-9 trans-11, C18:1 trans, and DHA concentrations.