Milk Conjugated Linoleic Acid Response to Fish Oil and Linseed Oil Supplementation of Grazing Dairy Cows

The effect of supplementing the diet of grazing dairy cows with fish oil (FO) and linseed oil (LSO) on milk conjugated linoleic acid (CLA) was investigated. Sixteen Holstein cows (170±19 DIM) were assigned into two groups and fed a grain supplement (8.0 kg/d; DM basis) containing 800 g of saturated animal fat (CONT) or 200 g FO and 600 g LSO (FOLSO). All cows grazed together on Sudan grass pasture ad libitum and were fed the treatment diets for 3 wks. Cows were milked twice a day and milk samples were collected during the last three days of the trial. Milk production (24.89 and 22.45 kg/d), milk protein percentage (2.76 and 2.82) and milk protein yield (0.68 and 0.64 kg/d) for the CONT and FOLSO diets, respectively, were not affected (p>0.05) by treatment diets. Milk fat percentage (3.90 and 2.86) and milk fat yield (0.97 and 0.64 kg/d) were lower (p<0.05) with the FOLSO diet compared with the CONT diet. The concentration and yield of milk cis-9 trans-11 CLA were higher (p<0.05) with the FOLSO diet (2.56% of total FA and 16.44 g/d, respectively) than the CONT diet (0.66% of total FA and 6.44 g/d, respectively). The concentrations of milk trans C18:1 and vaccenic acid (VA) were higher (p<0.05) with the FOLSO diet (13.53 and 7.48% of total FA, respectively) than the CONT diet (3.69 and 2.27% of total FA, respectively). In conclusion, supplementing the diet of grazing cows with FO and LSO increased milk cis-9 trans-11 CLA content but reduced milk fat content and yield. (


INTRODUCTION
Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds.CLA are natural occurring fatty acids (FA) in foods derived from ruminants.In milk fat, the cis-9 trans-11 CLA account for at least 82% of total CLA isomers (Sehat et al., 1998).The cis-9 trans-11 CLA is synthesized either in the rumen as an intermediate during the biohydrogenation of linoleic acid or in tissues by Δ-9 desaturase from vaccenic acid (VA), another important intermediate in ruminal biohydrogenation of C18 unsaturated FA (Grinari and Bauman, 1999;AbuGhazaleh et al., 2005).Dietary cis-9 trans-11 CLA is now receiving great attention because of its recognized health benefits as a cancer chemopreventive and antitherogenic (Berlury et al., 2002).
Concentration of CLA is higher in milk fat from cows offered fresh compared with conserved forages (Kelly et al., 1998;Dhiman et al., 1999;Couvreur et al., 2006).Dhiman et al. (1999) conducted a study in which cows consumed either 1/3, 2/3, or all of their daily feed from pasture.They showed that CLA concentration in the milk fat was highest for cows on 100% pasture (22.1 mg/g of FA), intermediate for cows on 2/3 pasture (14.3 mg/g of FA), and least for cows receiving 1/3 pasture (8.9 mg/g of FA).Recently, Couvreur et al. (2006) reported a linear relationship between the proportion of fresh grass in dairy cows' diets and milk cis-9 trans-11 CLA content.Milk cis-9 trans-11 CLA content can be also enhanced by supplementing dairy cows' diets with lipid sources (Schroeder et al., 2003;Boken et al., 2005;Flowers et al., 2007).Schroeder et al. (2003) reported 173% increase in milk CLA when corn grain in grazing dairy cows' diet was partially replaced with Ca-salts of unsaturated FA.When cows on annual ryeryegrass pasture were supplemented with a grain mix containing 0 or 500 g/d of soybean oil, milk CLA content increased by 46% with the soybean oil supplementation (Boken et al., 2005).Loor et al. (2005a) reported a significant increase in milk cis-9 trans-11 CLA content when dairy cows' diet was supplemented with linseed oil (LSO) at 3% of diet DM.Recently, Flowers et al. (2007) reported a linear increase in milk cis-9 trans-11 CLA content with increasing the level of LSO in grazing dairy cows' diet.
Inclusion of fish oil (FO) in dairy cows' diets has been also shown to increase milk cis-9 trans-11 CLA content (Donovan et al., 2000;Whitlock et al., 2002).Milk fat cis-9 trans-11 CLA response to FO is further enhanced when given in combination with linoleic or linolenic acid-rich oil sources (AbuGhazaleh et al., 2002(AbuGhazaleh et al., , 2003)).Despite the volume of research on the impact of dietary FO and plants oils on milk cis-9 trans-11 CLA content under confinement feeding system, there is surprisingly little data in the literature evaluating the effect that dietary FO and plants oils may have under grazing feeding system on milk cis-9 trans-11 CLA content.Therefore, the objective of this trial was to evaluate milk cis-9 trans-11 CLA response to FO and LSO supplementation when cows fed a pasture-based diet.

MATERIALS AND METHODS
All procedures for this study involving the use of animals were conducted at The Southern Illinois University Carbondale Dairy Center and were approved by the Southern Illinois University Intuitional Animal Care and Use Committee.Sixteen multiparous Holstein cows of mean live weight 610±23.3kg were divided into two groups (8 cows per group) based on milk yield (27±3.2 kg/d) and DIM (170±19), placed on Sudan-grass pasture, and offered 8 kg of grain supplement containing either 800 g saturated animal fat (rumo-fat; CONT) or 800 g of FO-LSO (1:3 w/w; FOLSO) for a total of 21 days.The first 18 days were used for adjustment to diets, and the last 3 days for milk samples collection.Grain supplements were formulated to be isocaloric and isonitrogenous (Table 1).Menhaden FO (Omega Protein Inc., Hammond, LA) and Rumo-fat (Robt Morgan Inc.Paris, IL), a source of saturated animal fat, were used in this study.Linseed oil was purchased from a local store.Cows were offered the grain supplement into two equal meals at 06:30 and 16:30 using Calan Broadbent feeder doors (American Calan, Inc., Northwood, NH) to monitor individual intakes.The Sudan-grass pasture area was a twelve ha area divided into fifteen 0.8 ha paddocks for rotational grazing where cows moved to a new paddock every day.Cows were on the experimental pasture for 21 h a day and had continuous access to water.Cows were milked twice daily at 06:00 and 16:00.
Milk yields were recorded daily, but measurements collected during the last 5 days were used for statistical analysis.Milk samples were collected from each cow during the last 3 days (d 19, 20, and 21) from the morning and afternoon milkings, made into daily composites based on milk yield at each milking, and then split into two portions for analyses.One aliquot was stored at 4°C and sent to Prairie Farms (Carlinville, IL) for analyses of fat, protein, total solids, and lactose (AOAC, 1997) by midinfrared spectrophotometry (Multispec; Foss Food Technology Corp., Eden Prairie, MN) and somatic cell counts (SCC; AOAC, 1997) using a Fossomatic 90 (Foss Food Technology Corp.).The second aliquot was stored at -20°C for FA analysis as described by AbuGhazaleh and Holmes (2007).

RESULTS AND DISCUSION
Linolenic acid was the main FA in pasture, it accounted for 37.66% of total FA.Both palmetic and linoleic acids were the second main FA in pasture; they accounted for approximately 13.46 and 13.95% of total FA, respectively (Table 2).As expected, the FA profile in the CONT diet was characterized by its high stearic acid content.In the FOLSO diet, linolenic acid was the main FA followed both linoleic and oleic acids.Stearic and Palmetic acids were the main FA in the rumo-fat.As expected linolenic acid was the main FA in LSO whereas; eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were the two main omega-3 FA in FO.
Milk yield, milk composition, and grain supplement intake are presented in Table 3. Inclusion of FO and LSO in the diet had no effect (p>0.05) on milk yield (24.89 and 22.45 kg/d for the CONT and the FOLSO diets, respectively).Similar results were reported under confinement (Boken et al., 2005;Loor et al., 2005a) and grazing (Rego et al., 2005b;AbuGhazaleh and Holms, 2007;Flowers et al., 2007) feeding conditions when diets were supplemented with oils.Supplementing diets with oils rich in polyunsaturated FA often results in a reduction in feed intake and therefore milk yield (Donovan et al., 2000;Lock and Shingfield, 2004).The equal intake of grain supplements in this experiment may explain the similar milk yield between treatment diets.
Milk fat percentages (3.90 and 2.86) and yields (0.97 and 0.64 kg/d) were higher (p<0.01) in cows fed the CONT diet compared with the FOLSO diet (Table 3).Milk fat depression is frequently seen in dairy cows fed oil supplements (AbuGhazaleh et al., 2002;Rego et al., 2005a,b;Shingfield et al., 2006;Holmes and AbuGhazaleh, 2007).After reviewing eighteen experiments involving 25 comparisons to describe the effects of lipid supplementation on milk production and composition with grazing dairy cows, Schroeder et al. (2004) concluded that the decrease in milk fat was a common result when unsaturated lipid supplements were added to grazing dairy cows' diets.Different explanations have been suggested for the milk fat depression associated with lipids feeding.Jenkins (1993) and Schroeder et al. (2004) suggested that the reduction in milk fat content with dietary lipids may results from their negative effects in rumen fiber digestion with a consequent reduction in acetate production, the precursor for short and medium-chain FA synthesis in mammary glands (Harfoot and Hazelwood, 1997).Others (Grummer, 1991;Chillard and Doreau, 1997) suggested that the increase uptake and incorporation of dietary long-chain FA into triglycerides inhibits the de novo FA synthesis in the mammary gland and therefore, reduce milk fat content.Recently, Bauman and Griinari (2001) suggested that milk fat depression was related to the direct action on the mammary gland of specific FA isomers derived from the ruminal metabolism of dietary unsaturated FA.Piperova et al. (2004) and Shingfield et al. (2006) reported a close association between milk fat depression and levels of milk trans-10 C18:1.In the current study, the concentration of trans-10 C18:1 in milk fat was 808% higher with the FOLSO diet than the CONT diet.
Treatment diets had no effect (p>0.05) on milk protein percentages (2.76 and 2.82) and yields (0.68 and 0.64 kg/d).Supplementing grazing cows' diet with 500 g of sunflower oil or soybean oil did not affect milk protein content or yield (Rego et al., 2005b).Similarly, AbuGhazaleh and Holmes (2007) and Flowers et al. (2007) reported no difference in milk protein content in pasture cows fed a diet with or without plant oils.However, others (Dhiman et al., 2000;Shingfield et al., 2006;Whitlock et al., 2006) reported a significant reduction in milk protein content with oil supplementation, changes that have often been attributed to increases in milk yield (dilution effect) rather than decreases in milk protein synthesis (DePeters and Cant, 1992).Similar milk yield in the current study may explain the similar milk protein content between treatment diets.Treatment diets had no effect (p>0.05) on grain supplement intake SCC, body weight, and BCS.
The FOLSO diet resulted in marked alterations in milk FA composition and yield relative to the CONT diet (Tables 4 and 5).The concentrations of milk short and medium chain FA (C4:0-C16:0) were significantly reduced (p<0.05)with the FOLSO diet compared with the CONT diet.Even short and medium chain FA (C4:0-C14:0) are derived from the de novo synthesis by the mammary gland and from the mammary uptake of preformed FA (Jenkins, 1993).In the current study, the lower concentrations of even-number saturated FA with the FOLSO diet compared with the CONT diet suggest less de novo FA synthesis occurred with cows fed the FOLSO diet.As stated earlier, feeding oils are typically associated with a decrease in the de novo synthesis of short and medium chain FA (Loor et al., 2005a,c;Rego et al., 2005b).
The objective of this study was to evaluate the effects of feeding a CLA-stimulating diet to grazing dairy cows on milk cis-9, trans-11 CLA content.Milk cis-9, trans-11 CLA concentration and yield increased by 288 and 155% with the FOLSO diet relative to the CONT diet, respectively (Tables 4 and 5).The concentration of cis-9, trans-11 CLA averaged 0.66 and 2.56% of total FA with the CONT and the FOLSO diets, respectively.Diets containing FO, plant oils or their combinations consistently increased milk cis-9, trans-11 CLA content under confinement (Whitlock et al., 2002;Loor et al., 2005a;Bu et al., 2007) and grazing (Boken et al., 2005;Rego et al., 2005b;AbuGhazaleh and Holmes, 2007) feeding conditions.Supplementing grazing dairy cows with 500 g/d of sunflower oil or soybean oil resulted in 68 and 53% increase in milk CLA compared with control cows (Rego et al., 2005b).Compared with pasture cows on control diet, supplementing grazing dairy cows diet with 160 and 320 g of FO/d resulted in 44 and 62% increase in milk CLA concentration, respectively (Rego et al., 2005a).The higher milk cis-9, trans-11 CLA concentration seen in this study with the FOLSO diet compared with that reported by AbuGhazaleh et al. (2003; 1.06% of milk total FA), Loor et al. (2005a; 1.34% of milk total FA) and Bu et al. (2007; 1.60% of milk total FA) when dairy cows fed LSO at 524, 588 and 636 g/d, respectively, may have resulted from grazing and/or FO supplementation.Cows on pasture diets have been shown to have higher levels of cis-9, trans-11 CLA (Boken et al., 2005;Couvreur et al., 2006) in their milk than those on conserved forages.Recently, Couvreur et al. (2006) reported a linear relationship between the proportion of fresh grass in dairy cows' diets and milk cis-9, trans-11 CLA.AbuGhazaleh and Jenkins (2004) showed that FO promotes VA accumulations in rumen by inhibiting the reduction of VA to C18:0.As mentioned earlier, cis-9, trans-11 CLA in milk originates from either the ruminal biohydrogenation of linoleic acid (Harfoot and Hazlewood, 1997) or from the endogenous synthesis in mammary gland from VA (Griinari and Bauman, 1999).The endogenous synthesis of cis-9, trans-11 CLA from VA has been proposed as the major pathway of cis-9, trans-11 CLA synthesis in lactating cows, accounting for an estimated 78 to 90% of the cis-9, trans-11 CLA in milk fat (Corl et al., 2001;Piperova et al., 2002).
Concentrations and yields of milk trans C18:1 were also affected by treatment diets (Tables 4 and 5).The concentration of trans C18:1 averaged 3.69 and 13.53% of total FA with the CONT and the FOLSO diets, respectively (Table 4).Vaccenic acid was the major trans C18:1 isomer in milk accounting for 61.5 and 55.3% of total trans C18:1 with the CONT and the FOLSO diets, respectively.Milk VA concentration was higher (p<0.05) with the FOLSO diet  The increase in concentrations of milk trans C18:1 isomers, VA in particular, with the FOLSO diet was expected.Similar increases in milk trans C18:1 and VA concentrations were observed under confinement (Loor et al., 2005a,b;Bu et al., 2007) and grazing (Flowers et al., 2007;Holmes and AbuGhazaleh, 2007) feeding conditions when cows were fed FO and/or LSO.AbuGhazaleh and Holmes (2007) observed a significant increase in milk VA content when partially grazed dairy cows were supplemented with a blend of sunflower oil (300 g/d) and FO (100 g/d) when compared to partially grazed dairy cows received control diet (5.11 vs. 2.20 g/100 g of milk total FA).Milk VA accounted for 5.43, 3.23 and 3.04% of milk FA when dairy cows fed LSO at 860, 588 and 636 g/d, respectively (Loor et al., 2005a,c;Bu et al., 2007).The higher milk VA concentration seen in this study (7.48%) may have resulted from grazing, FO or their combinations.Recently, Felton and AbuGhazaleh (2007) and Holmes and AbuGhazaleh (2007) showed that the increase in milk VA content was higher when oil supplements were fed to dairy cows managed under grazing than confinement feeding systems.Previously, AbuGhazaleh et al. (2002) and Whitlock et al. (2002) showed that combining FO with plant oils results in more increase in milk VA than when fed separately.Human body can convert some of the VA into cis-9, trans-11 CLA (Salminen et al., 1998); therefore, producing milk with high VA concentration could be also beneficial.

IMPLICATIONS
Supplementing grazing cows' diets with fish oil and linseed oil enhances milk conjugated linoleic acid content and may be used as a practical approach to increase conjugated linoleic acid as functional ingredient in milk.

Table 1 .
Ingredient and nutrient content of grain supplement and pasture measurements collected during the last 5 days were used for statistical analysis.Orts were removed and weighed after the morning feeding.Representative samples of Sudan grass (collected from 4 different locations per paddock then composited into one) and grain supplements were collected at d 7, 14 and 21 and stored at -20°C until analysis for chemical and FA composition.Grain supplements and pasture samples were freeze dried (Labconco Freeze Dry System, Labconco, Kansas City, MO) and then ground through a standard model No. 3 Wiley mill (Arthur H. Thomas Co., Philadelphia, PA) with a 2-mm screen.Samples were analyzed for CP and EE according to AOAC

Table 2 .
Fatty acid composition for grain supplements, pasture, rumo fat, linseed oil, and fish oil

Table 3 .
Effect of treatment diets on milk yield, composition and grain supplement intake.

Table 4 .
Effect of treatment diets on milk fatty acid composition

Table 5 .
Effect of treatment diets on milk fatty acid yield