Dairy products provide about 25% - 30% of total saturated fat in the human diet and some saturated FA like lauric (C12:0), myristic (C14:0) and palmitic (C16:0) may have a potential negative effect on human health if consumed in excess  . Milk also contains healthy FA such as RA, the main natural conjugated linoleic acid (CLA) which showed anticarcinogenic and antiatherogenic properties and VA that can be converted to RA in human  and animal body tissues. Milk RA originates from ruminal biohydrogenation of linoleic acid (cis-9, cis-12 C18:2) as an intermediate product and from endogenous synthesis in the mammary gland from VA. The RA and VA content of milk from ruminant animals can be increased by dietary factors as pasture intake and feeding polyunsaturated FA (PUFA) contained in vegetable oils like linoleic acid in soybean oil (SO) and linolenic (cis-9, cis-12, cis-15, C18:3) acid in linseed oil (LO)    . This practice is also effective to reduce the saturated FA content in milk fat   .
Studies in vitro suggested that the partial replacement of linoleic by linolenic acid in the diet increased the rate of conversion of linoleic to RA and from RA to VA in ruminal fluid. The higher rate of isomerization was obtained when linoleic was combined with linolenic acid  . Partial ruminal biohydrogenation of linolenic acid also yields VA and the inhibition in the conversion of VA to stearic acid (C18:0)   may also contribute to increase milk RA content avoiding a shift towards the formation of undesirable FAs like trans-10 C18:1  which may be detrimental to human health  . Supplementation with LO can also reduce (or contribute to maintain) the milk n − 6/n − 3 ratio close to 5 in cows  , goats  , ewes  and buffaloes  . The objective of the experiment was to quantify the effectiveness of the combination of SO and LO on productive performance and milk FA profile in order to obtain a functional bovine milk characterized by a reduction of its hypercholesterolemic FA fraction and enhanced RA content.
2. Material and Methods
2.1. Treatments, Animals and Experimental Design
The experiment was carried out at the National Institute of Agricultural Technology (INTA) in Balcarce (37˚45'S, 58˚18'W) during September and October of 2013. Total duration of the experiment was 38 days. Procedures and animal cares were approved by the Institutional Committee for the Care and Use of Experimental Animals (CICUAE, INTA CERBAS). Thirty-six multiparous Holstein cows (548 ± 56 Kg LW) in early lactation (77 ± 43 days postpartum) were grouped based on parity and milk production measured during the first 7 days of the experiment and randomly assigned to 1 of 6 treatments (6 cows/treat- ments) in a complete randomized design. The basal (Control) diet was composed (DM) by pasture (56%) and concentrate (44%) without supplementary oils. From day 8th of the trial, six cows remained in the Control diet while the cows in oil treatments were supplemented with SO (Glycine max), LO (Linum usitatitissimum), or their blends (%w/w) at 75 - 25 (SO75 - LO25), 50 - 50 (SO50 - LO50) and 25 - 75 (SO25 - LO75). The dose of supplemented materials was calculated to provide 4.0% of the total DM intake of cows  . Pure oils or blends (0.8 kg/cow.day) were manually-mixed to the concentrate during each milking time and thoroughly consumed by cows. Adaptation to oils proceeded gradually starting with 0.2 kg/cow.day over the first day, 0.3 kg during the following 2 days and 0.8 kg from day 4 until the end of the experiment including 28 days of full-dose oil supply. Cows were weighed on 2 consecutive days after the a.m. milking at the start (day 8th) and at the end of the period of lipid supplementation. Animals grazed together on mixed pastures of fescue (Festuca arundinacea), red clover (Trifolium pratense), white clover (Trifolium repens) and bromegrass (Bromus unioloides) in a daily-strip grazing system. The area of the strip was regulated using a temporary electric fence to provide an herbage allowance of 27 kg DM/cow.day. After grazing, each strip was clipped-out of non-grazed forage to about 6 cm to allow a lean and uniform regrowth. The concentrate (16% CP) was composed by ground corn grain (35%), malt brewery waste (10%), pelletized sunflower meal (20%), soybean (10%), wheatgrass (21.48%), calcium carbonate (2%), magnesium oxide (0.4%), salt (1%), rumensin (0.02%), and a vitamin-mineral mix (0.1%). It was offered at a rate of 8 kg/cow.day in two equal feedings during milking times (06.00 and 16.00).
2.2. Sampling Measurements and Laboratory Procedures
Milk production was daily recorded over the whole experiment. Milk samples (50 ml) were collected at a.m. and p.m. milkings twice a week on non-consecu- tive days, composited according to the corresponding volume measured at each milking time and analyzed for fat, total protein, lactose, total and not-fat solids by mid-infrared spectrophotometry (Milko Scan-Minor, Foss Electric, Hillerod, Denmark). Milk urea nitrogen (MUN) was determined using a commercial enzymatic kit (Wiener Lab., Rosario, Argentina). During the last 2 weeks of oil supplementation and from each composite sample collected to determine the chemical composition of milk, aliquots of 50 ml were frozen (−24˚C) to obtain a single pool sample per cow for the determination of milk FA composition by gas liquid chromatography (GLC) as described in  . Cows were weighed on two consecutive days after the morning milking at the start (days 6 and 7) and the end (days 38 and 39) of the experiment and the mean value of the 2 records was used to calculate changes in body weight (BW) gain.
The quality of the concentrate and herbage was estimated from samples taken weekly. Each sample was dried in a forced-air oven (60˚C, 48 hs), ground through a 1-mm screen (Willey mill, Philadelphia, PA) and analyzed for organic matter (OM), neutral detergent fiber (NDF)  , acid detergent fiber (ADF)  , crude protein (CP)  using an autoanalyzer (LECO FP-528, Leco Corp., Saint Joseph, MI, EE.UU.), water-soluble carbohydrate (WSC)  , ether extract (EE)  using an autoanalyzer (ANKOM Corp., Fairtport, NY, EE.UU.), In vitro DM digestibility (IVDMD) was estimated using the Ankom Tech. Daisy II incubator for 48 h and starch as described in  . Pasture DM intake was individually estimated during the last 3 days of weeks 4 and 5 of the trial by the difference method  . The average DM intake of the three consecutive days from each cow was computed for the statistical analysis.
2.3. Statistical Analyses
Milk production and composition were evaluated by the PROC MIXED procedure of SAS/STAT® program  using the following model:
where: Yijk = the dependent variable, μ: overall mean, Cov = covariate (milk yield and composition over the first 7 days), Ti = treatment effects, A(i)j = random effects of animal within treatments, Wj = effects of week, (Ti * Wj) = interaction effects between of treatment and sampling week, E(ijk) = the residual error associated with the ijk observation. Data from milk FA composition, DM intake and changes in BW gain were analyzed by the PROC GLM procedure of the SAS/STAT® (2002-2010) program using the following model:
where: Yij = the dependent variable, μ: overall mean, Ti = treatment effects, A(i)j = random effects of animal within treatments, E(ij) = the residual error associated with the ij observation.
Herbage mass in the pregrazing strips averaged 2253 ± 590 kg DM/ha and herbage allowance was 29 ± 1.1 Kg DM/cow.day. Chemical composition of the concentrate and the forage is shown in Table 1 while FA composition is presented in Table 2. On a DM basis, the estimated chemical composition for the basal Control diet was 912 g/kg OM, 161 g/kg CP, 364 g/kg NDF, 201 g/kg FDA, 157 g/kg of starch, 34 g/kg EE and 158 g/kg of water soluble carbohydrates.
As expected, the linoleic acid content in SO resulted high (53.55%) with a low level of saturated FA (SFA) and 21.55% of oleic (cis-9 C18:1) acid. Linolenic acid content resulted high in linseed oil (41.9%) and pasture (54.21%).
Table 1. Chemical composition and in vitro dry matter digestibility of pasture and concentrate1.
1Values are expressed as the mean ± standard deviation. Pasture and concentrate n = 10. 2Consociated pasture containing Bromus unioloides, Festuca arundinacea, Trifolium pratense and Trifolium repens.
Table 2. Fatty acid composition of feeds and oils.
1Consociated pasture containing Bromus unioloides, Festuca arundinacea, Trifolium pratense and trifolium repens. 2Soybean oil. 3Linseed oil. 4Not detected.
Compared to Control records (23.03 kg/cow∙day), supplementation with 4% oils increased (p < 0.05) milk yield (25.19 kg/cow∙day). Production of fat corrected milk (FCM) resulted greater for cows in AS75 - AL25 (Table 3). Milk fat content was reduced (p < 0.05) only in treatments that included 50% and 75% of LO with the lowest value in the AS50 - AL50 treatment. Milk protein content was not affected (p > 0.05). Compared to Control, the SO75 - LO25 blend was also the most effective to increase milk fat (0.886 kg/cow∙day) and milk protein (0.882 kg/cow.day) yields (p < 0.05). The result may be relevant in a context of the payment of milk per kg of fat and protein produced. Concentration of total solids resulted also higher (p < 0.05) in SO75 - LO25 (Table 3).
No significant differences were observed in BW gain of cows (Table 4).
Table 3. Milk production and composition in grazing dairy cows supplemented or not (Control) with combinations of soybean (SO) and linseed (LO) oils at different percentages (w/w).
1Values are expressed as least squares means and standard error of least squares means. Cows were fed a basal diet (Control) without oils or basal diet supplemented with pure oils or blends at 4% of total DM intake as follows: 0.8 kg SO, 0.6 kg SO and 0.2 kg LO (SO75 - LO25), 0.4 kg SO and 0.4 kg LO (SO50 - LO50), 0.2 kg SO and 0.6 kg LO (SO25 - LO75) and 0.8kg LO. 2Treatment effect. 3Not significant effects. 4FCM% = 4% Fat Corrected Milk. T = treatment effect. W = week effect. TxW = treatment for week interaction. a,dMeans in the same row with different superscripts differ significantly for treatment effect with P-value as mentioned in column for significance at p < 0.05 (Test Tukey-Kramer).
Table 4. Bodyweight (BW) changes in grazing dairy cows supplemented or not (Control) with combinations of soybean (SO) and linseed (LO) oils at different percentages (w/w).
1Values are expressed as least squares means and standard error of least squares means (SEM). Cows were fed a basal diet (Control) without oils or basal diet supplemented with pure oils or blends at 4% of total DM intake as follows: 0.8 kg SO, 0.6 kg SO and 0.2 kg LO (SO75 - LO25), 0.4 kg SO and 0.4 kg LO (SO50 - LO50), 0.2 kg SO and 0.6 kg LO (SO25 - LO75) and 0.8 kg LO. 2Treatment effect.
Pasture DM intake increased by 6% and 8% in SO100 and S75 - LO25 while it was reduced by 0.3%, 13% and 9% in SO50 - LO50, SO25 - LO75 and LO100 respectively (Table 5). Total DM intake was higher in SO100 and SO75 - LO25 while energy intake resulted higher in SO100, SO75, SO50 - LO50 and LO100 (Table 5). Feeding oils mixed with the concentrate (10% as fed) was an effective way to obtain the target lipid consumption avoiding refusals. Estimated intakes of linoleic and linolenic acids from supplementary oils were 407 - 179 g/d in SO100, 322 - 213 g/d in SO75 - LO25, 236 - 246 g/d in SO50 - SO50, 150 - 280 g/d in SO25 - LO75 and 65-313 g/day in LO100.
Milk content of butyric (C4:0) acid was not affected after oil intake (Table 6). The decrease in levels of de novo synthesized FA (C4:0 to C15:1) was not different between pure oils and their combinations. In SO50 - LO50 and SO25 - LO75 treatments, the lower synthesis of de novo FA was not apparently compensated for a correlative increase in the mammary uptake of preformed FA since milk fat content decreased when compared to Control (Table 3). Milk fat depression was maximum in the SO50 - LO50 treatment where the highest content of trans-10 C18:1 was also observed (Table 6). Content of the hypercholesterolemic FA of milk (C12:0 to C16:0) was reduced by oil intake (−27%) without differences between treatments (Table 6). The basal (1.85) atherogenic index (AI) and milk content of myristic acid (10.21 g/100g FA) were reduced by oil intake (40 and 33% respectively) without differences between SO-LO blends. Similar results were observed for lauric (−35%) and palmitic (−24%) acids (Table 6). After oil intake, content of stearic acid increased only when LO represented 75% and 100% of the supplementary blend suggesting a higher biohydrogenation because the estimated activity of the Δ9 desaturase enzyme did not differ between blends
Table 5. Pasture, concentrate and energy intake in grazing dairy cows supplemented or not (Control) with combinations of soybean (SO) and linseed (LO) oils at different percentages (w/w).
1Values are expressed as least squares means and standard error of least squares means (SEM). Cows were fed a basal diet (Control) without oils or basal diet supplemented with pure oils or blends at 4% of total DM intake as follows: 0.8 kg SO, 0.6 kg SO and 0.2 kg LO (SO75 - LO25), 0.4 kg SO and 0.4 kg LO (SO50 - LO50), 0.2 kg SO and 0.6 kg LO (SO25 - LO75) and 0.8 kg LO. 2Treatment effect. 3Consociated pasture containing Bromus unioloides, Festuca arundinacea, Trifolium pratense and trifolium repens. a,dMeans in the same row with different superscripts differ significantly for treatment effect with P-value as mentioned in column for significance at p < 0.05 (Test Tukey-Kramer).
Table 6. Milk fatty acid (FA) composition from grazing dairy cows supplemented or not (Control) with combinations of soybean (SO) and linseed (LO) oils at different percentages (w/w).
1Values are expressed as least squares means and standard error of least squares means (SEM). Cows were fed a basal diet (Control) without oils or basal diet supplemented with pure oils or blends at 4% of total DM intake as follows: 0.8 kg SO, 0.6 kg SO and 0.2 kg LO (SO75 - LO25), 0.4 kg SO and 0.4 kg LO (SO50 - LO50), 0.2 kg SO and 0.6 kg LO (SO25 - LO75) and 0.8 kg LO. 2Treatment effect. 3Short chain FA (C6:0 to C10:0). 4Medium chain FA: (C12:0 to C17:1). 5Long chain FA: (C18:0 to C22:6). 6Atherogenicity index: (C12 + 4 * C14 + C16)/(åUFA). UFA: cis-9 C14:1, C16:1, cis-9 C18:1, cis-11 C18:1, trans-11 C18:1, C18:3, C18:2, C18:2 cis-9 trans11 CLA. The detrimental FA trans-6-8, 9, 10 C18:1 were excluded. 7Index: ([å∆9Dproducts]/[å∆9D products + Susbstrates]). 8Substrates:C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + Trans11 C18:1. a,dMeans in the same row with different superscripts differ significantly for treatment effect with P-value as mentioned in column for significance at p < 0.05 (Test Tukey-Kramer).
(Table 6). Content of oleic acid resulted higher (+7%, p < 0.05) only in LO100. Compared to Control, the increase of the linoleic acid content in milk resulted high (62%, p < 0.05) in cows receiving supplementary oils without differences between blends. Linolenic acid gradually increased when LO replaced SO. The basal milk n − 6/n − 3 ratio (5.94) was increased (p < 0.05) up to 8.53 in SO alone and the inclusion of 25% LO in the blend allowed to maintain the ratio in values near to 5.66 and close to Control records. Concomitant increases in LO at 50%, 75% and 100% of the blend significantly reduced the n − 6/n − 3 ratio to 4.86; 3.47 and 2.76 respectively. Basal content (g/100g FA) of trans-9 C18:1 (0.23) and trans-10 C18:1 (0.46) were increased by oil intake (Table 6) reaching maximal values of 0.53 (trans-9) and 1.80 (trans-10) in SO100 and SO50-LO50 (Table 6). No differences (p > 0.05) between blends were detected for milk content of trans-9 C18:1 and a defined response-pattern in the case of trans-10 C18:1 was not observed.
In mik from Control cows, VA content represented 80.41% of the total trans- C18:1 remaining high (77% to 82%) after oil intake (Table 6). In Control treatment, trans-9 and trans-10 C18:1 represented 5.30% and 10.60% of the total trans-C18: 1 remaining low after oil intake (11.5% and 28.9%, respectively). VA and RA were highly correlated (r2 = 0.80) with an estimated rate of conversion of 32.8% (Figure 1) or 37.3% when the RA/VA ratio was used as an estimator (Table 6).
Content of VA in Control milk averaged 3.49 g/100g FA (Table 6) and increased (p < 0.05) after oil intake reaching maximal values in SO50 - LO50 (9.20 g/100 g FA) and SO100 (8.17 g/100 g FA). Basal RA content in milk (1.56 g/100g FA, Table 6) increased (p < 0.05) after oil intake showing the highest numerical value in SO50 - LO50 (3.24 g/100g FA) and the lower in SO25 - LO75 (2.33 g/100g FA). The SO50 - LO50 treatment also yielded the highest trans-9 (0.52) and trans-10 C18:1 (1.80 g/100g FA) contents. Milk VA and RA showed a high variable response to oil intake within treatments (Figure 1) without a well-de- fined response-pattern (Table 6). Total unsaturated FA content and the unsatu-
Figure 1. Relationship between rumenic (RA, cis-9, trans-11 C18:2) and vaccenic (VA, trans-11 C18:1) acids in milk from cows supplemented or not (Control) with combinations of soybean (SO) and linseed (LO) oils at different percentages (w/w).
rated/saturated/ratio in milk were higher in oil compared to Control (p < 0.05) treatment without differences between oil blends.
Plasma metabolite concentration (glucose, non-esterified fatty acids, triglyceride and urea) were not affected (data not shown). Compared to Control (199.4 mg/dl), circulating levels of plasma cholesterol increased (p < 0.05) in SO50 (229.3 mg/dl), SO25 (231.9 mg/dl) and LO 100 (236.4 mg/dl).
4.1. Pasture and Oil Characteristics
The daily strip-grazing system allowed to provide 29 kg DM/cow.day considered adequate to maximize pasture intake  . In grazing conditions, pasture intake should be maximal when herbage is offered at a rate of 45 g pasture OM/kg BW  . From the average BW of cows (563 kg) and the average OM content of pastures (90 g OM/100g DM, Table 1) it can be calculated that a non-limitant herbage allowance should be around 22.8 kg DM which resulted lower to that obtained in the present experiment. Maximal DM intake should be obtained when pasture allowance was 45 to 55 g DM/kg BW per day  . The average BW of cows (563 kg, Table 4) suggests an optimal range in pasture allowance of 25 to 31 kg DM. Thus, the herbage allowance obtained (29 kg DM/cow.day) was within the optimal range. Pasture DM content (21.85%) was over the critical range of 15% - 18% proposed to decrease voluntary intake  . In turn, NDF (46.23%) and CP (15.1%) contents were in the range of 40% - 50% (NDF) and 15% - 25% (CP) considered as adequate for well managed pastures  . In our experiment, pasture quality and quantity were sufficiently enough to maintain or increase total DM and energy intake of cows (Table 5).
SO represented a good source of oleic (21.55%) and essentially linoleic (53.55%) acids as reported in   . In LO, linolenic acid content (41.9%, Table 2) resulted lower to the 55% value reported by others    but near to values informed in   . Linolenic acid in pasture was nevertheless higher than reported by  and  .
4.2. Milk Yield and Composition and Changes in Body Weight
In  , supplementing SO or LO alone or in combination at 4% of DM intake increased milk yield (16.7%) compared to Control without differences between oils. In our trial, the average increase in oil-supplemented cows over Control was somewhat moderate (9.4%) and mainly explained by both oil blends at a ratio of 75:25 (Table 3). Since milk production at SO100 and LO100 did not differ from Control, a synergic effect on milk output of both 75:25 combinations can be expected. Comparison between oil blends did not reveal a specific effect on milk production. A high frequency of favorable effects on milk production after the inclusion of unprotected vegetable oils in the diet was reported by  . The lack of differences between SO100 and LO100 respect to Control (Table 3) was also observed in the meta-analysis by  suggesting the absence of any net advantage of one or another oil over milk production. Feeding LO at 3% or 4% DM intake increased milk production in  a result not observed in other trials   . Supplementary SO at 2.9% ± 1.2% of DMI (533 ± 228 g/day) did not affect milk production in the experiments reviewed by  or when SO was fed at 3.5% to 5% of DM intake    . In addition, LO supply (1% to 7% of DM intake) did not affect milk production in     . In our experiment, the higher yield of FCM from cows in SO75 - LO25 was explained by the higher volume of milk produced since milk fat content did not change (Table 3). These results suggest that energy excreted in milk was the same across treatments as reported by  . Unsaturated lipid supply generally has neutral effects on yield of FCM both in non-grazing  as in grazing experiments  . In a wide dose-range of lipid supplementation (0.2 to 1.0 kg/day) it has been observed that unsaturated lipids decrease milk fat content and fat yield by 8% in grazing dairy cows  . The lowest milk fat content observed in the 50 - 50 treatment (Table 3) was consistent with the higher levels of trans-10 C18:1 (Table 6) because both parameters were negative correlated (Figure 2). A direct relationship between increasing milk levels of trans-10 C18:1 and the reduction of de novo mammary synthesis has been previously reported  a fact that contributes to explain the lower milk fat content observed.
The lack of negative effects of oil supply on milk protein content (Table 3) is a relevant result as this parameter not only affects milk price but also determines the speed and quality of coagulation in cheese production. In confined production systems, supplementation with unprotected lipids often decrease milk protein content    while in pasture based diets this parameter is often not affected   . Feeding LO does not appear to affect either milk fat   nor milk protein contents or production    .
The lack of differences in BW gain (Table 4) was consistent with     and the similar plasma NEFA concentrations (data not shown). In fact, supplementation with unsaturated lipids does not appear to reduce BW loss in lactating cows or favor the reconstitution of body reserves in lactating cows  .
Figure 2. Relationship between milk fat content and trans-10 C18:1 in milk.
4.3. Dry Matter Intake
In our trial, the effect of supplemental fat on DM intake showed different responses (Table 5) depending on the specific oil blend consumed. Pasture intake slightly increased in SO100 and SO75 while it was decreased in others treatments (SO50 - LO50; SO25 - LO75 and AL100) while total DM intake resulted higher in the SO100 and SO75 (Table 5). The inclusion of LO (3.2% ± 1.7% DM intake) or SO (2.9% ± 1.2% DM intake) in the ration did not affect DM intake of cows in the meta-analysis by  nor in   . Feeding unsaturated FA is more likely to reduce feed intake than saturated FA owing to their potential negative effects on ruminal digestion. However the results are variable including negative  , neutral  or even positive   effects on rumen function. The forage concentrate ratio (F/C) is relevant because when LO was included at 3% of DM intake in a 65/35 F/C diet, positive effects were reported on FDN digestion with an opposite result when the F/C was 35:65  . In the present trial, the F/C averaged 54:46 (Table 5).
4.4. Milk Fatty Acid Profile
The increase in mammary uptake of circulating FA after oil supply  may explain the changes in milk FA composition compared to Control treatment (Table 6) confirming ruminant milk fat plasticity   . The consistency in content of butyric acid after oil intake (Table 6) is a frequently reported result   which is of interest for its potential beneficial role in human health  . Butyric acid can be synthesized by an independent malonyl-CoA pathway and therefore not dependent on the activity of the acetyl CoA carboxylase that is inhibited by the uptake of the exogenous FA supplied by oils   .
The decrease in the total content of de novo synthesized FA (C4:0 to C15:1) was similar between the pure oils and their mixtures (Table 6) as reported in  . The effect is explained by the inhibition in the activity of lipogenic mammary enzymes such as Acetyl-CoA carboxylase   . Antonacci et al.  also reported a reduction (−17.8%) in the total de novo synthesized FA content (from 22.49 to 18.48 g/100g FA) after feeding 0.7 kg of an SO70-LO30 blend to grazing dairy cows. In our study, the decrease in milk fat content (Table 3) was negatively correlated to trans-10 C18:1 content (Figure 2) in agreement with  . A high content of trans-10 C18: 1 or related metabolites like trans-10, cis-12 C18:2 in milk has been associated with dysfunctions in lipoprotein lipase (LPL) and stearoyl CoA desaturase (SCD) enzymes involved in milk fat uptake (LPL) and synthesis explaining the decrease in the fatty content of milk  . In our study, the reduction in milk fat content (Table 3) occurred in part at the expense of the amount of hypercholesterolemic FA (Table 6) which improves the healthy value of milk and contributes to decrease the atherogenic potential of milk fat. In grazing dairy cows, supplementation with 0.7 kg/cow/day of an SO70 - LO30 mixture reduced the atherogenicity index of milk from 1.6 to 1.25  .
The reduction (33%) in myristic acid content (Table 6) is an important result because the pro-atherogenic role of C14:0 is considered to be very potent  . The reductions of 35% for C12:0 and 24% for C16:0 (Table 6) were comparable to those obtained in  and contribute to avoid an excessive consumption of unhealthy saturated fat. In the experiment of  , the reductions of these three FA after supplementation at 4% DM intake with a SO50 - LO50 mixture or pure oils did not differ between treatments. In the present work, the reductions were within the range estimated from the meta-analysis performed by  for supplements with SO and LO with values of 42% - 37% (lauric), 23% - 24% (myristic) and 30% - 17% (palmitic).
Milk content of stearic acid increased only when LO was present at 75% and 100% of the blend without differences in treatments with a higher proportion of SO (Table 6). The results were consistent with that reported by  and could be linked to some possible inhibition of biohydrogenation from VA to C18:0 when high contents of linoleic acid are available   . The effect of the oil blends on the content of C18:0 in milk was inconsistent, which agrees with other experiments that reported the lack of differences in milk stearic content in dairy cows supplemented with oils rich in C18:2n − 6 or C18:3n − 3   .
In the meta-analysis by  all polyunsaturated FA supplements generate similar increases in the content of stearic and oleic acids in milk. In our trial, content of oleic acid numerically increased with oil supply but differed from Control only in LO100 (+7%). The increase in oleic acid after the addition of sunflower or soybean oils to the diet is a well-documented result    also observed when supplementing with LO     . Oleic acid content in milk did not increase after the intake of an SO70 - LO30 mixture at 0.7 kg/cow/day in grazing dairy cows  .
Linoleic acid content in milk from Control cows (2.96 g/100g FA, Table 6) was within the range (2% - 3%) suggested by  . In oil-enriched diets, linoleic acid content increased up to 2.74 - 3.50 g/100 g FA (Table 6) remaining below the 4% as reported by  and observed in  (3.25 to 3.92 g/100g FA) after supplying 0.7 kg/cow/day of an oil blend (SO70 - LO30).
The levels of RA achieved in treatments with pure oils (2.96 to 3.03 g/100g FA) were higher than those of 1.60 - 2.39 g/100g FA reported in  when rations with a high forage content (59%) were supplemented with oils at 4% of DM intake. These authors  obtained greater increases for both VA and RA using SO compared to LO with additive responses of the 50:50 blend but always lower to oils utilized in their pure form suggesting no synergistic effects. A higher and more complete ruminal biohydrogenation of PUFAs in animals that consumed LO would explain the response obtained  . In our trial, milk RA content in oil supplemented cows (2.33 to 3.24 g/100g) were higher than values reported in the meta-analysis by  when cows were suplemented with SO (1.02 ± 0.36 g/100g FA) or LO (1.75 ± 0.84 g/100g FA) and also to those obtained by  supplementing with 500 g/day of sunflower oil or SO (2.02 gRA/100g FA) to grazing dairy cows. They were also higher than observed in  using 0.9 kg/day of FA calcium salts (0.9 kg/cow/day ) containing 30% linoleic acid but close to those reported in  (3.13 g/100 g for AR) using the mixture SO70:LO30 (0.7 kg/cow.day) in grazing dairy cows.
The results confirmed the existence of a broad plasticity in milk FA composition in response to PUFA feeding to grazing dairy cows which constitutes an effective tool to the farmer in order to improve the healthy and added value of milk with a potential benefit to the consumer’s health. A net or well defined response over parameters linked to healthy value of milk was not detected after feeding soybean and linseed oils or blends at 4% of total DM intake. Taken together, the results suggest some advantage for the SO75:LO25 blend considering the relative costs of both oils, the positive effects on milk, fat and protein yields, the lower hypercholesterolemic FA content of milk and the increase in VA and RA content while maintaining a healthy n − 6/n − 3 ratio and very low levels of the detrimental trans-9 C18:1 and trans-10 C18:1 FA.
This work was supported by the National Institute of Agricultural Technology (INTA). This Institute is a decentralized state agency with operational and financial autarchy, under the Ministry of Agroindustry of the Argentine Republic. This publication is part of the requirements to access to the academic degree of Doctor in Agricultural Sciences by the Mar del Plata National University, Argentina.
 Turpeinen, A.M., Mutanen, M., Aro, A., Salminen, I., Basu, S., Palmquist, D.L. and Griinari, J.M. (2002) Bioconversion of Vaccenic Acid to Conjugated Linoleic Acid in Humans. American Journal of Clininical Nutrition, 76, 504-510.
 Chilliard Y., Ferlay A., Mansbridge R.M. and Doreau M. (2000) Ruminant Milk Fat Plasticity: Nutritional Control of Saturated, Polyunsaturated, Trans and Conjugated Fatty Acids. Annales de Zootechnie, 49, 181-205.
 Gagliostro, G.A. (2004) Nutritional Control of Conjugated Linoleic Acid (CLA) Content in Milk and Its Presence in Functional Natural Foods. 2. Production of High Cow’s Milk CLA. Revista Argentina de Producción Animal, 24, 137-163.
 Harvatine, K. and Bauman, D.E. (2006) SREBP1 and Thyroid Hormone Responsive Spot 14 (S14) Are Involved in the Regulation of Bovine Mammary Lipid Synthesis during Diet-Induced Fat Depression and Treatment with CLA. Journal of Nutrition, 136, 2468-2474.
 Chilliard Y. and Ferlay, A. (2004) Dietary Lipids and Forages Interactions on Cow and Goat Milk Fatty Acid Composition and Sensory Properties. Reproduction Nutrition Developement, 44, 467-492.
 Rego, O.A., Alves, S.P., Antunes, L.M.S., Rosa, H.J.D., Alfaia, C.F.M., Prates, J.A.M., Cabrita, A.R.J., Fonseca, A.J.M. and Bessa, R.J.B. (2009) Rumen Biohydrogenation-Derived Fatty Acids in Milk Fat from Grazingdairy Cows Supplemented with Rapeseed, Sunflower, or Linseed Oils. Journal of Dairy Science, 92, 4530-4540.
 Castillo Vargas, J.A. (2012) Kinetics of in Vitro Biohydrogenation of Polyunsaturated Fatty Acids in Ruminal Fluid. Master Science Thesis in Animal Production, Universidad Nacional de Colombia. Facultad de Medicina Veterinaria y de Zootecnia, Departamento de Producción Animal. Bogotá, Colombia.
 Hartoof, C.G., Noble, R.C. and Moore, J.H. (1973) Factors Influencing the Extent of Biohydrogenation of Linoleic Acid by Rumen Micro-Organisms in Vitro. Journal of Science Food Agriculture, 24, 961-970.
 Agazzi, A., Bayourthe, C., Nicot, M.C., Troegeler-Meynadier, A., Moncoulon, R. and Enjanbert, F. (2004) In Situ Ruminal Biohydrogenation of Fatty Acids from Extruded Soybeans: Effects of Dietary Adaptation and of Mixing with Lecithin or Wheat Straw. Animal Feed Science and Technology, 117, 165-175.
 Gómez-Cortés, P., Frutos, P., Mantecón, A.R., Juárez, M., De la Fuente, M.A. and Hervás, G. (2008) Milk Production, Conjugated Linoleic Acid Content, and in Vitro Ruminal Fermentation in Response to High Levels of Soybean Oil in Dairy Ewe Diet. Journal of Dairy Science, 91,1560-1569.
 Roy, A., Chardigny, J.M., Bauchart, D., Ferlay, A., Lorenz, S., Durand, D., Duffart, D., Faulconnier, Y., Sébédio, J.L. and Chilliard, Y. (2007) Butters Rich Either in Trans-10-C18:1 or in Trans-11-C18:1 plus cis-9-trans11 CLA Differentially Affect Plasma Lipids and Aortic Fatty Streak in Experimental Atherosclerosis in Rabbits. Animal, 1, 467-476.
 Gagliostro, G.A. (2004) Nutritional Control of Conjugated Linoleic Acid (CLA) Content in Milk and Its Presence in Functional Natural Foods. 3. Production of High Milk CLA through Strategic Supplementation of the Goat. Revista Argentina de Producción Animal, 24, 165-185.
 Gómez-Cortés, P. (2010) Effect of Supplementation of the Ovine Diet with Different Lipid Sources on the Fatty Acid Profile of Milk. Universidad Complutense de Madrid. Facultad de Ciencias Químicas. Departamento de Química Física I.
 Gagliostro, G.A., Patino, E.M., Sanchez Negrette, M., Sager, G., Castelli, L., Antonacci, L.E., Raco, F., Gallello, L., Rodríguez, M.A., Canameras, C., Zampatti, M.L. and Bernal, C. (2015) Milk Fatty Acid Profile from Grazing Buffaloes Fed a Blend of Soybean and Linseed Oils. Arquivo Brasileiro de Medicina Veteterinaria e Zootecnia, 67, 927-934.
 Masson, L., Alfaro, T., Camilo, C., Carvalho, A., Illesca, P., Torres, R., Tavares do Carmo, M., Mancini-Filho, J. and Bernal, C. (2015) Fatty Acid Composition of Soybean/Sunflower Mix Oil, Fish Oil and Butterfat Applying the AOCS Ce 1j-07 Method with a Modified Temperature Program. Grasas y Aceites, 66, e064.
 Komareck, A.R., Robertson, J.B. and Van Soest, P.J. (1994) Comparison of the Filter Bag Technique to Conventional Filtration in the Van Soest NDF Analysis of 21 Feeds. In: Fahey, G.C., Ed., Proceedings of National Conference on Forage Quality, Evaluation and Utilization, Nebraska University, Lincoln, 2.
 Komareck, A.R. (1993) An Improved Filtering Technique for the Analysis of Neutraldetergent Fiber and Acid Detergent Fiber Utilizing the Filter Bag Technique. Journal of Animal Science, 71, 824-829.
 Horneck, D.A. and Miller, R.O. (1998) Determination of Total Nitrogen in Plant Tissue. In: Kalra, Y.P., Eds., Handbook of Reference Methods for Plant Analysis, Soil and Plant Analysis Council, Inc. CRC Press, Boca Raton, 75-83.
 McRae, J.C. and Armstrong, D.G. (1968) Enzyme Method for Determination of Alpha-Linked Glucose Polymers in Biological Materials. Journal of the Science of Food and Agriculture, 19, 578-581.
 Verité, R. and Journet, M. (1970) Influence de la teneur en eau et de la deshydratation de l’herbe sur sa valeur alimentaire pour les vaches laitières. [Influence of Water Content and Dehydration of the Forage on Its Dietary Value for Dairy Cows]. Annales de Zootechnie, 10, 269-277.
 Glasser, F., Ferlay, A. and Chilliard, Y. (2008) Oilseed Lipid Supplements and Fatty Acid Composition of Cow Milk: A Meta-Analysis. Journal of Dairy Science, 91, 4687-4703.
 Martínez, M.G. (2010) Modulation of the Fatty Acid Composition of Bovine and Caprine Milk through Supplementation with Soybean and Fish Oil. Master Science Thesis, Faculty of Agrarian Sciences, National University of Mar de Plata, Argentina, 130 p.
 Martin, C., Rouel, J., Jouany, J.P., Doreau, M. and Chilliard, Y. (2008) Methane Output and Diet Digestibility in Response to Feeding Dairy Cows Crude Linseed, Extruded Linseed, or Linseed Oil. Journal of Animal Science, 86, 2642-2650.
 Pires, J.A.A., Pescara, J.B., Brickner, A.E., Silva del Rio, N., Cunha, A.P. and Grummer, R.R. (2008) Effects of Abomasal Infusion of Linseed Oil on Responses to Glucose and Insulin in Holstein Cows. Dairy Science, 91, 1378-1390.
 Loor, J.J., Ferlay, A., Ollier, A., Doreau, M. and Chilliard, Y. (2005) Relationship among Trans and Conjugated Fatty Acids and Bovine Milk Fat Yield Due to Dietary Concentrate and Lindseed Oil. Journal of Dairy Science, 88, 726-740.
 Bu, D.P., Wang, J.G., Dhiman, T.R. and Liu, S.J. (2007) Effectiveness of Oils Rich in Linoleic and Linolenic Acids to Enhance Conjugated Linoleic Acid in Milk from Dairy Cows. Journal of Dairy Science, 90, 998-1007.
 Morand-Fehr, P., Chilliard, Y. and Bas, P. (1986) Repercusions de l’Apport de Matieres Grasses dans la Ration sur la Production et la Composition du Lait de Ruminant. [Impact of Including Fats in the Ration on Yield and Composition of Ruminant Milk]. Institut National de la Recherche Agronomique, 64, 59-72.
 Dhiman, T.R., Satter, L.D., Pariza, M.W., Galli, M.P., Albright, K. and Tolosa, M.X. (2000) Conjugated Linoleic Acid (CLA) Content of Milk from Cows Offered Diets Rich in Linoleic and Linolenic Acid. Journal of Dairy Science, 83, 1016-1027.
 Alzahal, O., Odongo, N.E., Mutsvanqwa, T., Or-Rashid, M.M., Duffield, T.F., Baqq, R., Dick, P., Vessie, G. and McBride, B.W. (2008) Effects of Monensin and Dietary Soybean Oil on Milk Fat Percentage and Milk Fatty Acid Profile in Lactating Dairy Cows. Journal of Dairy Science, 91, 1166-1174.
 Huang, Y., Schoonmaker, J.P., Bradford, B.J. and Beitz, D.C. (2008) Response of Milk Fatty Acid Composition to Dietary Supplementation of Soy Oil, Conjugated Linoleic Acid, or Both. Journal of Dairy Science, 91, 260-270.
 Gagliostro, G.A. and Chilliard, Y. (1992) Use of Protected Lipids in Dairy Cow Nutrition. I. Effects on the Production and Composition of Milk, and on Intake of Dry Matter and Energy. Revista Argentina de Producción Animal, 12, 1-15.
 Schroeder, G.F., Gagliostro, G.A., Bargo, F., Delahoy, J.E. and Muller, L.D. (2004) Effects of Fat Supplementation on Milk Production and Composition by Dairy Cows on Pasture: A Review. Livestock Production Science, 86, 1-18.
 Piperova, L.L., Teter, B.B., Bruckental, I., Sampugna, J., Mills, S.E., Yurawecz, M.P., Fritsche, J., Ju, K. and Erdman, R.A. (2000) Mammary Lipogenic Enzyme Activity, Trans Fatty Acids and Conjugated Fatty Acids Are Altered in Lactating Dairy Cows Fed a Milk-Fat Depressing Diet. Journal of Nutrition, 130, 2568-2574.
 Palmquist, D.L., Beaulieu, A.D. and Barbano, D.M. (1993) Feed and Animal Factors Influencing Milk Fat Composition. Journal of Dairy Science, 76,1753-1771.
 Bargo, F., Muller. L.D., Kolver, E.S. and Delahoy, J.E. (2003) Invited Review: Production and Digestion of Supplemented Dairy Cows on Pasture. Journal of Dairy Science, 86, 1-42.
 AbuGhazaleh, A.A., Schingoethe, D.J., Hippen, A.R., Kalscheur, K.F. and Whitlock, A. (2002) Fatty Acid Profiles of Milk and Rumen Digesta from Cows Fed Fish Oil, Extruded Soybeans or Their Blend. Journal of Dairy Science, 85, 2266-2276.
 AbuGhazaleh, A.A., Schingoethe, D.J., Hippen, A.R.K.F. and Whitlock, A. (2002) Feeding Fish Meal and Extruded Soybeans Enhances the Conjugated Linoleic Acid (CLA) Content of Milk. Journal of Dairy Science, 85, 624-631.
 Angulo, J. (2012) Effects of Polyunsaturated Fatty Acids from Plant Oils and Algae on Milk Fat Yield and Composition Are Associated with Mammary Lipogenic and SREBF1 Gene Expression. Tesis, Universidad de Antioquia, Facultad de Ciencias Agrarias, Colombia.
 Gagliostro, G.A. and Chilliard, Y. (1992) Bibliographic Review. Use of Lipids Protected in Nutrition of Dairy Cows. II. Effects on Plasma Concentration of Metabolites and Hormones, Mobilization of Body Lipids and Metabolic Activity of Adipose Tissue. Revista Argentina de Producción Animal, 12, 17-32.
 Sutton, J.D., Knight, R., Mcallan, A.B. and Smith, R.H. (1983) Digestion and Synthesis in the Rumen of Sheep Given Diets Supplemented with Free and Protected Oils. British Journal of Nutrition, 49, 419-432.
 Gagliostro, G.A., Garciarena, D.A., Rodriguez, M.A. and Antonacci, L.E. (2017) Feeding Polyunsaturated Supplements to Grazing Dairy Cows Improve the Healthy Value of Milk Fatty Acids. Agricultural Sciences, 8, 759-782.
 Doreau, M. and Chilliard, Y. (1997) Effects of Ruminal or Postruminal Fish Oil Supplementation on Intake and Digestion in Dairy Cows. Reproduction Nutrition Development, 37, 113-124.
 Ueda, K., Ferlay, A., Chabrot, J., Loor, J.J., Chilliard, Y. and Doreau, M. (2003) Effect of Linseed Oil Supplementation on Ruminal Digestion in Dairy Cows Fed Diets with Different Forage: Concentrate Ratios. Journal of Dairy Science, 86, 3999-4007.
 Gagliostro, G.A., Chilliard, Y. and Davicco, M.J. (1991) Duodenal Rapeseed Infusion in Early and Midlactation Cows. 3. Plasma Hormones and Mammary Uptake of Metabolites. Journal of Dairy Science, 74, 1893-1903.
 Christie, W.W. (1981) The Effects of Diet and Other Factors on the Lipid Composition of Ruminant Tissues and Milk. In: Christie, W.W., Ed., Lipid Metabolism of Ruminant Animals, Pergamon Press, Oxford, 193-226.
 Antonacci, L.E., Rodríguez, A., Castelli, L., Zampatti, M., Castaneda, R., Ceaglio, J. and Gagliostro, G.A. (2013) Supplementation with a Blend of Vegetable Oils and the Fatty Acid Profile of Bovine Milk. Revista Argentina de Producción Animal, 33.
 Griinari, J.M. and Bauman, D.E. (2006) Milk Fat Depression: Concepts, Mechanisms and Management Applications. In: Sjersen, K., Hvelplund, T. and Nielsen, M.O., Eds., Ruminant Physiology: Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress, Wageningen Academic Publishers, Holand, 389-417.
 Ward, A.T., Wittenberg, K.M. and Przybylski, R. (2002) Bovine Milk Fatty Acid Profiles Produced by Feeding Diets Containing Solin, Flax and Canola. Journal of Dairy Science, 85, 1191-1196.
 Cruz-Hernandez, C., Kramer, J.K.G., Kennelly, J.J., Glimm, D.R., Sorensen, B.M., Okine, E.K., Goonewardene, L.A. and Weselake, R.J. (2007) Evaluating the Conjugated Linoleic Acid and Trans 18:1 Isomers in Milk Fat of Dairy Cows Fed Increasing Amounts of Sunflower Oil and a Constant Level of Fish Oil. Journal of Dairy Science, 90, 3786-3801.
 Chilliard, Y., Martin, C., Rouel, J. and Doreau, M. (2009) Milk Fatty Acids in Dairy Cows Fed Whole Crude Linseed, Extruded Linseed, or Linseed Oil, and Their Relationship with Methane Output. Journal of Dairy Science, 92, 5199-5211.
 Hurtaud, C., Faucon, F., Couvreur, S. and Peyraud, J.L. (2010) Linear Relationship between Increasing Amounts of Extruded Linseed in Dairy Cow Diet and Milk Fatty Acid Composition and Butter Properties. Journal of Dairy Science, 93, 1429-1443.
 Rego, O.A., Rosa, H.J.D., Portugal, P., Cordeiro, R., Borba, A.E.S., Vouzela, C.M. and Bessa, R.J.B. (2005) The Effects of Supplementation with Sunflower and Soybeans Oils on the Fatty Acid Profile of Milk Fat from Grazing Dairy Cows. Animal Research, 54, 17-24.
 Schroeder, G.F. and Gagliostro, G.A. (2007) Partial Replacement of Corn Grain with Calcium Salts of Fatty Acid in the Concentrate Fed to Grazing Primiparous and Multiparous Dairy Cows. New Zealand Journal of Agricultural Research, 50, 437-449.