Conjugated linoleic acid content in milk of Chilean Black Friesian cows under pasture conditions and supplemented with canola seed ( Brassica napus ) concentrate

At present, there is limited and contradictory information about the effects of the use of canola (Brassica napus) seed as supplement on the contents of conjugated linoleic acid (CLA) in milk of grazing cows. The objective of this study was to evaluate the effect of a dietary supplement with canola seed on the production and composition of milk, and CLA concentration in Chilean Black Friesian cows under pasture conditions. Three experiments were done. Experiment 1: control group was fed 5 kg d of commercial concentrate without canola (0-TC1) and treatment group that was fed 3.75 kg of commercial concentrate plus 1.16 kg of whole canola seed (1.16-TC1). Experiment 2: Control group was fed 8 kg d commercial concentrate without canola (0-TC2) and treatment group that was fed 6.2 kg of commercial concentrate plus 1.2 kg of ground canola seed (1.2-TC2). Experiment 3: control group was fed 6 kg d commercial concentrate without canola (0-TC3) and treatment group was fed 6 kg of commercial concentrate with 20% of whole canola seed (1.2 kg d, 1.2-TC3). The duration of each experiment was 60 days. No differences in milk production and quality were observed among the experimental groups in every assay. The CLA isomers trans-10, cis-12 and cis-10, cis-12 were higher than those normally found in the scientific literature. There was no effect of the inclusion of canola seed on total CLA content or the content of cis-9, trans-11, trans-10, cis-12 and cis-10, cis-12 isomers. Additional key words: CLA; dairy cows; grazing; milk quality. * Corresponding author: pertinez@us.es Received: 17-10-12. Accepted: 10-07-13. Abbreviations used: ADF (acid detergent fibre); BCS (body condition score); CLA (conjugated linoleic acid); DIM (days in milk); DM (dry matter); FA (fatty acid); FCM (fat corrected milk production); GLM (general linear model); ME (metabolizable energy); NDF (neutral detergent fibre); TC (treatment canola); TMR (total mixed ration); TVA (trans-vaccenic acid). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(3): 747-758 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2013113-3639 eISSN: 2171-9292


Introduction
Conjugated linoleic acid (CLA) has been indicated as one of the most beneficial fatty acids for human health (Pariza & Park, 2001).Of all possible isomers, only cis-9, trans-11 and trans-10, cis-12 have shown an interesting biological activity (Wahle et al., 2004).Studies in animal models and human cell lines have provided evidence that certain isomers of CLA, including cis-9, trans-11 CLA, exhibit potent antiinflammatory, immunomodulatory, antiobesity, and anticarcinogenic activities; and also improve biomarkers of cardiovascular health (Wahle et al., 2004;Shingfield et al., 2008).Levels of CLA in milk and dairy products are of particular nutritional interest, since the main food sources of CLA for humans are dairy and meat products from ruminant animals, with dairy products contributing up to 70% of total CLA intake (Lawson et al., 2001;Wahle et al., 2004).Diet has a major influence on milk fat CLA content and it has been extensively investigated (Bauman et al., 2001).The content of CLA in fat from ruminant-derived foods is dependent on the ruminal production of both CLA, trans-vaccenic acid (TVA), and on the tissue activity of Δ 9 -desaturase.For instance, it has been reported that fresh forage (Khanal et al., 2005;Dewhurst et al., 2006;Butler et al., 2008;Rego et al., 2008) and diets supplemented with plant oils, high in linoleic acid (Bauman et al., 2001;Stanton et al., 2003) increase CLA concentration in milk fat.Within the seeds of oilseeds, rapeseed (Brassica napus) has been used in TMR (total mixed ration) diets in dairy cows (Bayourthe et al., 2000, Chouinard et al., 2001;Ward et al., 2002;Chichlowski et al., 2005;Chen et al., 2008;Neves et al., 2009;Lerch et al., 2012b).However, the information about its utilization as a supplement under grazing conditions is limited and contradictory (Stanton et al., 1997;Lawless et al., 1998;Fearon et al., 2004;Lerch et al., 2012b).
In Chile, Chilean Black Friesian (Holstein × Black Friesian) is the most important dairy breed and produces almost 80% of cow's milk receptioned on dairy industry (INE, 2007).In addition, most of the Chilean dairy feeding systems are based on grazing due to the low cost of the diet (Balocchi et al., 2002).However, the quality of chilean southern pastures does not allow that milk productions being greater than 22 L cow -1 d -1 .Therefore, cows with high yield potential need to be supplemented with concentrates in the milking parlour.In recent years, canola has acquired a greater importance in the country, especially in the south, when compared with other oils crops such as the soybean.As result, a great proportion of this feedstuffs is being used for animal feeding as whole grain or its byproducts (e.g.canola bran) (INE, 2007).This oilseed is an excellent source of oleic and linoleic acids.The fatty acid profile of canola seed contains over 85% of total fatty acids (FA) as C18 unsaturated FA, primarily from 18:1 (> 60% of the total FA) and 18:2 (>20% of the total FA) (Aldrich et al., 1997).Hence, we hypothesized that a diet containing canola seeds would provide linoleic and linolenic acids stimulating TVA and CLA production in the rumen and in the milk.Therefore, the objective of this study was to evaluate the effect of a dietary supplement with canola seeds as (i) whole grain plus commercial concentrate, ii) ground canola grain plus commercial concentrate, and iii) different levels of canola seeds inclusion, on milk yield, CLA isomers content, and the metabolic profile of Chilean Black Friesian cows in grazing systems.

Animals and diets
Three experiments were conducted in three representative farms of southern Chile, characterized by different feed systems according to their technological level.All the experiments were done following the principles and specific guidelines on animal care and welfare as required by Chilean law (SAG, 2010).The genotype of dairy cows utilized in the experiments was Chilean Black Friesian.The supplements were formulated according to the animal requirements established by NRC (2001), and the ration supplements were made isoenergetic.Visual observation of feed intake indicated that cows consumed all concentrate offered.The duration of each experiment was 60 d, the first 15 d were for adaptation to the experimental diets and the experimental period was from day 15 to day 60.Cows were selected for the study based on previous milk production in order to make homogenous groups.
Experiment 1.This experiment was conducted in a farm with a herd of 122 dairy cows, located at Vilcún (38°39' 0" S and 72°14' 0" W), during months of November and December (end of spring-beginning of the summer).The farm lies in the pre-mountain range of the Chilean Andes, which is characterized by an average rainfall of 1,400 mm per year, with an average daily temperature of 23.6°C in January and 1.5°C in August.The altitude is 950 m asl.A total of 14 healthy multiparous cows (3.6 ± 0.8 calvings), with 45 ± 11 day in milk (DIM), and a body condition score (BCS) of 2.50 ± 1.0, were used in the study.Average milk production of cows was 13.8 ± 1.20 kg d -1 .Cows selected for this study were randomly assigned (n = 7 per group) into two groups (Table 1).The control group was fed with 5 kg head -1 d -1 of commercial concentrate without addition of canola seeds (0-TC1).The treatment group was fed with 3.75 kg of commercial concentrate plus the addition of 1.16 kg of whole canola seeds (1.16-TC1).The supplement (commercial concentrate + canola seeds) was supplied twice per day at the milking parlour during the milking time, at 06:00 h in the morning and at 14:30 h in the afternoon.The basic diet of both groups was natural -composed of Trifolium pratense, Dactylis glomerata, Bromus spp., Lolium multiflorum, Taraxacum officinale and Plantago lanceolata-and improved pastures -composed of Lolium perenne and Trifolium repens-.In addition, each animal received 6 kg DM d -1 of pasture silage -composed of Lolium perenne and Avena sativa-(Table 1).The cows grazed an area of 57 ha with 8 paddocks under continuous modality (1 paddock of 8 ha, 5 paddocks of 6 ha, and 2 paddocks of 9.5 ha), the criterium to switch cows among paddocks was the canopy height (5 cm).
Experiment 2. This experiment was carried out in a farm with a herd of 230 dairy cows, located at San Pablo sector (40°24' 0" S and 73°1' 0" W), between months of October and November (spring period).The farm lies in the central valley of southern Chile, which is characterized by an average rainfall of 1380 mm per year, with an average daily temperature of 23.4°C in January and 4.2°C in August.The altitude is 65 m asl.A total of 20 healthy multiparous cows (2.2 ± 0.8 calvings), with 44 ± 11 DIM, and a BCS of 2.75 ± 0.5 were selected for this study and randomly assigned into two groups (n = 10 per group).Average milk production of cows was 29.5 ± 5.84 kg d -1 .The control group was fed with 8 kg d -1 per animal of a commercial concentrate without adding canola seeds (0-TC2).The treatment group was fed with 6.2 kg of commercial concentrate plus 1.2 kg of ground canola seeds (1.2-TC2).Supplements were supplied twice per day at the milking parlour, at 04:30 h in the morning and at 15:30 h during the afternoon.The basic diet of all groups was an improved pasture composed of L. perenne and T. repens (Table 2).The animals grazed 10 paddocks of 0.5 ha each, that was handled under a rotational stripgrazing systems with an electric fencing.The animals were moved from one strip to the next one every 12 or 24 h.
Experiment 3.This experiment was conducted in a farm with herd of 342 dairy cows located at Vilcún sector (IX Region of Chile), between June and July (during the winter period).Twenty multiparous healthy cows (3.3 ± 0.5 calvings), with 32 ± 8 DIM, and a BCS of 2.75 ± 0.2 were selected for this study and randomly assigned into two groups (n = 10 per group).Average milk production of the cows was 15.4 ± 2.63 kg d -1 .The control group was fed with 6 kg d -1 per animal of commercial concentrate without the addition of canola seeds (0-TC3).The treatment group was fed with 6 kg d -1 of commercial concentrate with 20% of whole canola seed (1.2-TC3).Supplements were supplied twice per day at the milking parlour, at 05:30 h in the morning and at 15:30 h in the afternoon.The basic diet of both groups was a sown pasture composed of A. sativa, L. perenne, and T. repens (Table 3).In addition, each animal received 6 kg DM d -1 of silage (composed of A. sativa, L. perenne, and T. repens), and 1.5 kg DM d -1 of fodder cabbage (Brassica oleracea) (Table 3).The animals grazed 20 paddocks of 1-ha each, managed with a rotational system with the use of an electric fencing.The animals were moved from one strip to the next one every 12 h.

Laboratory analyses
A bromatological analysis, previous to the beginning of each experiment, was conducted by mean of a sampling of pastures and concentrates in order to determine their chemical and nutritional composition (Tables 1, 2 and 3).Representative samples of pasture were collected from each paddock before grazing with Cow's milk production was determined using a Waikato ® measuring equipment, on days 1, 15, 30, 45 and 60.At each control, a milk sample of 30 mL was collected (to which was added 0.03 g of potassium dichromate at 0.1% as a preservative).The contents of fat, protein, and urea were determined automatically as previously described by Avilez et al. (2012).Somatic cells counts were measured by flow cytometry, using a Fossomatic Electronic Cell Counter (Fossomatic 5000, Hillerod, Denmark).Additionally, another sample of 100 mL was also collected and sent to the laboratory in thermally insulated containers at 4°C for analysis of CLA isomers (cis-9, trans-11; trans-10, cis-12; cis-10, cis-12).Total lipids were extracted by the method of Folch et al. (1957), using a mixture of chloroform and methanol (2:1, v:v).The fatty acids (FA) contents were analyzed after transesterification of FA to FA methyl esters (Morrison & Smith, 1964), as previously described by Avilez et al. (2012).Briefly, FA methyl esters were analyzed by gas chromatography (HP 6890, Hewlett Packard, Surrey, UK), Flame Ionization Detector (FID), a capillary column SP-2560 (100 m, 0.25 mm i.d. with 0.20 µm thickness in the stationary phase; Supelco Inc., Bellefonte, PA, USA) using He as the tracer gas.Gas chromatography conditions were as follow: the injection volume was 0.5 µL, a split injection was used (70:1, v:v); ultrapure hydrogen was the carrier gas; and the injector and detector temperatures were 250 and 300°C, respectively.The initial temperature was 70°C (held for 1 min), increased by 5°C min -1 to 100°C (held for 3 min), increased by 10°C min -1 to 175°C (held for 40 min), and then increased by 5°C min -1 to 220°C (held for 19 min) for a total run time of 86.5 min.Data were then quantified using the HPCHEM Stations software, and expressed as a percentage of area according to the total FA identified.
On the other hand, at the beginning and at the end of the each experiment, blood samples were also collected (5 mL animal -1 ) by coccygeal venipuncture flow and placed in tubes with sodium heparin.The samples were then centrifuged for 3 min at 3,000 rpm and the plasma was aliquoted and frozen (-18°C) in microtubes of 1.5 mL.For each sample, the metabolic prof ile was determined as previously described by Avilez et al. (2012).

Statistical analysis
Data of milk production, milk's constituents, and metabolic profile of each experiment were analyzed with the repeated measures procedure, using the general linear model (GLM) of the SPSS for Windows 18.0 package (SPSS Inc., Chicago, IL, USA).The linear model used for each parameter was as follows: where Y ijk corresponds to each of the response variable; µ = overall mean; T i = fixed effect of the i treatment group; A ij = random effect of animal j for the i treatment; W k = f ixed effect of the k week of lactation; T × W = interactions among these factors for the i treatment and k week of lactation, and ε ijk = random effect of the residuals.Pairwise comparisons of means were carried out, where appropriate, using Tukey's significant difference tests.The level of significance for the analyses was 5%.

Milk yield and quality
There was no difference in none of the experiments between treatment and control groups for milk production or milk composition prior to the start of the experimental periods (data not shown).In the experimental period (from day 15 to day 60), there was no significant interaction effects (treatment × week) or main effects (Table 4).In this respect, no differences in milk production (p = 0.542 in exp.1; p = 0.622 in exp.2; p = 0.454 in exp.3), fat corrected milk production (FCM, kg d -1 ) (p = 0.361 in exp.1; p = 0.742 in exp.2; p = 0.677 in exp.3), fat content (%) (p = 0.891 in exp.1; p = 0.772 in exp.2; p = 0.214 in exp.3), fat yield (kg d -1 ) (p = 0.243 in exp.1; p = 0.901 in exp.2; p = 0.910 in exp.
The patterns of milk production and milk composition throughout lactation were affected by the lactation day (Tables 4 and 5).Differences in milk production, fat corrected milk production, fat content, fat yield, protein content and protein yield (this parameter only in exp. 1) were observed among the experimental weeks.Nevertheless, there were no differences in urea content and somatic cells.
The pattern of FA composition throughout lactation was affected by the lactation day for all components (Table 4 and Fig. 1).The total content of CLA and its isomers, showed a similar trend than those observed in all the treatments.In the experiment 2, the highest values of CLA isomers were obtained in the later weeks, except for the trans-10, cis-12 isomer.In the experiment 1, the lowest CLA values were obtained in the later weeks, while in the experiment 3 the first week had the highest CLA isomers concentration, except for the trans-10, cis-12 isomer.

Metabolic profile
Throughout each experiment, cows presented a good health status, and did not show any relevant pathology.All the metabolites evaluated, except blood urea at the  end of each experiment, were found to be within the normal range, with no significant differences observed between treatments (Table 6).

Milk yield and quality
Milk urea values were within the normal range, based on the report of Schüler et al. (1990).Similarly, somatic cells counts remained within a optimal range according to San Martin et al. (2002).Based on milk production and milk urea content, in every experiment, the diet contributed with an adequate level of protein and energy, in accordance with NRC (2001) recommendations.
Many of the studies with canola seeds refer to its use in diets as TMR, whereas that reports of its use on grazing systems are limited.Often fat supplements can result in decreased milk production, which may be due to inhibition of rumen fermentation or the low digestibility of FA in the rumen (NRC, 2001).Murphy et al. (1995) observed that cows on pasture and supplemented with concentrate containing 1.65 kg or 0.82 kg cow -1 d -1 of whole fat canola seeds, compared with cow's fed only with pasture, increased significantly milk yield.Nevertheless, as noted in this work in all experiments, in TMR [Delbecchi et al. (2001): using crushed canola seed well protected by pass or unprotected, 1 kg d -1 ; Chichlowski et al. (2005): ground canola seed compared with canola meal mechanically extracted, 1.20 kg d -1 ; Neves et al. ( 2009): feeding ex-truded versus non-extruded canola seed, 2.0 kg d -1 , treated with, or without, 50 g kg -1 lignosulfonate; Lerch et al. (2012a): cows received treatments for 2 consecutive lactations (treatments comprised the basal diet with no additional lipid, or supplements of extruded canola seeds, cold-pressed fat-rich canola seed meal, or whole unprocessed canola seeds to provide 2.5 to 3.0% of additional oil in diet dry matter; cows were housed during indoor periods, whereas cows were at pasture during outdoor periods)] and grazing diets [Lawless et al. (1998): cows on pasture and supplemented with 3.1 kg d -1 of unmolassed beet pulp or 3.0 kg d -1 of canola seed concentrate containing 1.65 kg of whole ground canola seed d -1 ; Fearon et al. (2004): animals on pasture and supplemented with four levels of canola seed oil inclusion, incorporated as whole canola seeds and offered as part of a dairy concentrate ration (control 0 g oil d -1 ; 200 g oil d -1 or 0.4 kg of canola seed d -1 ; 400 g oil d -1 or 0.8 kg of canola seed d -1 ; 600 g oil d -1 or 1.3 kg of canola seed d -1 ); Lerch et al. (2012a)], milk production were similar between treatments.
In relation to milk components, it is well known that low fiber diets, high rich concentrates diets, and/or high vegetable oils diets can cause a reduction in milk fat content in dairy cattle.Likewise, supplementation of dietary fat can decreasing the percentage of milk protein.Hence, presentation of the canola seeds as whole oilseeds is employed to minimise adverse effects of unsaturated dietary lipid on rumen microbial activity and possibly provide some measure of protection for the unsaturated oilseed fatty acids from ruminal biohydrogenation (Fearon et al., 2004).In this respect, Murphy et al. (1990)  intact canola seeds in an unpelleted concentrate to dairy cows resulted in the passage of a proportion of unbroken seeds through the digestive tract into the faeces and that, in this form, the oilseeds had less impact on milk fat composition than ground canola seeds.However, in the three experiments herein, the percentages and amounts of fat and milk protein were not different between the groups, despite the higher percentage of fat in the diet supplements in the groups with canola (Tables 1 and 3).This may be due to similar input fiber and energy of the total ration in each of the experiments, as was suggested by Murphy et al. (2008).
In other study that compared the use of canola seeds treated with alkaline peroxide vs. ground canola grain (2.4 kg d -1 ) in TMR diets, there was no differences on the percentage of milk fat (Aldrich et al., 1997), while the use of ground canola seed compared to canola meal extracted, decreased the percentage of fat (Chichlowski et al., 2005;Neves et al., 2009).Lerch et al. (2012a) observed higher milk fat contents in cows receiving cold-pressed fat-rich canola seed meal and whole unprocessed canola seeds compared with other treatments.On the other hand, in grazing studies, the inclusion of whole fat canola seeds resulted in a decreasing on milk fat content (Murphy et al., 1995;Lawless et al., 1998;Fearon et al., 2004), while the study reported by Lerch et al. (2012a) showed that whole unprocessed canola seeds increased milk fat content when compared with the control treatment.
Regarding to the protein content, some of these authors (Lawless et al., 1998;Chichlowski et al., 2005;Lerch et al., 2012a) reported a decreasing in the percentage of milk protein with the use of canola seeds.This reduction in the percentage of protein appears to be associated with negative effects on the growth of ruminal micro-organisms and the production of microbial protein (Solomon et al., 2000).The differences in feeding (mainly due to ingestion and nutritional composition of the herbage) and lactational effects can explain the changes on milk production and components across the weeks of the study.However, given that ingestion and nutritional composition of the herbage during the grazing period were not monitored in this study, this will have to be tested in future studies.

CLA content and composition
In the present study, CLA values obtained in milk were higher (0.995, 1.450 and 0.880% of total FA, in exp. 1, 2 y 3, respectively) than those found in TMR rations, but similar to others obtained in grazing studies.It is well established that milk coming from grazing cows contains higher concentrations of total CLA when compared with milk coming from cows fed with diets containing high proportions of grass silage or dried grass (Mohammed et al., 2009;Lerch et al., 2012b), corn silage (Couvreur et al., 2006), or concentrates (Kraft et al., 2003;Butler et al., 2008;Rego et al., 2008).Likewise, Lerch et al. (2012b) observed that the increase in milk CLA concentration from pasture is not solely explained by marginally higher dietary 18:2n-6 and 18:3n-3 concentration during outdoor than indoor periods.The underlying reasons for the higher enrichment of conjugated FA in milk from pasture vs. those coming from conserved forages are not fully understood, but may be related to the relatively high amount of rapidly fermentable sugars, soluble fiber, or possibly certain metabolites such as polyphenols and terpenes in fresh grass, higher rumen passage rate and fluid dilution rate in cows fed fresh grass, or possibly due to feeding behavior (meal size, feeding frequency and rumination; Kraft et al., 2003;Mohammed et al., 2009).
The use of canola seeds in dairy systems based on grazing, like in the present study, has not increased the CLA contents in milk.In TMR diets, comparing ground canola seed and canola meal mechanically extracted, the differences were not significant in the contents of total milk CLA and its cis-9, trans-11 isomer (Chichlowski et al., 2005;Neves et al., 2009), while cows received extruded canola seeds or cold-pressed fat-rich canola seed meal increased both parameters to a similar extent compared with the control treatment with no additional lipid (Lerch et al., 2012b).At pasture conditions, extruded canola seeds and whole unprocessed canola seeds in particular, decreased milk total CLA content and trans-10, cis-12 CLA concentrations, while all of the canola seed treatments, whole unprocessed canola seeds in particular, lowered milk fat cis-9, trans-11 CLA concentration (Lerch et al., 2012b).The responses to canola seeds supplementation in that study, and in the present experiments, differ from previous studies that reported that supplements of ground or pelleted canola seeds increased milk cis-9, trans-11 CLA content in grazing cows (Stanton et al., 1997;Lawless et al., 1998;Fearon et al., 2004).The different CLA values found in the experiments conducted in this study, and between this and earlier experiments, may be related to differences in the amount and quality of the pasture and forages ingested, derived from the different botanical and agronomic characteristics of the herbage used (Dewhurst et al., 2006), or by the level of fermentable starch in the diet (Lerch et al., 2012b).Diets rich in PUFA and starch are known to stimulate the accumulation of trans-9, cis-11 CLA, trans-10, cis-12 CLA, and trans 18:1 isomers in the rumen, alterations in ruminal biohydrogenation that are often accompanied by a shift from trans-11 18:1 toward trans-10 18:1 as the major intermediate leaving the rumen (Griinari & Bauman, 1999;Palmquist et al., 2005).
In the present study, in agreement with the results found in a previous work by Avilez et al. (2012), the trans-10, cis-12 and cis-10, cis-12 isomers presented higher values than normally reported in the literature.The regulation of isomer balance is largely unknown.Nevertheless, the cis-9, trans-11 isomer is mainly generated from vaccenic acid in the mammary gland (Palmquist et al., 2005;Mosley et al., 2006), while the trans-10, cis-12 is a minor intermediate of rumen biohydrogenation (Walker et al., 2004), and is relatively unaffected by changes in the diet, except at very high levels of concentrate feeding (Chilliard et al., 2007).Therefore, future studies are necessary to determine its biological function and metabolic production routes.
The pattern of FA composition throughout lactation (Fig. 1) may be related to differences in the nutritional composition of the herbage, which has also been shown to affect the FA composition of milk (Dewhurst et al., 2006).In this respect, lower CLA contents in milk have been observed with more mature pasture.This effect has been attributed to the declining quality and quantity of the herbage (Lock & Garnsworthy, 2003;Ward et al., 2003;Avilez et al., 2012).In exp.2, the highest CLA values were obtained in the later weeks, while in exp. 1 and 3 the lowest values were obtained in the later weeks.These results could be explained because the experiments 1 and 3 were done during the period in which the quality and quantity of the herbage was decreasing, while that experiment 2 was done in the period of major quality and quantity (Ruiz, 1996).

Metabolic profile
All the metabolites evaluated, except blood urea at the end of each experiment, were found to be within the normal range, in agreement with the values for healthy lactating dairy cows (Bertoni & Piccioli, 1999).Previous studies (Pulido, 2009;Avilez et al., 2012) have shown an increase in blood urea when diets present high levels of degradable protein, which is the case with animals fed to pasture on grass (L.perenne).

Conclusions
The addition of canola (B.napus) seed in whole grain or in ground grain to a supplement concentrate, considering quantities of 1.2 kg d -1 , fed to Chilean Black Friesian (Holstein × Overo Negro) cows, on pasture-based systems, did not influence milk production or total CLA contents or its cis-9, trans-11, trans-10, cis-12 and cis-10, cis-12 isomers.In the present study, high quantities of the trans-10, cis-12 and cis-10, cis-12 isomers were obtained in comparison to those normally found.The cis-10, cis-12 isomer does not appear in the scientific literature, therefore future studies are necessary to determine its biological function and metabolic production routes.The reasons of why the addition of canola seeds into the commercial supplement fed to Chilean Black Frison cows did not increase the concentration of cis-9, trans-11 CLA in milk fat, require further investigation.

Figure 1 .
Figure 1.Temporal patterns of the content of conjugated linoleic acid (CLA) isomers (g/100 g total fatty acids) in the milk during the experiments 1 (A, B and C), 2 (D, E, F and G) and 3 (H, I, J and K) (experiments explained in Tables 1, 2 and 3).Control group without canola seed ( ….. ); treatment group with canola seed ( ____ ).The mean values for each date were compared, and those with the same letter (a,b,c) do not differ (p > 0.05).

Table 1 .
CLA content in milk of Chilean Black Friesian cows 749 Ingredients and chemical composition of the supplemented dietary concentrates 1 and forages during experiment 1 a canopy height of 8 cm above the ground, using a 1-m 2 quadrant.Depending of the paddock size, 6 to 12 subsamples were taken in a diagonal pattern, forming an X.Subsequently, one composed sample was obtai-ned from these subsamples.All the samples (pasture and concentrates) were ground to pass a 1-mm screen in a Willey mill before analysis.Dry matter (method 934.01), ash (method 942.05), ether extract (method 750 J. P. Avilez et al. / Span J Agric Res (2013) 11(3): 747-758

Table 2 .
Ingredients and chemical composition of the supplemented dietary concentrates 1 and pasture during the experiment 2

Table 3 .
Ingredients and chemical composition of the supplemented dietary concentrates 1 and forages during the experiment 3

Table 4 .
Production and chemical composition (mean values) of the milk of Chilean Black Friesian cows supplemented with canola seeds 1 See Tables 1, 2 and 3. 2 Standard error of the mean. 3 week.In every experiment, no significant treatment main effects or treatment × week interactions were noted (p > 0.05).4For the statistical analysis, the values were transformed to logarithmic scale, base 10.

Table 5 .
Production and chemical composition (mean values) of the milk of Chilean Black Friesian cows supplemented with canola seeds during the weeks of lactation

Table 6 .
observed that feeding unground, Plasma metabolic profile of Chilean Black Friesian cows supplemented with canola seeds at the beginning and the end of every experiment