Effects of induced acidosis on milk fat content and milk fatty acids profile

Objective: The effect of wheat percentage in diets offered to lactating cows on variations of milk fat content and profile of milk fatty acids (FA) was studied, focusing on odd-chain FA and trans intermediates of ruminal biohydrogenation. Materials and methods: Two cows equipped with a ruminal canula successively received diets based on maize silage, and comprising 0, 20, 34 and again 0% wheat on a dry matter basis. Each diet was used during 12 days. The diet was distributed in two equal meals at 08:00 and 17:00, and wheat was top-dressed on silage. Milk samples were taken at the evening milking, and samples of ruminal contents were taken hourly from 08:00 to 16:00 on days 10 and 11 in the first period, and on days 5, 10 and 11 in the 3 subsequent periods. Results and discussion: Compared with the initial control, after 10 days adaptation, diet with 20% wheat significantly lowered mean ruminal pH (6.03 vs 6.77) and milk fat content (33.0 vs 44.1), and significantly increased the percentage of odd-chain FA in milk fat (2.38 vs 1.48). The trans-10C18:1 / trans-11C18:1 ratio increased from 0.34 to 0.82, but the difference was not significant. After 10 days adaptation, the diet with 34% wheat resulted in low ruminal pH (5.8), low milk fat content (22.4), and high percentage of odd-chain FA (3.03) and high trans-10C18:1 / trans-11C18:1 ratio (12.2). All these values were significantly different from both initial values and values observed with 20% wheat. After 10 days with the control diet following acidogenic diets, mean ruminal pH and milk fat content returned near initial value (6.98 and 41.1, respectively), but odd-chain FA and the trans-10C18:1 / trans-11C18:1 ratio (1.80 and 2.14, respectively) remained significantly higher than initial values. This suggests that the effects of a ruminal acidosis can remain a long time after returning to a non-acidogenic diet. Values observed after 5 days adaptation to the three diets were intermediate between values at the end of the previous period and values after 10 days adaptation, and significantly different from values after 10 days adaptation for all presented parameters except the trans-10C18:1 / trans-11C18:1 ratio (P = 0.25). The correlation coefficients between mean ruminal pH and milk fat content, proportion of odd-chain FA and the trans-10C18:1 / trans-11C18:1 ratio were significant (0.84, ־0.87 and ־0.62, respectively). However, this low latter value was due to a weak relationship when pH was over 6.2, and a large increase of the trans-10C18:1 / trans-11C18:1 ratio when mean ruminal pH was under 6.2. These results are consistent with present knowledge on trans-10 FA as a result of low ruminal pH and a cause of low milk fat content. They show that the trans-10C18:1 / trans-11C18:1 ratio can exhibit very large variations when the mean ruminal pH is under 6.2. Conclusion and perspective: Induced acidosis resulted in lowered milk fat content, and higher proportion of odd-chain FA and a higher trans-10C18:1 / trans-11C18:1 ratio. Milk fat content and proportion of odd-chain FA were linearly related to mean ruminal pH. On the contrary, trans-10C18:1 / trans-11C18:1 ratio only exhibited variations when mean ruminal pH was low, and these variations were in a large range, making this ratio a possible candidate for biochemical characterisation of acidosis

Effects of duration and temperature of heating of sunflower oil on ruminal linoleic acid biohydrogenation in vitro

Feeding cows with heat treated oilseeds results in an increase of milk conjugated linoleic acids (CLA) content compared to feeding raw seeds. Heating leads to a lipid peroxidation, which could reduce the amount of fatty acids (FA) subjected to biohydrogenation and produce peroxides which could act directly on this reaction. The effects of heating duration and temperature, on the biohydrogenation were examined. Sunflower oil was heated at 150°C during 0, 3, 14, or 22 h, or during 3 h at 70, 100, 130, or 160°C. Peroxides in oils were determined by the peroxide index (PI), and fatty acids in samples incubated during 6 h were analysed by gas chromatography. Increasing heating duration was more efficient than increasing heating temperature for peroxides production: PI = 15.9, 41.9, 93.0, and 162.8 mEq/kg for 0, 3, 14, and 22 h of heating, respectively, and PI = 40.9, 35.9, 84.0, and 88.2 mEq/kg for 70, 100, 130, and 160 °C of heating, respectively. For each mEq of PI/kg, FA content decreased by 0.11% (P=0.003; R2=0.177). After incubation, the percents of cis9,trans11-CLA (C18:2c9t11) and trans-vaccenic acid (C18:1t11) were significantly correlated with the heating duration: C18:2c9t11 (% of FA) = -0.040 DURATION (h) + 1.783 (P=0.000; R2=0.830) and C18:1t11 (% of FA) = -0.743 DURATION (h) + 3.711 (P=0.001; R2=0.396). The percent of C18:2c9t11 was significantly correlated with the heating temperature: C18:2c9t11 (% of FA) = -0.007 TEMPERATURE (°C) + 2.457 (P=0.006; R2=0.296), but this relationship was lower than for heating duration, and there was no effect of temperature on C18:1t11 percent, possibly because high heating temperature generated less peroxides than long heating duration. Across the two studied parameters, the relationship between the percent of C18:2c9t11 and the level of peroxides was significant: C18:2c9t11 (% of FA) = -0.007 PI (mEq/kg) + 1.998 (P=0.000; R2=0.494). This 0.35% relative decrease for one mEq of PI/kg, suggested that the effect was mainly due to the peroxides, not only to the decrease of FA content. In conclusion, increasing temperature and mainly heating duration of oil led to a decrease of C18:2c9t11 production in the rumen probably due to the lipid peroxidation. Increasing heating duration also decreased C18:1t11 production. These results suggest that peroxides cannot explain the increase of milk CLA noticed in the milk from cows eating heated oilseeds, and that a good quality fat, without lipids oxidation products, is necessary to increase CLA and C18:1t11 production in the rumen

Enzymatic approach of linoleic acid ruminal biohydrogenation

Ruminal biohydrogenation (BH) corresponds to a microbial reduction of dietary unsaturated fatty acid. The linoleic acid (C18:2) BH is divided into three steps: first an isomerisation into conjugated linoleic acids (CLA), then a reduction producing mainly trans-octadecenoic acids (trans-C18:1), and a final reduction producing stearic acid (C18:0). Isomerisations of CLA and trans-C18:1 can lead to a number of positional and geometrical isomers. The control of BH reactions is of interest for researchers because BH directly affects the composition of fatty acids of milk and meat. In order to better understand C18:2 BH and its variations, the development of an enzymatic approach is necessary to ascertain if the action of modulators affects the bacterial enzyme activity or ruminal bacteria. The aim of this study was to investigate the C18:2 BH capacity of ruminal content after inactivation of bacteria by chloramphenicol (Cm), an inhibitor of protein synthesis in prokaryotes. The BH of C18:2 produced mainly cis9,trans11-CLA and trans10,cis12-CLA, and trans11-C18:1 and trans10-C18:1 isomers, as previously described (Jouany et al., 2007). The increase in cis12-C18:1 came from reduction of trans10,cis12-CLA, that of trans6+7+8-C18:1 from the reduction of minor CLA isomers not quantified in this study, like trans8,trans10-CLA (Shingfield et al., 2008). The trans11 pathway was rapid: the cis9,trans11-CLA production was maximal at about 1h of incubation while trans11-C18:1 accumulated throughout incubation. On the other hand, trans10 pathway was slow: trans10,cis12-CLA regularly increased during incubation, so that it was more abundant than cis9,trans11-CLA after 3h incubation, and trans10-C18:1 only began to increase after 2h of incubation. The amount of C18:0 began to increase in the media when trans11-C18:1 concentration was over 0.05 mg/mL. Such evolution of fatty acids involved in C18:2 BH was similar to that reported in vitro with living ruminal microorganisms by Harfoot et al. (1973) and Jouany et al. (2007). So, this enzymatic approach using Cm could be an interesting and valid method to study C18:2 BH, however 3h of incubation were not sufficient to study the final reduction

Effects of heating process of soybeans on ruminal production of conjugated linoleic acids and trans-octadecenoic acids in situ

The effects of two thermal treatments of soybeans, i.e. roasting (150˚C dry heat) and extrusion (140-150˚C), on conjugated linoleic acids (CLA) and trans-octadecenoic acids (trans-C18:1) productions obtained throughout ruminal C18:2 biohydrogenation in cows were examined. Nylon bags containing raw, roasted or extruded soybeans were incubated in the rumen of dry fistulated cows, during 2, 4, 8, 16 or 24 hours. After incubation of 2-4 h, significantly greater amounts of linoleic acid (C18:2) remained in bags containing extruded and roasted soybeans than in those with raw soybeans, reflecting a lower biohydrogenation of C18:2 in both case. Furthermore, significant and marked accumulations of CLA and trans-C18:1 at a lesser extend were noticed in bags containing extruded soybeans compared to those with raw or roasted soybeans. By calculations of the efficiencies of the three reactions, an inhibition of the C18:2 isomerisation was evidenced with extruded and roasted soybeans, as well as an inhibition of the two reduction steps in presence of extruded soybeans. Consequently, the thermal treatment and the nature of heating process of fat are efficient ways to modulate the CLA and trans-C18:1 ruminal productions

Les acides linoléiques conjugués: 1. Intérêts biologiques en nutrition

Les CLA (Acides Linoléiques Conjugués) sont des isomères de position et géométriques de l'acide linoléique. Parmi eux, le c9t11-CLA et le t10c12-CLA, présentent des actions biologiques intéressantes, qui pourraient être exploitées en diététique humaine. Retrouvés en relativement grande quantité dans les productions animales issues de ruminants, et surtout dans les produits laitiers, ces deux isomères sont capables de limiter l'induction et le développement de tumeurs malignes, de stimuler les réponses immunes et de favoriser la croissance et le développement osseux. En outre, en orientant le métabolisme vers l'utilisation périphérique des substrats lipidiques et glucidiques aux détriments des voies de stockage ou de synthèse, en diminuant la cholestérolémie et la production des éïcosanoïdes, ces composés pourraient contribuer au traitement et/ou à la prévention de l'obésité, du diabète sucré et de l'athérosclérose. Bien que les effets des CLA aient été démontrés chez les animaux ou in vitro, à l'exception d’un rôle possible dans la prévention du cancer, ces activités biologiques potentielles restent encore très discutées chez l'homme

Polymers of triglycerides generated during heating of fat do not protect linoleic acid from ruminal biohydrogenation

Heating fats often induces a decrease of cis-9, cis-12 C18:2 and cis-9, cis-12, cis-15 C18:3 biohydrogenation (BH) in vivo (Gonthier et al. 2005), in situ (Troegeler-Meynadier et al. 2006) and in vitro (Privé et al. 2010). This is of interest because it could increase polyunsaturated fatty acids (PUFA) content of ruminant products. Temperature and duration of heating of sunflower oil affect ruminal BH of PUFA, in part due to peroxide value (Privé et al., 2010). Our hypothesis was that polymers of triglycerides (TG), formed during heating of TG but not of free FA, could be responsible for partial protection of PUFA from BH

Effects of fat source and dietary sodium bicarbonate plus straw on the conjugated linoleic acid content of milk of dairy cows

The effects of fat source (0.7 kg of fatty acids from extruded soybeans or palmitic acid), of sodium bicarbonate (0.3 kg) plus straw (1 kg) and the interaction of these treatments on the content of conjugated linoleic acid (CLA) in the milk of dairy cows were examined. During nine weeks a group of 10 cows received a ration with palmitic acid and bicarbonate plus straw (ration PAB). During three periods of three weeks a second group of 10 cows received successively a ration with extruded soybeans and bicarbonate plus straw (ration ESB), a ration with palmitic acid without bicarbonate or straw (ration PA), and a ration with extruded soybeans without bicarbonate or straw (ration ES). Rations ES and ESB increased the content of polyunsaturated fatty acids in milk, but decreased milk fat content, compared to rations PAB and PA. Ration ESB led to the greatest milk CLA content, by a synergy between the high amount of dietary fat, and the action of bicarbonate plus straw, favouring trans11 isomers of CLA and C18:1, presumably via a ruminal pH near neutrality. Ration ES favoured trans10 isomers, not desaturated in the mammary gland, so that the milk CLA content was lower than with ration ESB, and resulted in the lowest milk fat content. In conclusion, a ration supplemented with both extruded soybeans and bicarbonate plus straw, was an efficient way to increase the CLA content in the milk of dairy cows

Comparison of enzymatic activities of the reactions of linoleic and linolenic acids ruminal biohydrogenation

Introduction: Biohydrogenation (BH) is a microbial hydrogenation of dietary unsaturated fatty acids occurring in the rumen. BH is of interest because it directly affects the fatty acids composition of milk and meat. The linoleic acid (C18:2) BH is divided into three reactions: isomerisation into conjugated linoleic acids (CLA), reduction to trans-C18:1 and then to stearic acid (C18:0); that of alpha-linolenic acid (C18:3) into four reactions: isomerisation to conjugated linolenic acid (CLnA), reduction to trans11,cis15-C18:2, then to trans-C18:1 and finally to C18:0. The aim of this study was to compare enzymatic activities of the reactions of C18:2 BH to those of C18:3 BH. Materials and methods: Rumen fluid was collected from a dry dairy cow and strained on a metal sieve (1,6mm). Then, it was mixed with Chloramphenicol (Cm), an inhibitor of protein synthesis in prokaryotes, at a dose of 1mg/mL . Incubations were prepared by adding 1mL of rumen fluid + Cm, with 1mL of bicarbonate buffer and 1mg of C18:2 or C18:3 (purity ≥ 99%, Sigma), and were conducted in a waterbath at 39°C, with 3h agitation, in 3 replicates. Fatty acids were quantified by gas chromatography. Then rate (v, mg/L/h) and efficiency (E, %) of the reactions were calculated. Results and Discussion: The isomerisation of C18:3 was quicker and more efficient than that of C18:2, which was probably saturated2 (v = 129.6 vs. 94.4 mg/L/h; E = 80.2 vs. 52.7%, respectively. The reductions of conjugated isomers were rapid and efficient, mainly for CLnA (v = 123.7 mg/L/h; E= 95.5%) compared to CLA (v = 78.1 mg/L/h; E= 82.0%). However, for C18:2 BH, cis9,trans11-CLA disappeared quicker than trans10,cis12-CLA so that their respective production after 3h incubation was +0.016mg vs. +0.073mg. The last reduction of C18:2 BH was the slowest (v = 63.8 mg/L/h; E= 68.9%), and constituted the limiting step, resulting in trans-C18:1 accumulation. The second reduction of C18:3 BH was very slow and poorly efficient (v = 48.8 mg/L/h; E= 38.2%), so that trans11,cis15-C18:2 highly accumulated (+0.450mg produced). The last reduction of C18:3 BH was also a slow and poorly efficient reaction (v = 38.8 mg/L/h; E= 41.9%), so that trans-C18:1 would probably have accumulated with a longer incubation. Conclusion: The BH of C18:2 and C18:3 were not exactly similar. C18:2 BH was slower, its isomerisation seemed to be rapidly saturable and the limiting step was the final reduction inducing an accumulation of trans-C18:1. For C18:3 BH, first and second reactions were rapid, so that few CLnA was present in the media. Contrarily, the third and fourth reactions were slow so that trans11,cis15-C18:2 firstly accumulated. Such an evolution was previously reported in vitro with live mixed ruminal bacteria2, indicating that the evaluation of BH does not require live bacteria, and confirming the validity and interest of this enzymatic approach

Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations

Data from a previous study about the effects of pH and of linolenic acid (C18:3n-3) andlinoleic acid (C18:2n-6) concentrations on C18:2n-6 biohydrogenation in ruminal cultures were used to calculate the rates and efficiencies of the three reactions of C18:2n-6 biohydrogenation (isomerisation of C18:2n-6 to CLA; reduction of CLA to trans-octadecenoic acids; reduction of trans-octadecenoic acids to stearic acid). First, low pH was confirmed to inhibit isomerisation and was shown to inhibit the second reduction, leading to an accumulation of vaccenic acid. This later effect had only been observed in some in vivo studies using high concentrate diets, because in in vitro experiments, the very low pH frequently used depresses isomerisation which consequently generates very low amount of substrates for reductions whose variations become difficult to ascertain. Second, C18:2n-6 at high concentration was confirmed to saturate its own isomerisation and the increase of CLA production due to high initial C18:2n-6 was shown to inhibit the two subsequent reductions. Third, C18:3n-3 at high concentrations was confirmed to inhibit C18:2n-6 isomerisation. Moreover, the second reduction was shown to be saturated, probably by all trans-octadecenoic acids intermediates of C18:2n-6 and C18:3n-3 biohydrogenation, leading to an accumulation of trans-octadecenoic acids, especially vaccenic acid. This fatty acid is partly desaturated into CLA in the mammary gland, which explains the synergy between C18:2n-6 and C18:3n-3 for milk CLA noticed by others in vivo. This approach helped explain the actions of pH and of C18:2n-6 and C18:3n-3 concentrations on C18:2n-6 biohydrogenation and allows some explanations about differences noticed between studies

Les acides linoléiques conjugués : 2. Origines et effets sur les productions animales

Les aliments d’origine animale, surtout la viande et le lait des ruminants, contiennent en quantités non négligeables les deux principaux isomères biologiquement actifs des acides linoléiques conjugués (CLA), le c9t11-CLA et le t10c12-CLA. Ils sont obtenus au cours de la biohydrogénation de l’acide linoléique dans le rumen des polygastriques, et par désaturation tissulaire de l’acide trans-vaccénique chez tous les animaux. Comme cet acide gras est également synthétisé au cours de la biohydrogénation ruminale des acides gras polyinsaturés, et que chez la vache laitière, une activité intense de désaturation a lieu dans la mamelle, les concentrations en CLA dans les tissus et le lait des ruminants sont supérieures à celles des monogastriques. Chez ces derniers, l’enrichissement en CLA de leurs productions ne peut résulter que d’une supplémentation alimentaire directe en CLA ou en acide trans-vaccénique. Cependant, comme les CLA présentent des effets métaboliques systémiques, l’augmentation des apports en CLA chez les animaux de rente peut engendrer des conséquences positives ou négatives sur le niveau ou la qualité des productions : amélioration de la qualité des carcasses chez les monogastriques à l’exception des poulets, chute de la teneur en matière grasse du lait, altération de la ponte et de la qualité des oeufs