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, C18:2n-6, C18:3n-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 C18:2n-6, C18:3n-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

Milk fatty acid composition and associated rumen lipolysis and fatty acid hydrogenation when feeding forages from intensively managed or semi-natural grasslands

In order to evaluate the effect of replacing intensive forage by semi-natural grassland products on rumen lipid metabolism and milk fatty acid composition, four lactating and rumen canulated Holstein cows were used in a 4×4 Latin square design. Four different diets were fed: diet 100 IM - 100% intensively managed silage (IM), diet 20 SPP - 80% IM plus 20% semi-natural but species poor silage (SPP), diet 60 SPP - 40% IM plus 60% SPP and diet 60 SPR - 40% IM plus 60% semi-natural species rich silage (SPR). The silages showed significant differences in total fat content and in proportions of C18:2 n-6 and C18:3 n-3. Despite the reduced dietary supply of C18:3 n-3 with diets 60 SPP and 60 SPR, differences in milk C18:3 n-3 were small, suggesting higher recoveries of C18:3 n-3. Presumably, the latter are related to a higher transfer efficiency of C18:3 n-3 from the duodenum to the mammary gland, since rumen biohydrogenation, estimated from rumen pool size and first order rumen clearance kinetics, were similar among diets. CLA c9t11 in milk from cows fed diet 60 SPR were almost doubled compared to feeding one of the other diets. This has been related to the partial inhibition of rumen biohydrogenation of C18:3 n-3 and/or C18:2 n-6, as suggested by the increased proportions of hydrogenation isomers and reduced stearic acid proportions in rumen pool samples. In conclusion, the results suggest that the use of semi-natural grasslands in the diet of the animals reduce to some extent complete rumen biohydrogenation, which leads to an increase in milk CLA

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

Identifying and exploring biohydrogenating rumen bacteria with emphasis on pathways including trans-10 intermediates

Background Bacteria involved in ruminal formation oftrans-10 intermediates are unclear. Therefore, this study aimed at identifying rumen bacteria that producetrans-10 intermediates from 18-carbon unsaturated fatty acids. Results Pure cultures of 28 rumen bacterial species were incubated individually in the presence of 40 mu g/mL 18:3n-3, 18:2n-6 ortrans-11 18:1 under control or lactate-enriched (200 mM Na lactate) conditions for 24 h. Of the 28 strains,Cutibacterium acnes(formerlyPropionibacterium acnes) was the only bacterium found to producetrans-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 thetrans-11 totrans-10 shift, different biomass ratios ofButyrivibrio fibrisolvens(as atrans-11 producer) andC. acneswere incubated in different growth media (control, low pH and 22:6n-3 enriched media) containing 40 mu g/mL 18:2n-6. Under control conditions, atrans-10 shift, defined in the current study astrans-10/trans-11 >= 0.9, occurred when the biomass ofC. acnesrepresented between 90 and 98% of the inoculum. A low pH or addition of 22:6n-3 inhibitedcis-9,trans-11 CLA andtrans-10,cis-12 CLA formation byB. fibrisolvensandC. acnes, respectively, wherebyC. acnesseemed to be more tolerant. This resulted in a decreased biomass ofC. acnesrequired at inoculation to induce atrans-10 shift to 50% (low pH) and 90% (22:6n-3 addition). Conclusions Among the bacterial species studied,C. acneswas the only bacterium that have the metabolic ability to producetrans-10 intermediates from 18:3n-3 and 18:2n-6. Nevertheless, this experiment revealed that it is unlikely thatC. acnesis the only or predominant species involved in thetrans-11 totrans-10 shift in vivo

Failure of a dietary model to affect markers of inflammation in domestic cats

Background: Oxidative stress and inflammation can be altered by dietary factors in various species. However, little data are available in true carnivorous species such as domestic cats. As numerous anti-inflammatory and anti-oxidative additives become available and might be of use in cats with chronic low-grade inflammatory diseases, the current study aimed to develop a model of diet-induced inflammation by use of two opposite diets. It was hypothesized that a high fat diet enhanced in n-6 PUFA and with lower concentrations of antioxidants would evoke inflammation and oxidative stress in domestic cats. Results: Sixteen healthy adult cats were allocated to two groups. One group received a moderate fat diet, containing pork lard and salmon oil (AA:(EPA + DHA) ratio 0.19) (MFn-3), while the other group was fed a high fat diet, containing pork lard and chicken fat (AA:(EPA + DHA) ratio 2.06) (HFn-6) for 12 weeks. Prior to and 2, 4, 6, 8, 10 and 12 weeks after starting the testing period, blood samples were collected. Erythrocytic fatty acid profile showed clear alterations in accordance to the dietary fatty acid profile. Serum thiobarbituric acid reactive substances was higher when fed MFn-3 compared to the HFn-6, suggesting augmented oxidative stress. This was associated with a reduced serum vitamin E status, as serum a-tocopherol concentrations were lower with MFn-3, even with higher dietary levels of vitamin E. Serum cytokine and serum amyloid A concentrations were not influenced by diet. Conclusion: These results point towards a resistance of cats to develop dietary fat-induced inflammation, but also suggest a high susceptibility to oxidative stress when fed a fish oil-supplemented diet even with moderate fat level and additional vitamin E

Promising perspectives for ruminal protection of polyunsaturated fatty acids through polyphenol-oxidase-mediated crosslinking of interfacial protein in emulsions

Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions (<50% protection), which showed oiling-off after enzymatic crosslinking. This study demonstrated that alternative emulsifier proteins can be used in combination with PPO extract to protect emulsified PUFA-rich oils against ruminal biohydrogenation. By applying the new protocol, 6.5 times less PPO extract was required

Determining milk isolated and conjugated trans-unsaturated fatty acids using Fourier transform Raman spectroscopy

The feasibility of Raman spectroscopy in combination with partial least-squares (PLS) regression for the determination of individual or grouped trans-monounsaturated fatty acids (trans-MUFA) and conjugated linoleic acids (CLA) in milk fat is demonstrated using spectra obtained at two temperature conditions: room, temperature and after freezing at -80 degrees C. The PLS results displayed capability for direct semiroutine quantification of several individual CLA (cis-9,trans-11 and trans-10,cis-12 C18:2) and trans-MUFA (trans-4-15 C18:1) in minor concentrations (below 1.0 g/100 g of milk fat). Calibration models were based on reference data cross-correlation or determined by specific scattering signals in the Raman spectra. Distinct bands for trans-MUFA (1674 cm(-1)) and CLA (1653 cm(-1)) from the trans isolated and cis,trans conjugated C=C bonds were identified, as well as original evidence for the temperature effect (new bands, peak shifts, and higher intensities) on the Raman spectra of fatty acid methyl ester and triacylglyceride standards, are supplied