Milk fat composition can be modulated through feeding strategies. For example, feeding flaxseed increases the proportions of n-3 fatty acids (FA) and cis9,trans11-18:2 conjugated linoleic acid (CLA) in milk fat (Glasser et al. Reference Glasser, Ferlay and Chilliard2008) although the proportion of linolenic acid (cis9,12,15-18:3) in milk fat of cows fed whole flaxseed (Petit, Reference Petit2002) does not exceed 1% of total FA. However, protection against ruminal biohydrogenation by infusing in the small intestine a rich source of n-3 FA such as flaxseed oil has increased concentration of n-3 FA up to 14% of total FA in milk fat (Petit et al. Reference Petit, Dewhurst, Scollan, Proulx, Khalid, Haresign, Twagiramungu and Mann2002). Therefore, there is a potential to increase milk fat concentration of n-3 FA when linolenic acid is protected against ruminal biohydrogenation. Flaxseed hulls are a co-product obtained from flax processing and they contain 23·5% crude protein (% of dry matter), 29·8% total lipids (% of dry matter) and 53% of total FA as linolenic acid (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). Flaxseed hull is an interesting feed ingredient for dairy cow diets as shown by similar production of 4% fat-corrected milk for late-lactating cows fed 19·8% flaxseed hulls and those fed no flaxseed hulls (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). However, although flaxseed hulls are a rich source of n-3 FA, there is no information on their effects on milk and ruminal FA profile and ruminal fermentation characteristics.
Monensin, which is an ionophore, has been used extensively in the diet of dairy cows, and its effects on milk production and composition are well documented (Van der Werf et al. Reference Van der Werf, Jonker and Oldenbroek1998; Phipps et al. Reference Phipps, Wilkinson, Jonker, Tarrant, Jones and Hodge2000). Monensin has been shown to decrease in vitro ruminal biohydrogenation of polyunsaturated FA (Van Nevel & Demeyer, Reference Van Nevel and Demeyer1995) and to increase total CLA concentration in milk fat of dairy cows (Bell et al. Reference Bell, Griinari and Kennelly2008). In a previous experiment (da Silva et al. Reference da Silva, Santos, Branco, Damasceno, Kazama, Matsushita, Horst, dos Santos and Petit2007), monensin supplementation at 0·02% of the dry matter increased concentrations of CLA and decreased saturated FA in milk fat. In the same study, feeding ground flaxseed with monensin resulted in higher milk fat concentration of trans11-18:1 (precursor of CLA) than when feeding ground flaxseed without monensin or whole flaxseed with or without monensin. Collectively, these results showed that combination of monensin with a source of n-3 FA, such as flaxseed products may contribute to the modification of milk composition for better human health. However, there is no information on the effect of flaxseed hull supplementation on milk FA profile and ruminal fermentation. Therefore, the main objective of the experiment was to determine the effect of feeding a combination of monensin and flaxseed hulls on ruminal fermentation characteristics and FA profile of ruminal fluid and milk.
Materials and Methods
Cows and diets
The present study is part of a larger project concerning milk production, milk composition and concentration of the mammalian lignan enterolactone in the rumen and milk reported elsewhere (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). Four ruminally fistulated multiparous Holstein cows averaging 665±21 kg of body weight and 190±5 days in milk were assigned to a 4×4 Latin square design balanced for residual effect to determine the effects of monensin and flaxseed hulls supplementation on ruminal fermentation characteristics and FA profile of ruminal fluid and milk. Ingredients and chemical composition of the diets and feeding management were as described by Petit et al. (Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). Cows were assigned to a 2×2 factorial arrangement of treatments: 1) control, neither flaxseed hulls nor monensin (CO); 2) diet containing (dry matter basis) 19·8% flaxseed hulls (FH); 3) diet with monensin (16 mg/kg of dry matter; MO); 4) diet containing 19·8% (dry matter basis) flaxseed hulls and 16 mg/kg monensin (HM). Diets CO and MO contained, on a dry matter basis, 33·1% grass silage, 27·0% corn silage, 18·2% corn grain, and 9·4% ground barley. Diets FH and HM contained, on a dry matter basis, 30·1% grass silage, 24·3% corn silage, 8·8% corn grain, and 9·1% ground barley. Cows were housed in tie stalls, fed individually for ad libitum intake (10% refusals) twice a day (0830 and 1530 h), and milked twice daily in their stalls at 0800 and 1900 h. Cows were cared for in accordance with the guidelines of the Canadian Council on Animal Care (CCAC, Reference Olfert, Cross and McWilliam1993).
Experimental procedures
Each experimental period consisted of 28 d with 20 d of adaptation to the diets, one day of sampling ruminal contents, and 7 d of total milk collection. Samples of the total mixed diets were taken daily from d 22 to d 28 and pooled within period for each cow. All samples were frozen at −20°C for subsequent drying at 55°C. Milk samples were obtained from each cow for 14 consecutive milkings from d 22 to d 28 and pooled on a yield basis. One sample was kept frozen at −20°C without preservative for further analyses of milk FA profile. On d 21, a 200-ml sample of ruminal fluid was collected from each of five different sites within the rumen (anterior dorsal, anterior ventral, medium ventral, posterior dorsal, and posterior ventral locations) before feeding (0 h) and at 1, 2, 4, and 6 h after the am feeding. Ruminal fluid was strained through 2 layers of cheesecloth to separate the liquid and solid fractions and the pH (Accumet® pH meter; Fisher Scientific, Montreal, QC, Canada) was measured immediately in the liquid fraction. One sample of filtered ruminal fluid was acidified to pH 2 with 50% H2SO4 and frozen at −20°C for later determination of volatile fatty acids (VFA) and ammonia concentrations. Another subsample of filtered ruminal fluid was obtained after pooling all samples on an hour basis and frozen at −20°C for further analyses of FA profile.
Chemical analyses
Chemical analyses of the diets have been reported previously (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). Concentration of ammonia in ruminal fluid was determined by colorimetry using the indophenol-blue method (Novozamsky et al. Reference Novozamsky, Van Eck, Van Schouwenburg and Walinga1974). Concentration of VFA in ruminal fluid was measured using a HPLC (Waters Alliance 2695, Waters Milfort, Milford, MA, USA) with flame-ionization detection. Extraction of fat in milk and ruminal liquor samples, FA methylation and identification of individual FA in milk, diets and rumen fluid was done using methods described by Côrtes et al. (Reference Côrtes, da Silva, Kazama, Gagnon, Benchaar, Santos, Zeoula and Petit2010).
Statistical analysis
All results were analyzed using the MIXED procedure of SAS (2000) within a 2×2 factorial arrangement of treatments. Data were analyzed using a 4×4 Latin square design with the following general model:
where: Yijkl=the dependent variable, μ=overall mean, Ci=random effect of cow (i=1 to 4), Pj=fixed effect of period (k=1 to 4), Tk=fixed effect of treatment (k=1 to 4), and eijk=random residual error. Treatments were compared to provide factorial contrasts: 1) with vs. without monensin, 2) with vs. without flaxseed hulls, and 3) the interaction between monensin and flaxseed hulls. Data on ruminal fermentation characteristics (pH, VFA, ammonia) were analyzed as repeated measurements. The residual effect was initially included in the model but was removed when it was not significant. Results are reported as least squares means. When a significant F-test was detected for the interaction, multiple comparisons were done using a Tukey's adjustment for the probability. Significance was declared at P⩽0·05 and a trend at 0·05<P⩽0·10 unless otherwise stated.
Results and discussion
Feed composition and consumption
Results of the dry matter intake, milk yield and milk production have already been published (see Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009). Briefly, there was no interaction (P>0·10) between flaxseed hulls and monensin supplementation. Intake of dry matter was higher for CO and MO diets (20·1 and 20·0 kg/d, respectively) than for FH and HM diets (19·0 and 18·6 kg/d, respectively) and monensin had no effect. Yield of 4% fat-corrected milk and milk composition were similar (P>0·05) among diets.
Concentrations of 12:0, 14:0, 16:0, cis11-18:1, cis9,12-18:2, 20:0, 22:0 and 24:0 FA were lower and that of cis9,12,15-18:3 was higher in diets containing flaxseed hulls (Table 1) compared with diets with no flaxseed hulls. Flaxseed hulls contain 53% of total FA as cis9,12,15-18:3 (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009), thus contributing to increase omega 3 FA concentration in FH and HM diets and to decrease the omega 6 to omega 3 FA ratio.
Fatty acid (FA) profile of the total mixed diets fed to Holstein cows and containing no flaxseed hulls and no monensin (CO), flaxseed hulls and no monensin (FH), no flaxseed hulls with monensin (MO) or a mixture of flaxseed hulls and monensin (HM)
1 Mean of four cows; significant effect of flaxseed hulls (P<0·05)
2 Mean of four weekly samples prepared by compositing seven daily samples
3 cis9,12-18:2
4 cis9,12,15-18:3
Ruminal pH and concentrations of ammonia and volatile fatty acids
There was no interaction (P>0·10) between sampling time and treatment for ruminal fermentation characteristics; therefore, only average values and dietary effects are reported (Table 2). Feeding flaxseed hulls had no effect on ruminal pH and concentrations of ammonia and total VFA, which agrees with the lack of dietary effects when flaxseed is supplemented as whole seed (Petit et al. Reference Petit, Dewhurst, Scollan, Proulx, Khalid, Haresign, Twagiramungu and Mann2002) and micronized, extruded or ground flaxseed (Gonthier et al. Reference Gonthier, Mustafa, Berthiaume, Petit, Martineau and Ouellet2004). Feeding flaxseed hulls decreased and increased, respectively, proportions of acetate and propionate in the rumen, thereby decreasing the acetate to propionate ratio in ruminal fluid. This agrees with in vitro results (Chalupa et al. Reference Chalupa, Rickabaugh, Kronfeld and Sklan1994) where long-chain FA such as linolenic and linoleic acids decreased the acetate to propionate ratio. Similarly, Ueda et al. (Reference Ueda, Ferlay, Chabrot, Loor, Chilliard and Doreau2003) observed higher ruminal propionate proportion for cows supplemented with 3% flaxseed oil than for unsupplemented cows. A decrease in the acetate to propionate ratio was also observed by Gonthier et al. (Reference Gonthier, Mustafa, Berthiaume, Petit, Martineau and Ouellet2004) for cows fed flaxseed-based diets compared with those fed a diet without flaxseed, which was probably a result of higher concentration of linolenic acid in the former diets.
Ruminal fermentation characteristics of Holstein cows fed total mixed diets containing no flaxseed hulls and no monensin (CO), flaxseed hulls and no monensin (FH), no flaxseed hulls with monensin (MO) or a mixture of flaxseed hulls and monensin (HM)
Monensin supplementation had no effect on ruminal pH, mean concentration of ammonia, total concentration and proportions of individual VFA, and the acetate to propionate ratio. Similar results have been reported by Ramanzin et al. (Reference Ramanzin, Bailoni, Schiavon and Bittante1997) and Broderick (Reference Broderick2004) for ruminal pH and ammonia and by Ali-Haimoud et al. (Reference Ali-Haimoud, Vernay, Bayourthe and Moncoulon1995) for total concentration of VFA and the acetate to propionate ratio. There was no advantage in feeding a combination of monensin and flaxseed hulls as ruminal fermentation characteristics were similar for cows fed FH and those fed HM.
Fatty acid profile of ruminal fluid
Ruminal concentrations of 4:0 to 12:0 and cis9,12-18:2 were lower in cows fed flaxseed hulls compared with those fed no flaxseed hulls (Table 3). Flaxseed hull supplementation increased ruminal concentrations of cis9-16:1, 18:0, trans9-18:1, trans11-18:1, trans13/14-18:1+cis6/8-18:1, cis9-18:1, cis11-18:1, 19:0, cis7-19:1, cis11-20:1, cis9,12,15-18:3 and total trans. Cows fed flaxseed hulls had lower n-6 FA and higher n-3 FA concentrations than cows fed no flaxseed hulls. The effects of flaxseed hull supplementation on FA of ruminal fluid are in agreement with previous results. For example, Doreau et al. (Reference Doreau, Aurousseau and Martin2009) have reported higher duodenal flow of 18:0, cis-18:1, trans9-18:1, trans13/14-18:1+cis6-18:1 for cows fed 7·5% rolled flaxseed compared with those fed a diet without flax products.
Percentage of selected fatty acids in ruminal fluid (g/100 g of total fatty acids) of Holstein cows fed total mixed diets containing no flaxseed hulls and no monensin (CO), flaxseed hulls and no monensin (FH), no flaxseed hulls with monensin (MO) or a mixture of flaxseed hulls and monensin (HM)
† Means within a row without common subscript differ (P⩽0·05)
‡ cis9,12,15-18:3+cis5,8,11,14,17-20:5
§ cis9,12-18:2+cis5,8,11,14-20:4+cis6,9,12-18:3+cis11,14-20:2+cis8,11,14-20:3+cis5,7,10,13,16-22:5
Monensin supplementation had less pronounced effects on FA profile of ruminal fluid than flaxseed hull supplementation as previously reported for the effects of monensin and safflower oil supplementation on milk FA profile (Bell et al. Reference Bell, Griinari and Kennelly2008). Monensin decreased concentrations of 12:0, 17:0, trans9,12-18:2, cis9,12-18:2, polyunsaturated and n-6 FA in ruminal fluid. Similarly, Odongo et al. (Reference Odongo, Or-Rashid, Bagg, Vessie, Dick, Kebreab, France and McBride2007) reported lower concentrations of even-chain FA in the medium-chain range (e.g., 12:0, 14:0 and 16:0) with monensin supplementation. Although it is recognized that rumen outflow of trans-18:1 FA and stearic acid increases and decreases, respectively, as a result of monensin inhibiting the last step of biohydrogenation (Bauman & Griinari, Reference Bauman and Griinari2003), there was no effect of monensin supplementation on trans-18:1 and 18:0 FA proportion in ruminal fluid. This agrees with the lack of monensin supplementation effect on milk fat percentage previously reported (Petit et al. Reference Petit, Côrtes, da Silva, Kazama, Gagnon, Benchaar, dos Santos and Zeoula2009).
The only interactions between flaxseed hull and monensin were for proportions of 14:0, 15:0, 16:0, and medium-chain FA and the n-6 to n-3 FA ratio. Cows fed CO had higher proportions of these FA compared with those fed the other diets and there was no difference among cows fed FH, MO and HM. The lowest ratio of n-6 to n-3 FA was achieved with FH and HM diets and the highest ratio was obtained on the MO diet while cows fed the CO diet had intermediate ratio (interaction flaxseed hulls × monensin, P=0·02). Therefore, there was little advantage of feeding a combination of monensin and flaxseed hulls as feeding flaxseed hulls alone had more important effects on FA profile of ruminal fluid than when feeding only monensin.
Fatty acid profile of milk fat
There were no interactions between flaxseed hulls and monensin supplementation on milk proportions of individual FA with the exception of 4:0 and trans9,12-18:2 (Table 4). Feeding HM led to the lowest (P=0·04) 4:0 proportion in milk fat. Feeding flaxseed hulls compared with no flaxseed hulls decreased proportions of all short-chain FA (4:0 to 13:0) with the exception of cis11-12:1 and 13:0, which were similar among treatments. There was a decrease in proportions of most medium-chain FA (14:0, cis9-14:1, 15:0, 16:0, cis9-16:1 and 17:0) for cows fed flaxseed hulls with the exception of trans9-16:1, which increased when adding flaxseed hulls to the diet. Compared with cows fed no flaxseed hulls, those fed flaxseed hulls had higher proportions of 18:0, trans9-18:1, trans11-18:1, cis6-18:1, cis9-18:1, cis11-18:1, 19:0, trans9,12-18:2, cis9,12,15-18:3, cis9,trans11-18:2 and total trans FA. This agrees with the increase in all concentrations of trans-18:1 isomers of milk fat reported by Glasser et al. (Reference Glasser, Ferlay and Chilliard2008) when cows were supplemented with flax products.
Percentage of selected fatty acids (g/100 g of total fatty acids) in milk fat of Holstein cows fed total mixed diets containing no flaxseed hulls and no monensin (CO), flaxseed hulls and no monensin (FH), no flaxseed hulls with monensin (MO) or a mixture of flaxseed hulls and monensin (HM)
† Means within a row without common subscript differ (P⩽0·05)
‡ cis9,12,15-18:3+cis5,8,11,14,17-20:5
§ cis9,12-18:2+cis5,8,11,14-20:4+cis6,9,12-18:3+cis11,14-20:2+cis8,11,14-20:3+cis5,7,10,13,16-22:5
Changes in milk FA composition were generally similar to those observed when feeding whole flaxseed (Petit et al. Reference Petit, Germiquet and LeBel2004) and flaxseed oil (Glasser et al. Reference Glasser, Ferlay and Chilliard2008). However, proportion of cis9,12,15-18:3 was slightly higher than the average 1·56% value reported for cows fed different flax products (Glasser et al. Reference Glasser, Ferlay and Chilliard2008), which suggests that FA contained in flaxseed hulls may be somewhat protected against ruminal biohydrogenation. Indeed, greater concentrations of biohydrogenation intermediates of FA were obtained in milk fat of cows fed flaxseed hulls and Chilliard et al. (2009) reported that although the formation of trans FA was more important with extruded than whole flaxseed due to a rapid oil release, more cis9,12,15-18:3 might have been protected from ruminal biohydrogenation with extruded than whole flaxseed. The proportions of n-3 FA and n-6 FA were, respectively, higher and lower in milk fat of cows fed flaxseed hulls compared with those fed no flaxseed hulls, thus resulting in lower n-6 to n-3 FA ratio. A similar decrease in the n-6 to n-3 FA ratio in milk fat has been reported for cows were fed whole flaxseed compared with calcium salts of palm oil (Petit, Reference Petit2002).
Monensin supplementation had little effect on milk FA composition and the only effects were increased (P=0·03) concentrations of cis9-16:1 and cis9,12-18:2 in milk fat. Proportion of CLA in milk fat was similar for cows supplemented or not with monensin, which would agree with the results of Mutsvangwa et al. (Reference Mutsvangwa, Kramer, Blackadar, Duffield, Bagg, Dick, Vessie and McBride2003). There have been some suggestions that monensin can modify milk FA composition by inhibiting biohydrogenation of 18:2 and increasing the content of 18:1 (Fellner et al. Reference Fellner, Sauer and Kramer1997; Sauer et al. Reference Sauer, Fellner, Kinsman, Kramer, Jackson, Lee and Chen1998). Fellner et al. (Reference Fellner, Sauer and Kramer1997) observed higher concentrations of 18:2, trans-18:1 and CLA in continuous cultures of ruminal bacteria following infusion of ionophores (monensin, migericin, or tetronasin). Feeding monensin had similar effects on enhancing 18:2 and trans FA in milk of lactating cows (Sauer et al. Reference Sauer, Fellner, Kinsman, Kramer, Jackson, Lee and Chen1998). According to Jenkins et al. (Reference Jenkins, Fellner and McGuffey2003), the effect of monensin on concentration of unsaturated FA depends on the source of dietary starch with a decrease in the concentration of unsaturated FA (e.g. 18:2) in the rumen when the diet contained grains with lower rates of ruminal digestion such as corn as compared with barley. In the present study, the amount of starch supplied by corn (grain and silage) was higher in diets CO and MO than diets FH and HM. Differences in feed ingredient composition might then explain the lack of effect of monensin on the concentrations of 18:2 and 18:3 in milk fat.
In conclusion, the effects of feeding 19·8% flaxseed hulls in diet on ruminal fermentation and fatty acid profile of ruminal fluid and milk fat were more pronounced than those of monensin supplemented at 16 g/kg of dry matter. Flaxseed hull supplementation decreased the acetate to propionate ratio in ruminal fluid probably as a result of higher dietary concentration of linolenic acid while monensin had no effect. Concentrations of trans-18:1 isomers and n-3 FA in ruminal fluid and milk fat were higher and those of CLA in milk fat tended (P=0·05) to be higher when cows were supplemented with flaxseed hulls. Monensin had little effect on milk fatty acid profile. A combination of 19·8% flaxseed hulls and 16 g monensin/kg resulted in a similar milk fatty acid profile to that obtained when feeding only 19·8% flaxseed hulls.
