Accepted Manuscript Title: Effects of clays used as oil adsorbents in lamb diets on fatty acid composition of abomasal digesta and meat Author: Maria A. Oliveira Susana P. Alves Jos´e Santos-Silva Rui J.B. Bessa PII: DOI: Reference:
S0377-8401(16)30006-2 http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.01.006 ANIFEE 13451
To appear in:
Received date: Revised date: Accepted date:
2-9-2015 8-1-2016 9-1-2016
Please cite this article as: Oliveira, Maria A., Alves, Susana P., Santos-Silva, Jos´e, Bessa, Rui J.B., Effects of clays used as oil adsorbents in lamb diets on fatty acid composition of abomasal digesta and meat.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2016.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of clays used as oil adsorbents in lamb diets on fatty acid composition of abomasal digesta and meat
Maria A. Oliveira.a,b, Susana P. Alves a,c, José Santos-Silva c,d, Rui J.B. Bessa a,c*
– Faculdade de Medicina Veterinária, ULisboa, 1300-477 Lisboa, Portugal
– Escola Superior Agrária de Coimbra, Instituto Politécnico de Coimbra, 3045-601
Coimbra, Portugal. c
– CIISA, Centro de Investigação Interdisciplinar em Sanidade Animal, Avenida da
Universidade Técnica, 1300-477 Lisboa, Portugal d
– Unidade Estratégica de Investigação e Serviços em Produção Animal e Saúde
(UEISPSA-INIAV), 2005-048 Vale de Santarém, Portugal
* Corresponding author. Tel: +350 213652871; EM: [email protected]
Highlights Clays (bentonite and vermiculite) were used as oil adsorbents in lamb´s diets. Clays did not affect lamb growth or meat quality traits. Clays did not protect unsaturated fatty acids from rumen biohydrogenation. Clays did not prevent trans-10 18:1 accumulation in abomasal digesta and meat.
Abstract This experiment was designed to test the hypothesis that using clays as vegetable oil adsorbent results in a partial protection of polyunsaturated fatty acids from rumen biohydrogenation (BH). Complementarily, we also hypothesized that the addition of clays to high-concentrate high-oil diets attenuates the trans-10 shift of rumen BH pathways. Therefore, we compared the effect of using bentonite and/or vermiculite as dietary oil (60 g/kg of added sunflower and linseed oil blend, 1:2 v/v) adsorbent on the fatty acid (FA) composition of lamb´s abomasal digesta and meat. For that, we used 40 Merino Branco ram lambs, randomly allocated to 20 slatted boxes. The four established diets resulted from a completely randomized design: C (no clay), B (30 g/kg bentonite), V (30 g/kg vermiculite) and BV (15 g/kg bentonite plus 15 g/kg vermiculite). Pen was considered as the experimental unit. None of the diets affected (P>0.05) animal performance or meat quality traits although BV treatment resulted in higher proportions of high price joints in the carcass (P=0.024) and muscle (P=0.036) and lower proportions of kidney knob channel fat (P=0.014) and total dissectible carcass fat (P=0.046). No differences among diets were observed on rumen volatile FA and protozoa counts. Only minor effects of diets on FA composition of abomasal digesta were observed. Diet B reduced t11-18:1 (P<0.05) while diet BV reduced (P<0.05) t10-18:1 and tended (P=0.054) to increase 18:0. Total BH intermediates tended (P=0.060) to be reduced by diet BV while diet B clearly increased (P<0.001) the t10-18:1/t11-18:1 ratio. No effect was observed in BH of 18:2n6 or 18:3n-3. Treatments had no effect on total meat lipids and the FA profile of meat presented was very similar among treatments. Meat samples from animals from all diets presented high contents of t10-18:1 (≈ 11 g/100g of total FA) and t10,c15-18:2 (≈ 2.0 g/100g of total FA), whereas the contents of t11-18:1 (≈ 0.9 g/100g of total FA) and c9,t11-18:2 (≈ 0.2 g/100g of total FA) were quite low. No effect was observed in 18:2n6, 18:3n-3 and long chain PUFA contents in meat. Our results show that both bentonite and/or vermiculite used as vegetable oil adsorbents in high concentrate based diets were ineffective to protect PUFA from rumen BH as well as to prevent the trans-10 shift. Abbreviations: ADF, acid detergent fibre; B, diet with 30 g/kg of bentonite; BH, biohydrogenation; BV, diet with 15 g/kg of bentonite and 1.5 g/kg of vermiculite; C, control diet; DM, dry matter; FA, fatty acid; NDF, neutral detergent fibre; PUFA, polyunsaturated fatty acid; V, diet with 30 g/kg of vermiculite; VFA, volatile fatty acids. Keywords: Lamb meat, bentonite, vermiculite, trans-10 shift, rumen biohydrogenation
1. Introduction Edible fats from ruminants are characterized by being rich in saturated and trans fatty acids (FA) that have been associated with increased risk of cardiovascular diseases (FAO, 2010), despite the fact that individual saturated and trans FA might differ in its biological effects (Gebauer et al., 2011). Therefore, developing strategies to decrease saturated FA in ruminant products is an important research target. Modification of ruminant’s fat composition through nutrition is possible but is strongly limited by the fact that rumen microbial ecosystem extensively isomerises and hydrogenates dietary polyunsaturated FA (PUFA). During rumen biohydrogenation (BH), conjugated linoleic acid isomers and trans octadecenoates are formed. Some of these BH intermediates, like rumenic acid (c9,t11-18:2) but also vaccenic acid (t11-18:1) possess an anti-carcinogenic effect on several animal and cell culture models (Gebauer et al., 2011; Lim et al., 2014). The most effective way to increase meat c9,t11-18:2 content (the main conjugated linoleic acid isomer) is by supplementing forage based diets with unsaturated vegetable oils (Bessa et al., 2005; Bessa et al., 2015). In those conditions, dietary PUFA are extensively isomerised and partially hydrogenated, with a large accumulation of t11-18:1 that, after being absorbed in the small intestine, is extensively converted to c9,t11-18:2 by the action of delta-9 desaturase enzyme (Bessa et al., 2015). However, intensive animal production often uses diets based on compound feeds rich in starch, leading to modifications in BH pathways that result in an accumulation of t10-18:1 instead of t11-18:1 (i.e. trans-10 shift) (Aldai et al., 2013). As the t10-18:1 isomer cannot be endogenously converted into conjugated linoleic acid isomers, the establishment of the trans-10 shift results in a large accumulation of t10-18:1 and in a low content of c9,t11-18:2 in tissues (Bessa et al., 2015). Furthermore, contrasting with t11-18:1, the t10-18:1 isomer is potentially harmful to consumer’s health (Aldai et al., 2013; Mapiye et al., 2015). Thus, strategies to mitigate the production of t10-18:1 in animals fed diets based on compound feeds need to be developed. Bentonite and vermiculite are three-layer minerals with an expanding lattice belonging to the smectite group (Murray, 2006). The special properties of this group of clay minerals, such as high layer charge, medium to high cation exchange capacity and high surface area, are responsible for their high absorptive capacity and medium to high swelling capacity, making then valuable materials for a wide range of applications in industrial and farming systems. Bentonite has been used extensively to improve the binding quality of feed pellets, but also tentatively as rumen buffering agent to mitigate 3
acidosis and milk fat depression (Bringe and Schultz, 1969). Vermiculite has been used as a fat carrier in order to mitigate the negative impact of fat supplements on rumen digestion (Tamminga et al., 1983; Jenkins and Palmquist, 1984). More recently, Sinclair et al. (2005) reported that using vermiculite as a carrier for linseed oil resulted in partial protection against rumen BH. The putative effects of clays in protecting PUFA from rumen BH might occur due to a slower release of triacylglycerols or through PUFA saponification with Mg ions, released due to its high exchangeable ion capacity. Our team tested the effects of bentonite inclusion in highforage high-oil lamb´s diets and did not detect any major effect on lamb meat FA profile, apart from a significant reduction of t10-18:1 (Jerónimo et al., 2010). In that experiment, besides using only bentonite, all diet ingredients were mixed together and thereafter pelleted. Thus, we hypothesized that, in order to achieve a protective effect against rumen BH, the dietary oil needs to be previously adsorbed into the clays and that vermiculite differs from bentonite regarding to their potential to protect the dietary oil from rumen BH. The classic literature explored the effects of bentonite on mitigation of diet-induced milk fat depression in dairy cows (Bringe and Schultz, 1969; Rindsig et al., 1969). It is now well established, that diet-induced milk fat depression is closely linked to the occurrence of the trans-10 shift in the rumen (Bauman and Griinari, 2003). Moreover, our team had reported that bentonite reduced t10-18:1 concentration in lamb meat (Jerónimo et al. (2010), thus we hypothesized that inclusion of clays into high starch diets might mitigate the trans-10 shift that frequently occurs with those types of diets. Therefore, in the present experiment we compared the effect of using bentonite and/or vermiculite as dietary oil adsorbents on FA composition of lamb´s abomasal digesta and meat.
2. Material and Methods
2.1. Animals and diets The trial was conducted at the facilities of Escola Superior Agrária de Coimbra, Portugal. Animal handling followed EU Directive 86/609/EEC concerning animal care. Forty Merino Branco ram lambs, with an average initial live weight of 12.2 ± 1.61 kg and ageing 60 ± 6.2 d (mean ± SD) were randomly assigned to 20 slatted pens. After one week of adaptation to the experimental conditions, in which lambs were dewormed with 4
Ivomec® (Merial Portuguesa, Portugal), lambs stayed on trial for 45 d. Lambs were weighed at the beginning of the trial and then weekly before the morning feeding without fasting. During the trial, animals had fresh water always available and were fed once a day (at 09:00) with a diet containing 9 parts of a compound feed and 1 part of wheat straw. The four compound feeds used were named as C (no clay), B (30 g/kg of bentonite), V (30 g/kg of vermiculite) and BV (15 g/kg of bentonite + 15 g/kg of vermiculite) and were randomly allocated to the pens following a completely randomized design. Compound feed were produced in a commercial feed mill; the oil blend (linseed and sunflower oil, 2:1) was mixed with clays and then added to the compound feed in a vertical mixer before being pelleted (3 mm diameter, cold pelleting with no steam added). Ingredients and chemical composition of the feeds used in this trial are presented on Table 1. Straw and concentrate were given separately and the amounts offered and refused were recorded daily. Diets were provided ad libitum (110% of the consumption of the day before).
2.2. Sample collection After 45 d of trial, lambs were transported to the INIAV experimental abattoir to be slaughtered. Slaughter was performed in two consecutive days with half of animals of each treatment slaughter in each day. Animals arrived 24 h before the first slaughter day and were housed in 4 pens according to its allocation to the treatments and continue to be fed with the previous diet. At the day of slaughter, lambs were weighed and slaughtered without fasting by stunning and exsanguination. Samples of abomasum content (≈ 50 mL) were collected and frozen, freeze-dried and stored at -20ºC until further analysis. Rumen fluid (about 500 mL) was immediately collected and strained through 4 layers of cheesecloth. Subsamples of 4 mL were transferred to test tubes containing 80 L of MgCl2 (saturated solution) to be analysed for volatile FA (VFA) and immediately stored at -20ºC. For protozoa counting, subsamples of 4 mL were transferred to test tubes containing 4 mL of formaldehyde and stored in the dark at 4ºC until enumeration procedures. The carcasses were weighed in order to obtain hot carcass weight and cooled at 10ºC for 24 hours, and weighed again to obtain cold carcass weight. Carcasses were then refrigerated for 48 h at 2ºC. At the third day after slaughter kidney knob channel fat and kidneys were removed, weighed and the carcasses were cut in halves along the spine. The left side of each carcass 5
was separated in 8 joints according to Santos-Silva et al. (2002) and used for further carcass and meat quality traits analysis. The weight of each joint was registered and used to estimate the proportion of high price joints (leg + chump + loin + ribs). Chumps and shoulders were packed in vacuum, refrigerated at 0ºC and dissected into muscle, bone, subcutaneous and intermuscular fat in a maximum period of 48 h. The rib joints of the left half of the carcasses, containing the Longissimus thoracis muscle, were vacuumpacked and frozen at −20°C until shear force determinations. The Longissimus lumborum muscle was removed from loin joints of the left halves of the carcasses and, after the removal of the epimysium, was minced with a food processor (3 × 5 s), vacuum-packed, freeze-dried, and stored at −80°C until further fatty acid analysis. Colour of the left longissimus thoracis muscle was determined after 1 hour of exposure to air to allow blooming at the left 13th thoracic vertebra using a Minolta CR-300 (Konica Minolta, Portugal) chromometer through the system L* (lightness), a* (redness) and b* (yellowness). Meat shear force was determined in left longissimus thoracis muscle samples following the procedures described by Bessa et al. (2005), and using a WarnerBratzler shear device, mounted in a Texture Analyser (TA-XT2i) (TA-tx2i Texture Analyser, Stable Micro Systems, Surrey, UK).
2.3. Analytical methods Samples from feed distributed to lambs, were collected weekly (± 1 kg), pooled and a subsample (± 1 kg) grounded (1 mm). Feed samples were analysed for dry matter (ISO 6496, 1999), crude ash (ISO 5984, 2002), crude protein (ISO 5983, 1997), starch (Clegg, 1956) and acid detergent fibre (ADF) (Van Soest et al., 1991). Neutral detergent fibre (NDF) was determined using the procedure described by Mertens (2002), with no added sodium sulphite or α-amylase, and the results were expressed including residual ash. Fatty acid methyl esters from feed lipids were prepared by one-step extraction transesterification using toluene, as described by Sukhija and Palmquist (1988). Abomasal digesta samples were lyophilized and FA methyl esters prepared by 2-step methylation procedure using sodium methoxide and methanolic HCl, adapted from Jenkins (2010). Nonadecanoic acid (19:0) was added at the start of methylation and used as internal standard. Total lipids from lyophilized meat samples were extracted using dichloromethane and methanol (2:1, v/v). Total lipids were measured gravimetrically, in duplicate, weighing the residue after evaporation of solvents in a vacuum oven at 37oC. Extracted meat lipid were converted to fatty acid methyl esters using sodium methoxide 6
in anhydrous methanol (0.5 mol/L) followed by hydrochloric acid in methanol (1:1, v/v). Nonadecanoic acid (19:0) was used as internal standard. Fatty acid methyl esters from feeds, meat and abomasum samples were then analyzed by gas chromatography using a Shimadzu 2010Plus (Shimadzu, Kyoto, Japan), equipped with a flame-ionization detector a fused silica capillary column (TR-CN100, 100 m, 0.2 mm internal diameter, and 0.20 µm ﬁlm thickness; Teknokroma, Barcelona, Spain). The injector and detector temperatures were maintained at 250°C. The initial oven temperature of 50°C was held for 1 min, increased at 50°C/min to 150°C and held for 20 min, increased at 1°C/min to 190°C and then increased at 2°C/min to 220°C and held for 18 min. Helium was used as carrier gas at a flow rate of 1 mL/min and 1 L of sample was injected. In addition, samples were run using a SLB-IL111 capillary column (Supelco Inc., Bellefont, PA, USA) in order to separate the t10,c15-18:2 from the t11,c15-18:2 as described by Alves and Bessa (2014) and the c9,t11-18:2 from t7,c9-18:2 as described by Turner et al. (2011). Moreover, identification of FA methyl esters was achieved by comparison of their retention times with those of commercial standard mixtures (FAME mix 37 components from Supelco Inc., Bellefont, PA, USA) and by electron impact mass spectrometry using a Shimadzu GC-MS QP2010 Plus (Shimadzu, Kyoto, Japan). Strained rumen fluid samples (1 mL) used for VFA analysis, were prepared by adding 170 L of orthophosphoric acid solution (25/100, v/v) and 130 L of internal standard (iso-6:0 at 50 mmol/L) and then centrifuge at 15000 g for 15 min. The supernatant was analysed for VFA using a gas chromatograph Shimadzu 2010Plus (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and fused capillary column (Nukol, Supelco, Bellefonte, PA, USA) with 30 m, 0.25 mm internal diameter and 0.25 µm film thickness. Helium was the carrier gas and the split ratio was 1:50. The injector and detector temperatures were 250oC and 280oC, respectively. The column operated isothermally at 180ºC for 10 min. Volatile FA were quantified using calibration curves, which were prepared for each VFA at concentrations ranging from 0.2 to 30 mmol/L and using iso-6:0 as internal standard at 5 mmol/L. Rumen protozoa were counted using an optical microscope and a Neubauer improved counting chamber after adding 100 L of brilliant green dye to the preserved rumen fluid and allowed to stand for one hour. The numbers of Entodiniomorphida and Holotricha were recorded.
2.4. Calculations and statistical analysis The BH estimates for 18:2n-6 and 18:2n-3 were obtained using the diminishing abundance of these FA, proportional to the sum of 18-carbon FA, between diet and abomasal digesta, assuming that no losses of C18 FA occur in the gastric compartments (Fievez et al., 2007). The calculations, exemplified here for 18:2n-6 BH, were as follows: BH18:2n-6 (g/100g) = (18:2n-6 Diet –18:2n-6Abomasum)/ 18:2n-6 Diet ×100 Where, - BH18:2n-6 (g/100g) is the estimate of the disappearance of 18:2n-6 between the diet and the abomasal digesta (i.e. BH); - 18:2n-6Diet is the 18:2n-6 in diet as g/100g of total C18 FA; - 18:2n-6Abomasum is the 18:2n-6 in abomasal digesta as g/100g of total C18 FA. Data were analysed using MIXED procedure from SAS 9.4 software (SAS Institute Inc., Cary, NC). Pen was considered as experimental unit and lambs as sampling units within the pens with a compound symmetry covariance structure. Diet was considered as fixed effect. Least square means were estimated and when a significant (P<0.05) diet effect was detected, the differences between means were determined using the PDIFF adjusted by Tukey option. The protozoa counts data were analysed using a similar model but with the GLIMMIX procedure from SAS 9.4 software (SAS Institute Inc., Cary, NC) using the negative binomial distribution and the log link function.
3. Results 3.1. Feed intake, productive performance, carcass traits and meat quality traits Feed intake and productive performance data as well as carcass and meat quality traits data are presented in Table 2. Lambs fed the control diet tended (P=0.10) to had lower compound feed intake than the ones fed with clay diets. This resulted in slightly but significantly lower (P<0.05) NDF intake in C compared with BV treatments. The control compound feed contained more fatty acids than all the clay containing compound feeds, although no significant differences were observed in total FA intake among diets. No differences were observed (P>0.10) on average daily gain (252 g/d), feed conversion ratio (2.9 kg of DM intake/kg of live weight gain), live slaughter weight (23.2 kg) and hot carcass weight (10.9 kg). On the contrary, treatments had impact on carcass quality traits (Table 2) with a significant (P<0.05) increase in high priced joints in lambs fed with BV diet compared with those fed with B diet, mainly due to a trend for a higher proportion of ribs (data not shown). Lambs fed with BV diet also had higher (P < 0.05) 8
muscle proportions in the chumps+shoulders than lambs fed C diet. Fat partition was also affected by diets, with lambs fed BV diet having lower (P<0.05) kidney knob channel fat than those fed with C and B diets, and lower (P<0.05) dissectible fat compared to those fed with the C diet. The B and V diets resulted in intermediate results between control and BV diets for muscle and dissectible fat. No differences were observed between treatments in shear force or colour parameters measured in longissimus muscle.
3.2. Rumen fermentation and protozoa counts Volatile fatty acid concentration averaged 20.9 ± 2.80 mmol/L and did not differ between treatments. Molar proportions of major VFA (mol/100 mol) also did not differ between treatments, with acetic, propionic, isobutyric, butyric, isovaleric and valeric acids comprising 41.3 ± 0.97, 30.6 ± 1.36, 5.8 ± 0.40, 14.2 ± 0.87, 3.2 ± 0.26 and 5.0 ± 0.33, respectively. The protozoa counts in rumen contents were also not affected by treatments, with an average count per ml of 4.88×105 ± 0.859×105 holotrich protozoa and 3.54×106 ± 0.393×106 entodiniomorphs protozoa.
3.3. Fatty acid composition of abomasal digesta Total FA concentration and FA composition of abomasal digesta from lambs fed the experimental diets are presented in Table 3. The concentration of FA in abomasal digesta averaged 57.6 mg/g DM and did not differ between diets. Only a few FAs were affected by treatments. For lambs fed with Control, B and V diets, the major FA in abomasal digesta was t10-18:1 (≈ 30 g/100g of total FA) followed by 18:0 (≈ 22 g/100g of total FA), whereas for lambs fed with BV diet the major FA was 18:0 (34.2 g/100g of total FA) followed by t10-18:1 (20.1 g/100g of total FA). The proportions of 18:2n-6 and 18:3n-3 were notably constant throughout treatments, averaging 5.5 and 2.6 g/100g of total FA, respectively. Compared to diet V, control diet decreased (P<0.05) 14:0 and diet B decreased 16:0. Diet B reduced t11-18:1, compared to control diet. Diet BV reduced (P<0.05) t10-18:1, compared to C and B diets, and tended (P=0.054) to increase 18:0, compared to the other diets. Moreover, diet BV decreased (P<0.05) c9-18:1, compared to diet V, and c11-18:1, compared to control diet. The c9,t11-18:2 was very low and not detectable in the 13 of the 40 samples.
Total BH intermediates tended (P=0.060) to be reduced by diet BV, compared to the other diets, and diet B clearly increased (P<0.001) t10/t11 ratio compared to the other diets.
3.4. Total lipids and fatty acid composition of meat Lipid content and FA composition of meat presented no differences between treatments, except for 3 minor FA (anteiso-15:0, c9-14:1 and 20:0) that presented slightly but significant (P<0.05) differences. Thus, in Table 4 the general means and SD of the forty lambs are presented. Quantitatively, the main FA were c9-18:1, 16:0 and 18:0, averaging across diets 23.6, 19.8 and 13.6 g/100g of total FA, respectively. Meat samples from animals from all diets presented very large contents of t10-18:1 (≈ 10.7 g/100g of total FA) and t10,c15-18:2 (≈ 2.0 g/100g of total FA), whereas the contents of t11-18:1 (≈ 0.89 g/100g of total FA) and the c9,t11-/t7,c9-18:2 (≈ 0.21 g/100g of total FA) were quite low. The c9,t11-18:2 comprises about 60 to 80% of the common c9,t11-/t7,c9-18:2 peak as confirmed by complementary GC analysis with an ionic capillary column which means that c9,t11-18:2 concentration is estimated to be as low as 0.15 g/100g of total FA. No effect was observed on the 18:2n-6, 18:3n-3 and on the very long chain PUFA, but the high content of 18:3n-3 (≈2 g/100g of total FA) clearly reflects the fact that animals were fed an oil blend containing linseed oil.
4. Discussion The present results clearly show that both bentonite and vermiculite, used as vegetable oil adsorbents in high concentrate diets, were ineffective to protect PUFA from rumen BH as well as to prevent the trans-10 shift. In fact, none of the data presented here points to a reduction on 18:2n-6 and 18:3n-3 rumen BH, as no differences between treatments for these FA were detected in abomasal digesta or in meat. The FA flow to abomasum was not measured, which prevents a definite rumen balance, but if oil adsorption to clays resulted in any degree of protection against PUFA’s BH it should have increased the proportion of C18 PUFA in total FA present in abomasal digesta and, ultimately, in meat FA. As discussed by Fievez et al. (2007), the BH of C18 PUFA can be estimated, with fairly good accuracy, from 18:2n-6 and 18:3n-3 concentrations present in diet and in abomasal digesta, assuming a neutral rumen balance of C18 FA. Our BH estimates for 18:2n-6 and 18:3n-3 showed no effect of treatments, confirming our previous results with bentonite incorporated in high forage high oil diets fed to lambs 10
(Jerónimo et al., 2010) but contrast with the results from Sinclair et al. (2005) that found a protective effect of vermiculite against rumen BH of the C18 PUFA from linseed oil. Despite Sinclair et al. (2005) findings, that adsorption of linseed oil to vermiculite resulted in a higher flow of 18:3n-3 to the duodenum, they did not detect an increased concentration of 18:3n-3 in lamb´s blood plasma, suggesting that absorption might be diminished. We did not detect any difference between treatments in 18:2n-6 and 18:3n-3 concentrations in meat. Moreover, clay containing diets presented lower total FA concentrations than the control diet, but total FA concentrations in abomasal digesta were similar among treatments. The lower FA content in clay diets probably results from oil losses due to the additional step of mixing oil with clay powder before being mixed with the other feed ingredients. Thus, adsorption of FA to clays did not protect dietary FA from BH and no indication of lower post-absorptive PUFA availability could be detected. The other hypothesis tested was that clays might attenuate the occurrence of a trans10 shift in the rumen of lambs fed high oil, high concentrate diets. Data presented here clearly refute that hypothesis. All lambs in all treatments exhibit a profound trans-10 shift, evaluated by the concentrations of t10-18:1 and t11-18:1 in abomasal digesta and in meat. The amount of t10-18:1 in abomasal digesta was quite variable, ranging from 3 to 48 g/100g of total FA. However, this variability was not linked to increases in t11-18:1, which remained always low, but with the amount of 18:0 as displayed in Figure 1. The lower mean value of t10-18:1 observed in abomasum digesta with BV treatment is just a reflex of a more completeness of BH process, with a larger 18:0 production, rather than to an increase in t11-18:1 production. Incomplete BH patterns, with large accumulation of BH intermediates, has been interpreted as a stress response of rumen microbial ecosystem to low pH or high PUFA concentrations in the rumen (Bessa et al., 2000) or as the toxic effect of these factors on the microbial community specialized in the formation of stearic acid (Maia et al., 2007). As PUFA concentration in clay diets did not differ, the more completeness of BH observed with BV could be linked to a buffer effect, as dietary buffers supplementation can reduce the amount of BH intermediates (Kalscheur et al., 1997; Cabrita et al., 2009). However, it is not clear why the two clays combined promoted the completeness of BH and when fed isolated, did not. Our data on rumen pH were collected just after slaughter and, therefore, did not reflect what would have been the circadian pH variation. The differences on rumen BH patterns observed with BV treatment in the abomasal digesta are not clearly reflected in meat FA profile. This suggests that the effect of BV 11
treatment on BH pattern was not sustained during the 6 weeks of the experiment, as the BH intermediates deposited in meat lipids must reflect the sustained rumen conditions during the whole finishing period. The occurrence of the trans-10 shift in ruminants has been more clearly associated with the dietary supply of 18:2n-6 rather than with the dietary supply of 18:3n-3 (Zened et al., 2011; Aldai et al., 2013). The trans-10 shifted pathways for 18:3n-3 BH are not well established as recently discussed by Alves and Bessa (2014) but the large accumulation of both t10-18:1 and t10,c15-18:2 observed in abomasal digesta and in meat lipids strongly suggests that dietary 18:3n-3 was extensively biohydrogenated through a trans-10 shifted pathway. As extensively discussed by Bessa et al. (2015), ruminants exhibiting such a clear trans-10 shift as observed here, present a very low content of c9,t11-18:2 in meat. Nutritional strategies to modulate meat FA composition in ruminants may have implications on animal productive performance and meat quality traits. The results presented here do not show any significant change on animal performance or on meat quality between diets, but point to a higher proportion of muscle in shoulder+chump and a lower dissectible fat accumulation in the carcass with diet BV. It is not clear why the combination of both clays might alter the composition of body weight gain by promoting the protein deposition in detriment of fat deposition. Comparable results were reported by Khadem et al. (2007) with lambs fed bentonite displaying higher longissimus muscle area and reduced subcutaneous fat thickness and fat-tail depot than control lambs. To our knowledge, no similar work was performed with vermiculite. Bentonite has been, although inconsistently, associated with increased wool growth in sheep (Murray et al., 1992) supposedly by affecting the rumen protozoa population and thus increasing the microbial N flow to the intestine (Ivan et al., 1992). It is doubtful that this type of explanation can be applied to our results, as we do not detect any effects of clays on protozoa counts in the rumen, and as bentonite and vermiculite alone had no effect of muscle and fat composition of the carcass. 5. Conclusions Adding clays as oil adsorbents to high concentrate diets supplemented with vegetable oils do not protect the PUFA from rumen BH nor prevented trans-10 shifted BH pathways in lambs.
Conflict of interest The authors declared that there is no conflict of interests.
Acknowledgments The authors would like to thank the staff of Unidade Estratégica de Investigação e Serviços em Produção Animal e Saúde (UEISPSA-INIAV), particularly Paula Santos for her cooperation in slaughter, carcass and meat physical quality traits procedures and to José Batista for the assistance in the feed analytical determinations. The authors would also like to thank Fundação para a Ciência e a Tecnologia (FCT) through research grants to MAO (SFRH/PROTEC/67566/2010), SPA (SFRH/BPD/76836/2011) and through the PTDC/CVT/120122/2010 and UID/CVT/00276/2013 projects
Aldai, N., de Renobales, M., Barron, L.J.R., Kramer, J.K.G., 2013. What are the trans fatty acids issues in foods after discontinuation of industrially produced trans fats? Ruminant products, vegetable oils, and synthetic supplements. Eur. J. Lipid Sci. Technol. 115, 1378-1401. doi: 10.1002/ejlt.201300072 Alves, S.P., Bessa, R.J.B., 2014. The trans-10,cis-15 18:2: A missing intermediate of trans-10 shifted rumen biohydrogenation pathway? Lipids 49, 527-541. doi: 10.1007/s11745-014-3897-4 Bauman, D.E., Griinari, J.M., 2003. Nutritional regulation of milk fat synthesis. Annu. Rev. Nutr. 23, 203-227. doi: 10.1146/annurev.nutr.23.011702.073408 Bessa, R.J.B., Alves, S.P., Santos-Silva, J., 2015. Constraints and potentials for the nutritional modulation of the fatty acid composition of ruminant meat. Eur. J. Lipid Sci. Technol. 177, 1325-1344. doi: 10.1002/ejlt.201400468 Bessa, R.J.B., Portugal, P.V., Mendes, I.A., Santos-Silva, J., 2005. Effect of lipid supplementation on growth performance, carcass and meat quality and fatty acid composition of intramuscular lipids of lambs fed dehydrated lucerne or concentrate. Livest. Prod. Sci. 96, 185-194. doi: 10.1016/j.livprodsci.2005.01.017 Bessa, R.J.B., Santos-Silva, J., Ribeiro, J.M.R., Portugal, A.V., 2000. Reticulo-rumen biohydrogenation and the enrichment of ruminant edible products with linoleic acid conjugated isomers. Livest. Prod. Sci. 63, 201-211. doi: 10.1016/S03016226(99)00117-7 Bringe, A.N., Schultz, L.H., 1969. Effects of roughage type or added bentonite in maintaining fat test. J. Dairy Sci. 52, 465-471. doi: 10.3168/jds.S00220302(69)86589-6 Cabrita, A.R.J., Vale, J.M.P., Bessa, R.J.B., Dewhurst, R.J., Fonseca, A.J.M., 2009. Effects of dietary starch source and buffers on milk responses and rumen fatty
acid biohydrogenation in dairy cows fed maize silage-based diets. Anim. Feed Sci. Technol. 152, 267-277. doi: 10.1016/j.anifeedsci.2009.04.020 Clegg, K.M., 1956. The application of the anthrone reagent to the estimation of starch in cereals. J. Sci. Food Agric. 7, 40-44. doi: 10.1002/jsfa.2740070108 FAO, 2010. Fats and fatty acids in human nutrition (FAO report of an expert consultation). FAO, Rome. Fievez, V., Vlaeminck, B., Jenkins, T., Enjalbert, F., Doreau, M., 2007. Assessing rumen biohydrogenation and its manipulation in vivo, in vitro and in situ. Eur. J. Lipid Sci. Technol. 109, 740-756. doi: 10.1002/ejlt.200700033 Gebauer, S.K., Chardigny, J.M., Jakobsen, M.U., Lamarche, B., Lock, A.L., Proctor, S.D., Baer, D.J., 2011. Effects of ruminant trans fatty acids on cardiovascular disease and cancer: A comprehensive review of epidemiological, clinical, and mechanistic studies. Adv. Nutr. 2, 332-354. doi: 10.3945/an.111.000521 ISO 5983, 1997. Animal feeding stuffs - Determination of nitrogen content and calculation of crude protein content- Kjeldhal method. International Organization for Standardization, Geneva, Switzerland. ISO 5984, 2002. Animal feeding stuffs - Determination of crude ash. International Organization for Standardization, Geneva, Switzerland. ISO 6496, 1999. Animal feeding stuffs - Determination of moisture and other volatile matter content. International Organization for Standardization, Geneva, Switzerland. Ivan, M., Dayrell, M.D., Mahadevan, S., Hidiroglou, M., 1992. Effects of bentonite on wool growth and nitrogen-metabolism in fauna-free and faunated sheep. J. Anim. Sci. 70, 3194-3202. doi: 1992.70103194x
Jenkins, T.C., 2010. Technical note: Common analytical errors yielding inaccurate results during analysis of fatty acids in feed and digesta samples. J. Dairy Sci. 93, 11701174. doi: 10.3168/jds.2009-2509 Jenkins, T.C., Palmquist, D.L., 1984. Effect of fatty acids or calcium soaps on rumen and total nutrient digestibility of dairy rations. J. Dairy Sci. 67, 978-986. doi: 10.3168/jds.S0022-0302(84)81396-X Jerónimo, E., Alves, S.P., Martins, S.V., Prates, J.A.M., Bessa, R.J.B., Santos-Silva, J., 2010. Effect of sodium bentonite and vegetable oil blend supplementation on growth, carcass quality and intramuscular fatty acid composition of lambs. Anim. Feed Sci. Technol. 158, 136-145. doi: 10.1016/j.anifeedsci.2010.04.010 Kalscheur, K.F., Teter, B.B., Piperova, L.S., Erdman, R.A., 1997. Effect of dietary forage concentration and buffer addition on duodenal flow of trans-C18:1 fatty acids and milk fat production in dairy cows. J. Dairy Sci. 80, 2104-2114. doi: 10.3168/jds.S0022-0302(97)76156-3 Khadem, A.A., Soofizadeh, M., Afzalzadeh, A., 2007. Productivity, blood metabolites and carcass characteristics of fattening Zandi lambs fed sodium bentonite supplemented total mixed rations. Pak. J. Biol. Sci. 10, 3613-3619. doi: 10.3923/pjbs.2007.3613.3619 Lim, J.N., Oh, J.J., Wang, T., Lee, J.S., Kim, S.H., Kim, Y.J., Lee, H.G., 2014. trans-11 18:1 vaccenic acid (TVA) has a direct anti-carcinogenic effect on MCF-7 Human mammary adenocarcinoma cells. Nutrients 6, 627-636. doi: 10.3390/nu6020627 Maia, M.R.G., Chaudhary, L.C., Figueres, L., Wallace, R.J., 2007. Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen. Antonie Van Leeuwenhoek 91, 303-314. doi: 10.1007/s10482-006-9118-2 Mapiye, C., Vahmani, P., Mlambo, V., Muchenje, V., Dzama, K., Hoffman, L.C., Dugan, M.E.R., 2015. The trans-octadecenoic fatty acid profile of beef: Implications for global food and nutrition security. Food Res. Int. 76, 992-1000. doi: 10.1016/j.foodres.2015.05.001 16
Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: Collaborative study. J AOAC Int. 85, 1217-1240. Murray, H.H., 2006. Applied clay mineralogy. Elsevier Science, New York. Murray, P.J., Winslow, S.G., Rowe, J.B., 1992. Effect of dry or hydrated bentonite on the wool growth and liveweight gain of sheep fed wheat chaff. Aust. J. Exp. Agric. 32, 595-600. doi: 10.1071/ea9920595 Rindsig, R.B., Schultz, L.H., Shook, G.E., 1969. Effects of addition of bentonite to highgrain dairy rations which depress milk fat percentage. J. Dairy Sci. 52, 1770-1775. doi: 10.3168/jds.S0022-0302(69)86839-6 Santos-Silva, J., Mendes, I.A., Bessa, R.J.B., 2002. The effect of genotype, feeding system and slaughter weight on the quality of light lambs - 1. Growth, carcass composition and meat quality. Livest. Prod. Sci. 76, 17-25. doi: 10.1016/S03016226(01)00334-7 Sinclair, L.A., Cooper, S.L., Chikunya, S., Wilkinson, R.G., Hallett, K.G., Enser, M., Wood, J.D., 2005. Biohydrogenation of n-3 polyunsaturated fatty acids in the rumen and their effects on microbial metabolism and plasma fatty acid concentrations in sheep. Anim. Sci. 81, 239-248. doi: 10.1079/ASC50040239 Sukhija, P.S., Palmquist, D.L., 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36, 12021206. doi: 10.1021/Jf00084a019 Tamminga, S., Vanvuuren, A.M., Vanderkoelen, C.J., Khattab, H.M., Vangils, L.G.M., 1983. Further studies on the effect of fat supplementation of concentrates fed to lactating dairy cows. 3. Effect on rumen fermentation and site of digestion of dietary components. Neth. J. Agric. Sci. 31, 249-258. Turner, T., Rolland, D.C., Aldai, N., Dugan, M.E.R., 2011. Rapid separation of cis9, trans11- and trans7,cis9-18:2 (CLA) isomers from ruminant tissue using a 30 m 17
SLB-IL111 ionic column. Can. J. Anim. Sci. 91, 711-713. doi: 10.4141/Cjas2011071 Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597. doi: 10.3168/jds.S0022-0302(91)78551-2 Zened, A., Troegeler-Meynadier, A., Nicot, M.C., Combes, S., Cauquil, L., Farizon, Y., Enjalbert, F., 2011. Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids. J. Dairy Sci. 94, 5634-5645. doi: 10.3168/jds.2011-4491
Figure 1 - Relationship between t10-18:1 and 18:0 (g/100 g of total fatty acids) in abomasal digesta.
30 20 10 0 0
Table 1 – Ingredients (g/kg feed), chemical composition (g/kg DM) and fatty acid composition (g/kg DM) of the experimental compound feeds and of the wheat straw. Compound feeds1 C B Ingredients Wheat 400 388 Corn 52 49 Corn DDGS 155 150 Wheat bran 35 33 Barley sprouts 103 100 Palm kernel meal 103 100 Soybean meal 46 44 2 Others 46 46 3 Oil blend 60 60 Bentonite4 0 30 5 Vermiculite 0 0 Chemical composition Dry matter (DM) 854 867 Crude ash 54 83 Crude protein 151 147 NDF 206 203 ADF 90 103 Starch 353 303 Fatty acids 68 55 Fatty acid (g/kg DM) 14:0 0.64 0.49 16:0 4.85 3.81 18:0 2.18 1.73 c9-18:1 11.8 9.34 c11-18:1 0.41 0.32 18:2n-6 21.9 17.2 18:3n-3 16.2 12.9 1 C, no added clay; B, 30 g/kg of bentonite; V, 30 g/kg
388 49 150 33 100 100 44 46 60 0 30
388 49 150 33 100 100 44 46 60 15 15
859 83 148 200 102 317 54
855 83 147 239 99 321 58
866 54 24 802 500 3 18.2
0.47 0.52 3.66 4.00 1.67 1.82 8.98 9.80 0.30 0.34 16.6 18.1 12.4 13.6 of vermiculite; BV,
0.22 0.84 0.22 0.07 0.29 0.10 0.08 15 g/kg of
bentonite plus 15 g/kg of vermiculite. 2
Others - all compound feeds had sugar cane syrup=20.5 g/kg, Calcium carbonate=13.3 g/kg, Sodium bicarbonate=5.1 g/kg, Salt=4.1 g/kg, Minerals and vitamins=3.0 g/kg;
Oil blend = 2 parts of sunflower oil + 4 parts of linseed oil;
Bentonite – 582 g/kg SiO2 + 336 g/kg Al2O3 + 47 g/kg Fe2O3 + 2 g/kg CaO + 6 g/kg MgO + 1 g/kg Na2O + 18 g/kg K2O + 8 g/kg TiO2 + 1 g/kg P2O5;
Vermiculite – 423 g/kg SiO2 + 131 g/kg Al2O3 + 96 g/kg Fe2O3 + 30 g/kg CaO + 257 g/kg MgO + 35 g/kg K2O + 20 g/kg TiO2 + 1 g/kg others non identified.
Table 2 – Feed intake, animal performance, carcass and meat quality traits of lambs fed control diet (C, no clay added) or diets with bentonite (B), vermiculite (V) and both clays (BV) used as oil absorbent.
Dry matter intake (g/d) Compound feed Straw Total Nutrient intake (g/d) Crude protein NDF ADF Fatty acids Starch Slaughter weight (kg) Average daily gain (g/d) Feed conversion1 Carcass quality traits Hot carcass weight (kg) Dressing proportion High price joints3 Kidney knob channel fat 2 Chumps + Shoulders 3 Muscle Bone Intermuscular fat Subcutaneous fat Total dissectible fat 4
Treatments C B
589 65 655
696 61 757
682 58 740
90 173b 86 48 208 22.4 234 2.8
104 188ab 101 41 207 23.5 265 2.9
103 181ab 98 40 214 23.9 265 2.8
45.9 0.57b 4.4a 45.9
46.7 0.57b 4.4ab 46.7
47.3 0.58ab 3.7ab 47.3
57.7b 21.5 10.4 10.4
57.7b 22.5 9.8 9.9
663 65 728
30.3 3.8 31.5
0.10 0.49 0.15
99 212a 97 43 212 23.0 244 3.0
4.5 7.8 3.9 2.1 9.6 0.88 13.3 0.08
0.16 0.016 0.07 0.44 0.95 0.66 0.27 0.43
47.1 0.59a 3.4b 47.1
0.51 0.009 0.42 0.22
0.58 0.70 0.036 0.014
58.5ab 22.7 9.1 9.8
60.8a 22.2 8.9 8.1
0.77 0.57 0.55 0.76
0.036 0.56 0.23 0.19
Meat quality traits Shear force (kg/cm2) 7.0 7.1 6.6 6.8 0.38 0.74 Meat colour L* 41.1 42.9 43.6 42.0 0.85 0.21 a* 14.4 12.9 14.1 13.3 0.45 0.11 b* 4.3 3.7 5.1 4.6 0.47 0.29 1 kg dry matter intake/kg live weight gain 2 Dissection data expressed as percentage of chumps and shoulders weight and adjusted to cold carcass weight 3 Expressed as g/100 g of chumps+shoulder 4 Intermuscular fat plus subcutaneous fat
Table 3 – Fatty acid composition of abomasum digesta from lambs fed control diet (C, no clay added) or diets with bentonite (B), vermiculite (V) and both clays (BV) used as oil absorbent. Treatments C B 60.7 60.3
1.51 1.42ab 0.07 0.40 0.22 10.9ab 0.27 34.2
0.162 0.059 0.009 0.033 0.018 0.18 0.027 3.58
0.69 0.031 0.77 0.39 0.24 0.023 0.66 0.054
2.04 1.12 27.5ab 3.82ab 0.45 1.84 1.02 0.55 9.29a 0.91ab 0.24 0.14 0.15 1.00 0.14
2.15 1.31 20.1b 2.41ab 0.74 2.28 1.33 0.62 6.29b 0.69b 0.40 0.13 0.15 1.77 0.11
0.267 0.171 2.96 0.560 0.107 0.266 0.225 0.087 0.606 0.064 0.083 0.024 0.023 0.397 0.025
0.34 0.28 0.048 0.017 0.14 0.63 0.64 0.66 0.023 0.030 0.44 0.99 0.81 0.70 0.49
0.67 0.08 0.19 2.63 5.83 0.10 2.79
0.35 0.07 0.27 1.51 5.10 ND5 2.36
0.115 0.020 0.053 0.410 0.507 0.021 0.275
0.37 0.39 0.71 0.16 0.67 0.53 0.72
1.76 1.46ab 0.07 0.34 0.18 10.6b 0.22 24.8
1.69 1.59a 0.07 0.41 0.22 11.5a 0.25 20.4
2.36 1.33 31.0a 3.32a 0.75 1.99 1.00 0.48 7.50ab 0.99a 0.34 0.13 0.17 0.78 0.10
1.65 0.90 31.8a 1.96b 0.68 1.84 0.97 0.49 7.58ab 0.86ab 0.22 0.13 0.14 0.83 0.10
0.37 0.09 0.22 2.04 5.73 0.11 2.59
0.35 0.04 0.19 1.45 5.21 0.07 2.54
Total FA (mg/g DM) Fatty acids (g/100g total FA) 1.53 12:0 1.36b 14:0 0.08 i-15:0 0.42 a-15:0 0.22 15:0 10.8ab 16:0 0.26 17:0 21.1 18:0 18:1 t6 + t7 + t8 t9 t10 t11 t12 t13 + t14 t15 t16 c9 c11 c12 c13 c14 c15 c16 18:2 tt + cyclo-171 t8,c13 / c9,t12 t9,c12 t10,c152 c9,c12 (n-6) c9,t11 18:3n-3
0.33 0.34 0.36 0.39 20:0 0.43 0.39 0.41 0.46 22:0 0.27 0.27 0.30 0.32 24:0 Partial sums 0.21 0.13 0.23 0.21 Dimethylacetals 46.0 43.2 43.5 35.0 Total BI3 b a b 9.7 16.7 11.4 8.0b t10-18:1/t11-18:1 Biohydrogenation4 83.6 85.0 83.0 85.3 18:2n-6 90.0 90.2 89.2 90.9 18:3n-3 1 tt, trans, trans 18:2 isomers ; Cyclo-17, 11-cyclohexyl-11:0
0.020 0.026 0.016
0.23 0.23 0.085
0.031 2.74 1.13
0.19 0.060 <0.001
peak contains about 90-94% of t10,c15-18:2 and 6 to 10% of t11,c15-18:2
expressed as g /100 g of each C18 PUFA.
Table 4 – Total lipids and fatty acid (FA) composition of meat from lambs averaged across diets Item
Mean ± SD
Mean ± SD
0.35 ±0.13 0.34 ±0.14 10.7 ±2.67 0.89 ±0.96
t8,c13 + c9,t12
0.14 ±0.10 23.6 ±2.42
c9-17:1 20:0 c11-20:1
0.18 ±0.11 7
c9,t11 / t7,c9
Sums DMA1 2
Total lipid (g/100 g meat)
Total FA (mg/g DM)
5.32 ±1.00 69.6 ±23.5
Dimethylacetals Branched chain fatty acids, including iso (i-), anteiso (a-) and non terminal branched chain FA 3 Saturated fatty acids 4 Monounsaturated fatty acids with cis double bonds 5 Contains c10-, t13-, t14-18:1 as minor components. 6 Contains t11,c15-18:2 as minor component (less than 8%). 7 t7,c9 isomer comprised about 20 to 40% of the co-eluted peak 8 Biohydrogenation intermediates 9 Monounsaturated fatty acids with trans double bonds 2