Animals, diet and treatments

Six multiparous Holstein cows were used in this study, with 602±45 kg average live weight, 23.0±2.9 kg of average daily milk production, and 220±54 lactation days. Cows were distributed randomly in a 3×3 repeated Latin square with three experimental periods of 21 d each: 16 d for adaptation, and 5 d for measurements. The cows were managed according to the University’s internal bioethics and welfare regulations, which are based on official standards (NOM-062-ZOO-1999; NOM-051-ZOO-1995). The cow feeding consisted of grazing (12 h) and -in the barn- a partial TMR (pTMR; Table 1) formulated to meet the lactating cows’ needs (^{NRC, 2001}).

Table 1 Partial TMR ingredients. 

^†pTMR-0: total mixed rations without soybean oil; pTMR-3: total mixed rations with 3 % soybean oil; pTMR-6: total mixed rations with 6 % soybean oil.^¶Multitec, bovine dairy®: vitamin A: 231 IU; vitamin D3: 58.5 IU: vitamin E: 566 mg; Cu 400 mg; Fe: 2.560 mg; Mn: 1,860 mg; Co: 5.85 mg; I 19.84 mg; Zn: 16 mg; Se: 12 mg; P: 38.220 mg; Mg: 39,959.92 mg; calcium carbonate: 194 g; salt: 236.621g; sodium bicarbonate: 150 g; Na: 1.851.60 mg; K: 2.439 mg.

At the end of adaptation period, the following treatments were randomly assigned to the cows: 1) total mixed rations without soybean oil (pTMR-0); 2) total mixed rations with 3 % soybean oil (pTMR-3); and, 3) total mixed rations with 6 % soybean oil (pTMR-6).

Grazing was rotational in prairies, composed mainly of fescue (Festuca arundinacea), cocksfoot (Dactylis glomerata), perennial ryegrass (Lolium perenne), Kikuyu grass (Pennisetum clandestinum), and white clover (Trifolium repens), fertilized with 50 kg ha^-1 of urea every month.

In the barn, cows were kept in 3.50×4.50 m individual pens, equipped with food and water dispensers, with ad libitum water available. The milking was automatized at 06:00 and 15:00 h.

Development of the experiment

The cows remained 12 h in prairies (07:00-15:00 and 16:00-20:00 h) respecting the milking time. The rest of the time the cows remained in the barn, where the pTMR was provided freely, according to the treatment assigned.

In the prairie enclosed with an electric fence, 22 kg MS d^-1 were assigned to each cow: two thirds were offered between milkings, and the rest, after the evening milking. Forage production in the prairie was determined every day by cutting 2 m² at ground level, randomly distributed at eight points. A 0.25 m² quadrant was used. Then, the sample was dried in a microwave oven, in order to determine MS (^{Teuber et al., 2007}).

Soybean oil was added every third day to the concentrate portion (cereals and oilseeds) used to prepare pTMR; this also helped to avoid rancidity. Corn silage was included in the concentrate one hour before the cows entered the barn.

Each cow’s daily pTMR intake was measured by the offer-refuse difference. Pasture intake was estimated by the difference between the cow’s net energy requirements (EN) for lactation (ENL, Mcal d^-1) minus the ENL consumed with the pTMR, according to the method described by ^{Macoon et al. (2003}). The pTMR’s lactation EN was calculated using the equations described by ^{Menke and Steingass (1988}), based on the acid detergent fiber content. ENL requirements were estimated using the prediction equations by ^{NRC (2001}), including the EN requirements for lactation, maintenance, body weight change, grazing activity, and movement to or from the prairie to the barn. At the beginning and end of the measurement phase, the cows were weighed after the morning milking.

Three consecutive days during each period’s measurement phase, the pTMR of each treatment (as well as the prairie forage) was sampled at the moment it was offered, following the simulated grazing technique described by ^{Wayne (1964}). The samples were frozen (at -4°C) until they were analyzed.

Every day, individual milk production was recorded in both milkings. Milk was sampled at each milking and an aliquot (100 mL) per cow was obtained for analysis in the laboratory.

Laboratory analysis

Food samples (pTMR and fodder) were dried in a forced air oven at 60 °C during 24 h, and were subsequently ground using a 2 mm mesh. The content of dry matter and ashes was determined by weight loss after the sample was dried at 100±1 °C in a forced air oven during 24 h, followed by incineration in a muffle furnace at 600 °C during 4 h. The crude protein content was determined using the Kjeldalh method. The ether extract (EE) content was determined according to the ^{AOAC (2012}). The analysis of acid detergent fiber (FDA), neutral detergent fiber (FDN), and acid detergent lignin (LDA) was carried out using the method described by ^{Van Soest et al. (1991}).

The food’s AG content was determined after the samples were freeze dried (LABCONCO, Free Zone 2.5) by means of the ^{Sukhija and Palmquist technique (1988}), as modified by ^{Palmquist and Jenkins (2003}), using methanolic hydrochloric acid at 10 % for esterification and hexane as organic solvent.

The fat, protein, and lactose content of the milk was determined with a Lactoscan analyzer (Milkotronic, LTD). For the analysis of AG in milk, fat was extracted by ultracentrifugation (^{Feng et al., 2004}); methylation was performed according to the methodology described by ^{Christie (1982}), with the modifications made by ^{Chouinard et al. (1999}).

The AG methyl esters of the food and milk were separated and quantified by gas chromatography (Perkin Elmer Clarus 500), with a 100 m×0.25 mm×0.2 µm capillary column (SUPELCO TM-2560); nitrogen was used as carrier gas. Both the detector and the injector were kept at 260 °C; the initial oven temperature was 140 °C during 5 min, and it was increased 4 °C per minute until it reached 240 °C. Each peak was identified according to the methyl ester standards retention times (Supelco 37 Component FAME Mix, trans-vaccenic acid and linoleic acid conjugated by SIGMA-LADRICH). AGs are recorded in g 100 g^-1 of the AG total.

Statistical analysis

The pTMR chemical composition was analyzed with SAS’ GLM procedure (1999). The results of fed intake, and production, composition and AG profile in the milk were analyzed using the MIXED procedure (^{SAS, 1999}), averaged per cow and period, according to the 3×3 replicated Latin square model:

where Y_ijkl: is the response of the variables; µ: the overall mean; C_i: the random effect of i-th square (1, 2); P_(i)j: the fixed effect of the period (1, 2, 3); A_(i)k: the random effect of the animal (1, 2, 3); T_xl: the fixed effect of the treatment (1, 2, 3); E_ijkl: the residual error.

The significance was p≤0.05. In addition, orthogonal polynomial analyses were carried out in order to evaluate the linear and quadratic effects of the treatments.

Chemical composition and fatty acids content of the diet

Table 2 shows the chemical composition and AG content of the pTMR and fodder consumed by the cows. The EE content was 2.0 and 4.4 % greater in pTMR-3 and pTMR-6 diets, compared to pTMR-0 (as a result of adding soybean oil). The LA and LAN acids represented 79 % of the total AG; LAN represented 53 % of the total AG of fresh fodder consumed by the cattle.

Table 2 Chemical composition and fatty acid content of experimental diets and fodder. 

^†pTMR-0: total mixed rations without soybean oil; PTMR-3: total mixed rations with 3 % soybean oil; PTMR-6: total mixed rations with 6 % soybean oil. ^¶Value estimated using the equation proposed by ^{Menke and Steingass (1988}). ENL=(9.07-0.0097*FDA (g kg^-1 MS). The result was divided y 4.184 in order to obtain Mcal.

Feed intake, production, and chemical composition of milk

There was no difference (p>0.05) in pasture intake (2.11 kg MS cow^-1 d^-1) (Table 3). ^{Morales-Almaráz et al. (2010}) report -on dairy cattle with 12 h access to pasture and a 39.7 kg MS d^-1 forage availability per cow- a pasture intake of 8.56 kg MS cow^-1 d^-1 when a corn silage-based TMR was offered ad libitum in the barn. ^{Castro-Hernández et al. (2014}) report 3.36 and 4.63 kg MS cow^-1 d^-1 pasture intake, when 4.5 and 2.7 kg MS of cereal-based concentrate plus corn silage were supplied ad libitum in the barn, and when the cows remained in the prairie 12 h with a forage allowance of 25 kg MS d^-1. In our research, the TMR supply to cows in the barn was not limited, and this decreased pasture intake in the prairie -even though they remained 12 h in the prairie with an allowance of 22 kg MS cow^-1 d^-1. Grazing dairy cows eat more forage in the prairie when the feed offered in the barn is restricted (^{Palladino et al., 2014}).

Table 3 Daily feed intake, and milk production and composition of grazing Holstein cows plus a supplement of pTMR with different contents of soybean oil. 

^ab Mean values in a row with different letters are statistically different (p≤0.05) (n=90). ^†Treatments, pTMR-0: total mixed rations without soybean oil; PTMR-3: total mixed rations with 3 % soybean oil; PTMR-6: total mixed rations with 6 % soybean oil. ^¶ EEM: standard error of the mean. ^§Effects, L: linear; Q: Quadratic. ÞCMS: dry matter consumption.^¤Estimated by difference between the net energy requirements for lactation (ENL) minus the ENL consumed with pTMR (^{Macoon et al., 2003}).

The pTMR and total MS intake decreased linearly (p≤0.05) when the soybean oil content in the diet increased. In our study, TMR intake had an inverse relation to the EE content in the diets. According to ^{Chamberlain and Wilkinson (1996}), including more than 6 % of unsaturated lipids in the diet may reduce microbial activity in rumen and would be reflected in lower feed intake and lower fat synthesis in milk.

Feeding efficiency (FE=kg milk cow^-1 d^-1/ MS total intake d^-1) was greater for soybean oil treatments. Milk production in pTMR-3 was 1.9 and 4.8 % higher (p≤0.05) than in pTMR-0 and pTMR-6 (Table 3). A production unit of grazing dairy cows can be more efficient when an additional source of lipids is included in their diet. Cows fed by grazing only showed an EA of 1.12 (^{Palladino et al., 2014}) and 0.95 when they received 6.3 kg of concentrate (^{Roca-Fernández et al., 2012}), with a milk production of 22.7 and 22.6 kg d^-1.

The treatments had a quadratic effect (p≤0.05) on milk production and its fat and protein content; the lowest values were observed in pTMR-6. The diet’s influence on fat content in milk depends on the fiber and lipid content (^{Bauman and Griinari, 2001}). According to ^{Veira et al. (2001}), the content of fat (3.24 vs. 2.6 %) and protein (3.19 vs. 3.18 %) in milk decreased when 0 and 3 % soybean oil is provided. This could be explained because cellulolytic activity in rumen decreases when vegetable oil is consumed, therefore decreasing the production of acetate and the synthesis of short-chain AG in the mammary gland (^{Griinari et al., 1998}). In addition, the consumption of unsaturated oils may inhibit the synthesis of milk fat, as a result of the production of partially hydrogenated AGs, specifically AG trans (^{Griinari et al., 1998}). Both mechanisms could have caused the reduction of milk fat.

The protein content in the milk of cows fed with pTMR-6 was lower, which may be due to the probable negative effect of AGI on rumen microorganisms (^{Buccioni et al., 2012}). The excess of these acids in the rumen can affect the activity of the microorganisms, therefore decreasing the synthesis of microbial protein (^{Chamberlain and Wilkinson, 1996}).

Profile of milk fatty acids

Including soybean oil in the cow’s diet linearly decreased (p≤0.05) the saturated AG (AGS) concentration, and therefore the AGS/AGI relation, mainly as a result of the increase of the monounsaturated AGs (AGMI) and polyunsaturated AGs (AGPI) content in milk (Table 4). According to ^{Dewhurst et al. (2006}), including vegetable oils in diet reduces short- and medium-chain AGs, and increases long-chain AGs, with a response set apart by a change towards C18 at the expense of C16, and a decrease in the AGS proportion and an increase of AGMI and AGPI in milk. Accordingly, cows fed with pTMR-6 and pTMR-3 produced milk with higher nutritional values, compared with pTMR-0, because they had 26.7 and 9.1 % less AGS. ^{Ulbricht and Southgate (1991}) suggest that humans should consume more AGMI and AGPI, and less AGS, in order to reduce coronary heart disease risks.

Table 4 Fatty acid profile of the milk extracted from grazing Holstein cows plus a supplement of pTMR with different contents of soybean oil. 

^ab Mean values in a row with different letters are statistically different (p≤0.05) (n=18). ^†Treatments, pTMR-0: total mixed rations without soybean oil; PTMR-3: total mixed rations with 3 % soybean oil; PTMR-6: total mixed rations with 6 % soybean oil. ^¶EEM: standard error of the mean. ^§L: linear; Q: Quadratic. ÞCategory, AGS: saturated fatty acids; AGMI: monounsaturated fatty acids; AGPI: polyunsaturated fatty acids; AGI: unsaturated fatty acids.

The content of short-chain (C4, C6, C8, C10) and medium-chain AGs (C11, C12, C13, C14, C15, C16, and C17) was lower in the milk of cows that were provided a diet with 6 % soybean oil. Meanwhile, the milk content of medium-chain AGs C12, C14 and C16 was reduced by 56.7, 43.2, and 18.2 %, with regard to the control treatment. The studies quoted in the review carried out by ^{Martínez et al., (2013}) show that short- and medium-chain AG content decreases in milk fat when oils are added to the ruminants’ diet. In our study, adding soybean oil to the diet could have affected the total production of volatile AGs in the rumen; therefore, acetic acid was reduced -which is the main substrate necessary for the de novo synthesis of short- and medium-chain AGS (^{Chilliard and Ferlay, 2004}). A lower de novo synthesis can occur as a consequence of the inhibitory effect on the activities of the acetyl- CoA carboxylase and fatty acid synthetase enzymes (^{Martínez et al., (2013}) -as a result of the higher content of long-chain AG, absorbed in the small intestine, with greater flow and availability for the mammary gland. C14:1, C16:1 and C17:1 AGs experienced a quadratic effect (p≤0.05), while the C14:1 AG content decreased drastically with the pTMR-6 diet, and the C16:1 and C17:1 AGs increased. This may be the result of the activity of desaturase in the organism, as observed in some AG pairs (C14:1/C14; C16:1/C16; C18:1/C18, RA/VA) (^{Bauman and Griinari, 2001}). But their activity rate depends on each animal (^{Soyeurt et al., 2008}), as well as on the supplement or diet features (^{Shi-jun et al., 2007}).

Figure 1 shows the linear and quadratic effects (p≤0.05) on the content of stearic and oleic AGs in milk. In pTMR-3 and pTMR-6 diets, the concentration of stearic AG increased 13.8 and 26.1 %, compared to the pTMR-0 diet; meanwhile, oleic AG increased 14.3 and 40.6 %. The increase of stearic AG with the addition of soybean oil may be due to the action of rumen microorganisms, which saturate the AGIs of 18 carbons (^{Buccioni et al., 2012}); meanwhile, the increase of oleic AG may be associated with the action of the delta9 desaturase, which uses the stearic AG as a substrate for oleic AG synthesis (^{Griinari et al., 2000}).

Figure 1 Effect of adding soybean oil to the diet on the content of oleic (C18:1 c9), and stearic (C18:0) fatty acids in the milk fat of grazing Holstein cows. L: Linear, Q: quadratic. 

The content of AG LAN in milk fat decreased slightly (p≤0.05), when the level of soybean oil in TMRs increased (Figure 2). The LAN levels in milk are lower than those reported by other authors who allowed the same prairie time (^{Castro-Hernández et al., 2014}; ^{Morales-Almaraz et al., 2010}). Fresh forage is one of the major LAN sources, avoids the ruminal BH process, reaches the mammary gland, and goes into milk. In our study, the lower fresh forage intake by cows helps to explain its slightly lower content in milk. According to ^{Huang et al. (2008}), LAN is lower (0.24 vs. 0.30 g 100 g^-1 AG) in the milk of Holstein cows in barns with a diet that includes 5 % soybean oil, compared to the control group.

Figure 2 Effect of adding soybean oil to the diet on the content of vaccinic (C18:1 t11), linoleic (C18:2 c9c12), rumenic (C18:2 c9t11), and linolenic (C18:3 c9c12c15) fatty acids in the milk fat of grazing Holstein cows. L: Linear, Q: quadratic. 

Including soybean oil in the diet increased (p≤0.05) LA in milk fat, which may be the result of its greater contribution. A similar response was observed in other studies in which soybean oil was added to the diet of dairy cows (^{Huang et al., 2008}; ^{Rego et al., 2005}).

VA is the largest AG trans produced by the biohydrogenation of AG LA and LAN (^{Bauman and Griinari, 2001}). In our research, we showed that augmenting the soybean oil content in the diet increased the VA concentration in milk (p≤0.05), which was 50.3 and 128.7 % higher in pTMR-3 and pTMR-6 compared to pTMR-0 (Figure 2). ^{Sun and Gibbs (2012}) conclude that providing cows with diets with high content of AGPI can inhibit the last phase of the biohydrogenation process in the rumen. Consequently, there would be a greater concentration of intermediate AGs and a greater flow of these to the small intestine, where they are absorbed and transported to the mammary gland, before they are excreted in the milk. An important aspect of VA content in cow’s milk is that this acid is a precursor of RA synthesis in humans (^{Turpeinen et al., 2002}). Both VA and RA have been associated with the reduction of coronary heart disease and atherosclerosis risk in animal models, and probably in humans (^{Wang et al., 2012}). In our research, the RA content underwent a quadratic effect (p≤0.05) and we observed a higher content in the pTMR-3 diet (Figure 2). In contrast, the pTMR-6 treatment reduced the RA content in milk, which may be the result of a possible inhibition of delta9 desaturase activity by the AG C18:2 t10c12 -perhaps due to a lower expression of the gene responsible for this enzyme (^{Choi et al., 2000}). Likewise, the CLA’s C18:2 t10c12 isomer is the result of the isomerization of LA, and a higher content has been observed when the proportion of the concentrate in the diet is greater than the fodder’s (^{Bauman et al., 1999}).