III – Results and discussion
2. Effects on in vitro fatty acid composition of rumen digesta
H. Benhissi, G. Hervás, P.G. Toral, A. Belenguer and P. Frutos1
Instituto de Ganadería de Montaña (CSIC-ULE), Finca Marzanas s/n, 24346 Grulleros, León (Spain)
1E-mail: p.frutos@csic.es
Abstract. Supplementing the diet of ruminants with tannins has been suggested as a strategy to modulate ruminal biohydrogenation (BH) of dietary polyunsaturated fatty acids (FA) and enhance the accumulation of vaccenic acid (VA) due to an inhibition of the last step of BH. The ultimate goal of this strategy is to increase the content of some bioactive FA (such as CLA or VA) in the ruminant products. This experiment was conducted in sheep to study the effect of the addition of tannins to a total mixed ration supplemented with 2% DM of sunflower oil on ruminal BH. The assay was carried out in vitro using batch cultures of rumen microorganisms. Four commercial extracts of tannins [2 hydrolysable (HT: chestnut and oak) and 2 condensed (CT: quebracho and grape) tannins] × 2 doses of each one (2 and 5% DM) were added to the supplemented diet. After 24 h incubations, none of the four tannin extracts and none of the two doses tested were able to modify the rumen contents of total CLA, VA or stearic acid. However, some increases were detected in the concentration of linolenic, linoleic and oleic acids, which may suggest a general inhibition of the ruminal BH rather than a specific inhibition of the last step. Most significant results were observed with HT and at 5%. Overall, the results would not allow to recommend this strategy to modulate ruminal BH and improve the nutritional quality of the ruminant products.
Keywords.CLA – Condensed tannin – Hydrolysable tannin – Lipid supplementation – Vaccenic acid.
Utilisation des tanins pour modifier la biohydrogénation ruminale chez les ovins. 2. Effets sur la composition en acides gras du digesta ruminal in vitro
Résumé. Afin de modifier la biohydrogénation (BH) ruminale des acides gras polyinsaturés (AG) et d'améliorer l'accumulation de l'acide vaccénique (VA) par une inhibition de la dernière étape de la BH, une des stratégies proposées est d’incorporer des tanins dans les régimes destinés aux ruminants. Le but ultime de cette stratégie est d'augmenter la teneur en certains AG bioactifs (tels que le CLA ou VA) dans les produits animaux. Une expérience a été menée sur le mouton pour étudier l'effet de l'incorporation des tanins dans une ration complète enrichie d'huile de tournesol (2% de la matière sèche) sur la BH ruminale.
Le test a été réalisé in vitro en utilisant des cultures non renouvelées de micro-organismes du rumen.
Quatre extraits commerciaux de tanins ont été ajoutés à la ration supplémentée [2 extraits de tanins hydrolysables (HT) : châtaignier et chêne et 2 extraits de tanins condensés (CT) : quebracho et raisin] à raison de 2 doses de chacun (2 et 5% de la matière sèche). Après une incubation de 24 h, aucun des 4 extraits de tanins et aucune des deux doses testées n’ont modifié la teneur ruminale en CLA totaux, en VA ou en acide stéarique. Toutefois, une augmentation des concentrations des acides linolénique, linoléique et oléique a été observée, ce qui suggère une inhibition générale de la BH ruminale plutôt qu'une inhibition spécifique de la dernière étape. Les résultats les plus significatifs ont été observés avec les HT et à une dose de 5%. Dans l'ensemble, ces résultats ne permettent pas de recommander cette stratégie pour modifier la BH ruminale et améliorer la qualité nutritionnelle des produits issus de ruminants.
Mots-clés. CLA – Tanins condensés – Tanins hydrolysables – Supplémentation lipidique – Acide vaccénique.
Options Méditerranéennes, A no. 107, 2013
24
I – Introduction
Recent studies have suggested that supplementing the diet of ruminants with tannins may be a good strategy to modulate the process of ruminal biohydrogenation (BH) of dietary polyunsaturated fatty acids (FA) and enhance the accumulation of vaccenic acid (VA) due to an inhibition of the last step of BH (Khiaosa-ard et al., 2009; Vasta et al., 2009). The ultimate goal of this strategy would be to increase the content of some potencially healthy FA (such as conjugated linoleic acid [CLA] and VA) in ruminant-derived products (Vasta and Luciano, 2011).
However, reports in this regard are still limited and inconsistent.
This experiment was therefore conducted to study, in sheep, the effect of the addition of tannins to a total mixed ration (TMR; forage:concentrate ratio 40:60) supplemented with 2% of sunflower oil (as a source of linoleic acid for the synthesis of VA in the rumen; Shingfield et al., 2008; Toral et al., 2012), on ruminal BH, with special attention to the accumulation of some FA of special interest (e.g., linolenic and linoleic acids, CLA and 18:1 metabolites).
II – Materials and methods
The experiment was conducted in vitro using batch cultures of rumen microorganisms following a 4 × 2 + 2 (controls) design. Treatments were: 4 types of tannins [2 hydrolysable (HT: chestnut and oak) and 2 condensed (TC: quebracho and grape)] × 2 doses of each one (2% and 5% DM) were added to a diet supplemented with 2% of sunflower oil (on a DM basis; SO). In addition to the positive control (i.e., the supplemented diet), a negative control (i.e., the diet without lipid supplementation) was also included to monitor the effect of the inclusion of lipids in the diet. No tannins were added to the negative control.
Four ewes cannulated in the rumen and fed a TMR similar to that used in the cultures (forage:concentrate ratio 40:60; 173 g of CP, 260 g of NDF, and 29 g of ether extract/kg DM) were used as donors of ruminal inoculum. Rumen fluid was collected before the morning meal.
Sixty samples of the diet [10 treatments × 3 flasks/treatment × 2 replicates (i.e., repetition in 2 different days)] ground through a 1-mm screen (≈ 500 mg), plus 6 blanks (3 flasks without substrate/day), were incubated for 24 hours at 39ºC with 10 mL strained rumen fluid and 40 mL phosphate-bicarbonate medium (Goering and Van Soest, 1970).
For FA composition analysis, lipids in 200 mg of freeze dried in vitro ruminal digesta were extracted with 4 mL of a mixture (3:2, v/v) of hexane and isopropanol following the adjustment of digesta pH to 2.0. Lipids were then converted to fatty acid methyl esters (FAME) using a base-acid catalyzed transesterification procedure with freshly prepared 0.5 M sodium methoxide in methanol followed by reaction with 1% sulphuric acid in methanol, as outlined by Toral et al.
(2010). Tridecanoic acid (Sigma-Aldrich, Madrid, Spain) was used as internal standard. Methyl esters were separated and quantified using a gas chromatograph (Agilent 7890A GC System, Santa Clara, CA, USA) equipped with a flame-ionization detector and a 100 m fused silica capillary column (0.25 mm i.d., 0.2-μm film thickness; CP-SIL 88, Chrompack 7489, Varian Ibérica S.A., Madrid, Spain) and He as the carrier gas. Total FAME profile in a 2 μL sample volume at a split ratio of 1:50 was determined using a temperature gradient programme (Shingfield et al., 2003). Isomers of 18:1 were further resolved in a separate analysis under isothermal conditions at 170ºC (Shingfield et al., 2003). Peaks were identified based on retention time comparisons with authentic standards (from Nu-Chek Prep., Elysian, MN, USA;
Sigma-Aldrich, Madrid, Spain; and Larodan Fine Chemicals AB, Malmö, Sweden). Identification of FA was verified based on FAME standard mixtures when available, chromatograms reported in the literature (e.g., Shingfield et al., 2003, 2006) and by comparison with milk samples for which the FA composition was determined based on GC analysis of FAME and GC-MS analysis of corresponding 4,4-dimethyloxazoline derivates (Toral et al., 2010).
Feeding and management strategies to improve livestock
productivity, welfare and product quality under climate change
25
All data were analysed as a one-way analysis of variance using the MIXED procedure of SAS (Version 9.2). Means were compared to the positive control using the ‘pdiff’ option of the
‘lsmeans’ statement of the MIXED procedure.
III – Results and discussion
Table 1 shows the content of several long-chain FA in the ruminal digesta, and Tables 2 and 3 a partial profile of cis 18:1 and trans 18:1 isomers, respectively. In all cases, tables show the mean and standard error of the negative (TMR) and positive (TMR+sunflower oil) controls, and the percentage of variation caused by each tannin treatment (i.e., quebracho, grape, chestnut and oak tannin extracts at 2 and 5%) with respect to the positive control. When the inclusion of sunflower oil (i.e., TMR vs. TMR+sunflower oil) caused a significant effect, this is indicated on the positive control value.
As expected (Hervás et al., 2008; Shingfield et al., 2008; Toral et al., 2012), diet supplementation with sunflower oil significantly increased (P<0.05) the content of total 18:2 non-conjugated linoleic acid, cis-9 cis-12 18:2, total CLA, cis 18:1 and trans 18:1, without effect on stearic acid (P>0.10).
Table 1. Content (mg/g digesta) of several long-chain fatty acids in the ruminal digesta. Mean and standard error of the negative (TMR) and positive (TMR+sunflower oil) controls, and percentage of variation caused by each tannin treatment with respect to the positive control
TMR 0.19±0.008 1.07±0.115 0.75±0.051 0.19±0.005 4.83±0.809 12.4±0.98 TMR+sunflower
oil
0.17±0.009 2.59±0.150** 2.31±0.128** 0.64±0.022** 7.75±0.481** 14.2±0.10 + Quebracho
The level of significance of each percentage of variation (tannin treatment vs. positive control) appears on its right: ns= non-significant (P>0,10); * = P<0,10 and ** = P<0,05.
On the contrary, none of the four tannins at any of the doses used were able to modify the rumen contents of cis-9 trans-11 18:2, total CLA or trans-11 18:1. The levels of trans-10 18:1 and 18:0 were not changed either. However, significant increases, due to the action of tannins, were detected in the concentration of some FA that were added with the sunflower oil, such as linoleic, linolenic and oleic acids, which would suggest a general inhibition of the ruminal BH of dietary polyunsaturated FA rather than a specific inhibition of the reduction of 18:1 to 18:0.
These in vitro results are in line with those observed previously by Kronberg et al. (2007) but not with the specific inhibition of the last step of the BH process suggested by Khiaosa-ard et al.
(2009) and Vasta et al. (2009).
Options Méditerranéennes, A no. 107, 2013
26
The higher content of linoleic acid due to the effect of tannins was accompanied in some cases by increases in non-conjugated 18:2 and decreases in their biohydrogenation products (e.g., trans-9 18:1, cis-12 18:1 and cis-16 18:1; Shingfield et al., 2008). A similar response occurred with other 18:1 deriving mainly from the isomerization of the oleic acid (Jenkins et al., 2008).
Table 2. Content (mg/g digesta) of some cis 18:1 fatty acids in the ruminal digesta. Mean and standard error of the negative (TMR) and positive (TMR+sunflower oil) controls, and percentage of variation caused by each tannin treatment with respect to the positive control
cis-9 18:1 cis-11 18:1 cis-12 18:1 cis-16 18:1
TMR 1.13±0.260 0.21±0.022 0.10±0.016 0.04±0.002
TMR+sunflower oil 2.13±0.162* 0.31±0.013* 0.22±0.007* 0.05±0.003*
+ Quebracho 2% -1.1 ns 3.3 ns -10.7 ns 4.6 ns
The level of significance of each percentage of variation (tannin treatment vs. positive control) appears on its right: ns= non-significant (P>0,10) and * = P<0,05.
Table 3. Content (mg/g digesta) of some trans 18:1 fatty acids in the ruminal digesta.
Mean and standard error of the negative (TMR) and positive (TMR+sunflower oil) controls, and percentage of variation caused by each tannin treatment with respect to the positive control TMR 0.14±0.018 0.12±0.012 0.16±0.015 1.80±0.298 0.27±0.028 TMR+sunflower oil 0.20±0.010** 0.21±0.008** 0.34±0.021** 2.63±0.223** 0.36±0.008**
+ Quebracho 2% -3.4 ns -2.0 ns 0.4 ns 0.4 ns -2.0 ns
The level of significance of each percentage of variation (tannin treatment vs. positive control) appears on its right: ns= non-significant (P>0,10); * = P<0,10 and ** = P<0,05.
In general, the effects on the ruminal biohydrogenation of dietary polyunsaturated FA varied remarkably depending on the dose and type of tannin, which would probably be accounted for by the great diversity in the structural features, and consequently in the reactivity, of different tannins (Mueller-Harvey, 2006). Most of the significant results were observed in treatments with hydrolysable tannin extracts (chestnut at 2 and 5% and oak at 5%).
Feeding and management strategies to improve livestock
productivity, welfare and product quality under climate change
27
These large variations in the effect of different tannins would explain the controversy in the literature surrounding the use of these phenolic compounds to modulate the fatty acid profile of ruminant-derived products (Vasta and Luciano, 2011).
IV – Conclusion
Overall, the results of this study do not allow to recommend the treatment of a diet enriched in linoleic acid (namely, supplemented with 2% of sunflower oil) with tannin extracts when the ultimate goal is to modulate ruminal BH in order to improve the nutritional quality of sheep-derived products.
Acknowledgments
This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO;
AGL2011-23700). H. Benhissi was granted a fellowship from the IAMZ-CIHEAM.
References
Goering M.K. and Van Soest P.J., 1970. Forage fiber analysis (apparatus, reagents, procedures and some applications). In: Agriculture Handbook No 379. USDA, Washington, DC, USA.
Hervás G., Luna P., Mantecón A.R., Castañares N., de la Fuente M.A., Juárez M. and Frutos P., 2008.
Effect of diet supplementation with sunflower oil on milk production, fatty acid profile and ruminal fermentation in lactating dairy ewes. In: J. Dairy Res., 75, p. 399-405.
Jenkins T.C., Wallace R.J., Moate P.J. and Mosley E.E., 2008. Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem. In: J. Anim. Sci., 86, p. 397-412.
Khiaosa-ard R., Bryner S.F., Scheeder M.R.L., Wettstein H.R., Leiber F., Kreuzer M. and Soliva C.R., 2009. Evidence for the inhibition of the terminal step of ruminal alpha-linolenic acid biohydrogenation by condensed tannins. In: J. Dairy Sci., 92, p. 177-188.
Kronberg S., Scholljegerdes E., Barcelo-Coblijn G. and Murphy E., 2007. Flaxseed treatments to reduce biohydrogenation of alpha-linolenic acid by rumen microbes in cattle. In: Lipids, 42, 1105-1111.
Mueller-Harvey I., 2006. Unravelling the conundrum of tannins in animal nutrition and health. In: J. Sci.
Food Agric., 86, p. 2010-2037.
Shingfield K.J., Ahvenjärvi S., Toivonen V., Äröla A., Nurmela K.V.V., Huhtanen P. and Griinari J.M., 2003. Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows. In:
Anim. Sci., 77, p. 165-179.
Shingfield K.J., Ahvenjärvi S., Toivonen V., Vanhatalo A., Huhtanen P. and Griinari J.M., 2008. Effect of incremental levels of sunflower-seed oil in the diet on ruminal lipid metabolism in lactating cows. In:
Br. J. Nutr., 99, p. 971-983.
Shingfield K.J., Reynolds C.K., Hervás G., Griinari J.M., Grandison A.S. and Beever D.E., 2006.
Examination of the persistency of milk fatty acid composition responses to fish oil and sunflower oil in the diet of dairy cows. In: J. Dairy Sci., 89, p. 714-732.
Toral P.G., Belenguer A., Shingfield K.J., Hervás G., Toivonen V. and Frutos P., 2012. Fatty acid composition and bacterial community changes in the rumen fluid of lactating sheep fed sunflower oil plus incremental levels of marine algae. In: J. Dairy Sci., 95, p. 794-806.
Toral P.G., Shingfield K.J., Hervás G., Toivonen V. and Frutos P., 2010. Effect of fish oil and sunflower oil on rumen fermentation characteristics and fatty acid composition of digesta in ewes fed a high concentrate diet. In: J. Dairy Sci., 93, p. 4804-4817.
Vasta V., Makkar H.P.S., Mele M. and Priolo A., 2009. Ruminal biohydrogenation as affected by tannins in vitro. In: Br. J. Nutr., 102, p. 82-92.
Vasta V. and Luciano G., 2011. The effects of dietary consumption of plants secondary compounds on small ruminants’ products quality. In: Small Rum. Res., 101, p. 150-159
Options Méditerranéennes, A no. 107, 2013 – Feeding and management strategies
to improve livestock productivity, welfare and product quality under climate change