Enriching milk fat with n−3 polyunsaturated fatty acids by supplementing grazing dairy cows with ruminally protected Echium oil

Animal Feed Science and Technology 170 (2011) 35–44

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Enriching milk fat with n−3 polyunsaturated fatty acids by supplementing grazing dairy cows with ruminally protected Echium oil Soressa M. Kitessa a,b,∗ , Paul Young a,b a b

CSIRO Livestock Industries, Private Bag 5, Wembley, WA 6913, Australia Food Futures Flagship, Riverside Corporate Park, 5 Julius Avenue, North Ryde, NSW 2113, Australia

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 2 August 2011 Accepted 14 August 2011

Keywords: Echium oil Stearidonic acid Dairy cow Milk n−3 Fatty acid Pasture

a b s t r a c t Echium oil is a naturally rich source of stearidonic acid (SDA; C18:4n−3), an n−3 polyunsaturated fatty acid (n−3 PUFA) which is a precursor to the long chain (LC) n−3 polyunsaturated fatty acids (LC n−3 PUFA) eicosapentaenoic (EPA, C20:5n−3) and docosahexaenoic (DHA, C22:6n−3). The latter are LC n−3 PUFA for which there is accumulating evidence for positive cardiovascular health claims in human consumers. To determine the extent to which SDA supplementation can enrich milk fat with EPA and DHA, we supplemented 5 dairy cows on irrigated pasture with Echium oil for 10 d. The oil supplement was ruminally protected against biohydrogenation by using a protein–aldehyde matrix. Milk samples were collected during supplementation and 1, 2 and 30 d after supplementation ceased. Echium oil supplementation had no effect on levels of milk crude protein, fat, lactose and solids-not-fat. Average daily milk yield gradually declined as the period of supplementation progressed, and returned to pre-supplementation period levels after withdrawal of the supplement. The proportions of ␣-linolenic acid (ALA, 18:3n−3), SDA, EPA and total n−3 PUFA in milk fat increased in response to supplementation. The initial (Day 1) and final (Day 10) concentrations (in mg/l) of ALA, SDA, EPA and DPA in whole milk were 463 ± 29.2 versus 877 ± 63.1, 38 ± 8.6 versus 144 ± 12.4, 13 ± 6.2 versus 76 ± 9.0 and 45 ± 4.5 versus 65 ± 4.3, respectively. ALA, SDA, EPA and total n−3 PUFA concentrations increased linearly with increasing days of supplementation, while increases in DPA concentrations were curvilinear with a 3–4 d delay in their rise. DHA was not detected in milk fat. In terms of fatty acid yield/cup (i.e., 250 ml) of whole milk, enrichment of milk with EPA amounted to an increase from around 3.1–13.9 mg. As there was no change in DHA content, the long-chain n−3 PUFA content based on EPA alone was less than half of the cut-off point for omega-3 “source” claim (30 mg EPA + DHA per human serving) for foods in Australia. However, the total n−3 PUFA content of milk increased from 559 ± 41.0 to 1162 ± 82.4 mg/l. Data suggest that this oil containing SDA enriched milk with EPA, but not DHA. Dose response and large scale studies are needed to determine the optimal dietary inclusion rate and the commercial feasibility of SDA-containing oils as a means of increasing n−3 fatty acids in dairy cow milk. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

Abbreviations: ALA, ␣-linolenic acid; CP, crude protein; DHA, docosahexaenoic acid; DM, dry matter; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; FSANZ, Food Standards Australia and New Zealand; GC, gas chromatograph; LA, linoleic acid; NHMRC, National Health and Medical Research Council; MUFA, mono-unsaturated fatty acids; PEO, ruminally protected Echium oil; PTO, ruminally protected tuna oil; PUFA, polyunsaturated fatty acids; SDA, stearidonic acid; SFA, saturated fatty acids. ∗ Corresponding author. Current address: CSIRO Food and Nutritional Sciences, P.O. Box 10041, Adelaide BC SA 5000, Australia. Fax: +61 8 83038841. E-mail address: [email protected] (S.M. Kitessa). 0377-8401/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.08.007

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1. Introduction Cardiovascular diseases (CVD) are one of the major sources of morbidity and mortality in humans (Mitka, 2004; Yach et al., 2004). A number of eminent international bodies such as the World Health Organisation (WHO) and The International Society for Study of Fatty Acid and Lipids (ISSFAL), as well as several national heart foundations and associations recommend that people consume long chain (≥C20) n−3 polyunsaturated fatty acids (LC n−3 PUFA) to reduce CVD risk. In most cases these recommendations specifically advise consumption of LC n−3 PUFA, eicosapentaenoic acid (EPA, C20:5n−3) and docosahexaenoic acid (DHA, C22:6n−3). These fatty acids are commonly sourced from oily fish and seafood. In Australia, the Suggested Dietary Targets (SDT) for adult men and women are 610 and 430 mg of EPA plus DHA/d (NHMRC, 2006). The average value is consistent with the American Heart Association’s recommended value of 500 mg of EPA plus DHA/d (Kris-Etherton et al., 2002). Such recommendations of consumption of LC n−3 PUFA for cardiovascular health, and additional evidence for the roles of LC n−3 PUFA in modulation of inflammatory disorders (Calder, 2006), mood disorders (Colangelo et al., 2009; Lucas et al., 2009) and positive effects on visual acuity and cognition early (Jacobson et al., 2008) and late (Connor and Connor, 2007) in life, have resulted in sustained growth of consumer demand for LC n−3 PUFA ingredients over the last decade (Bimbo, 2009). It has been recognised for some time now that this continuing increased consumer demand for LC n−3 PUFA may not be matched by the diminishing supply of wild catch fish due to the deterioration of the marine ecosystems (Worm et al., 2006a,b). In their recent review, Brunner et al. (2009) called for urgent policy action to address this dichotomy between increasing human demand for fish and seafood, and the declining marine ecosystem health. The search for alternative/complementary sources of LC n−3 PUFA through land plant biotechnology has ensued in earnest, and groups from Australia (Robert et al., 2005), the USA (Damude and Kinney, 2007) and Canada (Truksa et al., 2009) have reported on the feasibility of production of LC n−3 PUFA in substantive quantities in land plants. These approaches, which are based on genetic engineering of oilseed plants, remove the likelihood of heavy metal poisoning of humans potentially associated with fish consumption, especially prenatal exposure in high fish diet populations (Dewailly et al., 2008). While this genetic modification option points to a safe (from methyl mercury poisoning), sustainable, land-based LC n−3 PUFA supply over the long term, wide scale development and potential use is yet to overcome agronomic and regulatory issues. In a previous article (Kitessa and Young, 2009), we showed the merits of using precursors of EPA and DHA from vegetable oils in livestock feed in production of animal derived foods with enhanced levels of EPA and DHA. Alpha linolenic acid (ALA, C18:3n−3), which is abundantly found in linseed oil, and stearidonic acid (SDA, C18:4n−3), which constitutes 120–140 g/l of Echium oil, are on the same biosynthetic pathway as EPA and DHA, and can act as precursors for the latter two. ALA is very inefficiently converted to EPA and DHA, and there are serious reservations about use of increased consumption of ALA for CVD risk reduction (Burdge and Calder, 2006). Evidence for the role of SDA in CVD risk reduction, and other chronic diseases, is at an emerging stage, probably due to the lack of naturally rich sources of SDA. A recent critical review by Whelan (2009), which focused on direct comparison of the biological activities of SDA with other dietary n−3 PUFA, concluded that “SDA could become a prominent surrogate for EPA in the commercial development of foods fortified with n−3 PUFA.” In a recent publication (Kitessa and Young, 2009), we demonstrated that, per unit of supplemental oil, an oil containing ALA and SDA (Echium oil) was more effective in enhancing the EPA content of thigh muscle in broiler chickens than that containing ALA alone (i.e., rapeseed oil). In that study, we also observed that, while Echium oil supplementation improved the mg EPA and DPA (docosapentaenoic acid, C22:5n−3) per human serving in both thigh and breast muscles of chicken, only the change in the thigh muscle (i.e., 6.0 mg with ALA diet versus 19.9 mg with SDA diet/100 g muscle) was nutritionally meaningful. The difference in the comparative levels in breast muscle from rapeseed and Echium oil supplemented groups, at 0.6 and 3.2 mg/100 g muscle respectively, is of little nutritional consequence. This brought into question the cost effectiveness of using Echium oil in poultry diets where only half the product will have meaningful amounts of LC n−3 PUFA per human serving. In contrast, using milk as the vehicle for enhanced supply of n−3 PUFA to humans does not pose such a problem. The current study was initiated to determine the degree to which milk can be enriched with LC n−3 PUFA using Echium oil as a supplement in dairy cows grazing irrigated pasture and if such enrichment can be achieved without a detrimental effect on milk yield and composition. 2. Materials and methods All animal handling, feeding, and sampling procedures were approved by the Common Wealth Scientific and Industrial Research Organisation’s Animal Ethics Committee according to guidelines of the National Health and Medical Research Council (NHMRC) for ethical care and handling of animals under experimental conditions (NHMRC, 2004). 2.1. Supplement preparation and ingredients The ruminally protected supplement was produced following the method (Scott et al., 1971) used in a previous study (Kitessa et al., 2004). Briefly, a protein–oil emulsion was produced by mixing Echium oil and casein in 1 kg batches in a GiffordWood colloid mill (Model W200 79, Fallsdell Machinery Pty Ltd., Condell Park, NSW, Australia). The emulsion was treated with formaldehyde and dried using a bench top Fluid Bed Dryer (Extech Equipment Pty. Ltd., Boronia, VIC, Australia). Dried material was pulverised using a household blender and stored at −20 ◦ C until the beginning of the feeding experiment. Two days before the start of feeding the ruminally protected Echium oil powder was mixed with vitamin E (3000 IU/kg

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37

Table 1 Fatty acida composition (g/100 g total fatty acids)b of lipid supplement, pellets and pasture fed to the dairy cows.

C14:0 (Myristic) C16:0 (Palmitic) C18:0 (Stearic) C18:1 cis-9 (Oleic) C18:2 n−6 (LA) C18:3 n−6 (GLA) C18:3 n−3 (ALA) C18:4 n−3 (SDA) C20:5 n−3 (EPA) C22:5 n−3 (DPA) C22:6 n−3 (DHA) Total SFA Total MUFA Total n−6 Total n−3

Echium oil

Protected Echium oil (PEO)c

PEO + molasses

Pellets

Pasture

0.00 7.76 4.06 17.2 15.8 12.0 32.6 10.3 0.00 0.00 0.00 0.00 17.3 27.7 42.9

0.00 10.4 5.30 21.0 15.2 10.5 28.4 8.98 0.00 0.00 0.00 0.00 21.1 25.6 37.4

0.00 11.5 5.94 23.5 14.7 9.65 26.1 8.22 0.00 0.00 0.00 0.00 23.6 14.3 34.4

0.00 19.21 3.72 50.80 22.32 0.00 3.34 0.00 0.00 0.00 0.00 0.00 51.1 22.3 3.34

2.02 27.0 2.68 3.95 10.5 0.00 39.2 0.00 0.00 0.00 0.00 0.00 17.0 10.5 39.2

a ALA, alpha linolenic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; GLA, gamma linolenic acid; LA, linoleic acid; MUFA, monounsaturated fatty acids; SDA, stearidonic acid; SFA, saturated fatty acids. b Each value is the average of duplicate samples with the assay repeated where duplicates varied by more than 5%. c PEO, ruminally-protected Echium oil supplement containing a 500:500 mixture of Echium oil-casein treated with formaldehyde.

supplement) and coated with molasses in kg batches (i.e., 800 g of supplement powder and 200 g of molasses) to enhance the palatability of the supplement. Echium oil was purchased from Croda Australia, Villawood, NSW, Australia (Crossential SA14, Product Code SR03959, Batch No. 262415). It is derived from Echium plantagineum, an annual weed plant commonly known as Paterson’s Curse, Salvation Jane, Blueweed, Lady Campbell Weed, or Riverina Bluebell.in Australia. Edible casein (Product No. 1600672) was obtained from Murray Goulburn Nutritionals (Murray Goulburn Cooperative Ltd., Brunswick, VIC, Australia). Vitamin E (d-alpha tocopherol acetate, 500 IU/g) was purchased from International Animal Health (IAH Sales Pty. Ltd., Blacktown, NSW, Australia).

2.2. Cows - supplement feeding and milk sampling procedures Due to the limited availability of Echium oil, we used prior data from Kitessa et al. (2004) to determine the minimum number of cows required to detect a significant change (P<0.05) in EPA content of milk fat. Using a power of 80% and significance level of 5% power analysis showed that we could expect to detect an absolute difference of 0.124 g/100 g total fatty acids by Day 3 using 5 cows. Hence, we selected 5 multiparious cows (i.e., 3 to 5 lactations) of good temperament from a dairy herd on a private farm in Harvey, Western Australia (PB & JR Maughan, Australia). Average days in milk was 213 ± 6.7. At the monthly herd test before the start of the experiment, cows did not show any anomaly with respect to milk composition or cell counts. At each milking (i.e., 06:00–08:00 and 15:00–17:00 h) cows were fed about 3 kg of commercial pellets for contentment during milking. The commercial pellet was a wheat–barley grain blend with canola meal with135 g/kg crude protein (CP) and 13 MJ metabolisable energy/kg dry matter (DM). All cows were grazed on an irrigated perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) pasture (700:300). The FA profiles of the pasture, pellets and ruminally protected Echium oil are in Table 1. On Days 1, 2, 3, 4 and 5 of the 10 d supplement period, each cow was offered 0.25, 0.40, 0.60, 0.80 or 1.00 kg of Echium oil (PEO). From Days 6–10 the supplement offered was maintained at 1.10 kg. Over the first 5 d, the computed oil intake increased from 100 g to 400 g/d and was 440 g/d for Days 6–10. This was equivalent to an oil inclusion rate of 22 g/kg DM for cows with a daily intake of 20 kg DM. Cows were drafted at each milking to come into the milking area at the end of each milking and were offered the amount of supplement for the day at the morning milking. Residue was collected into labelled buckets and re-presented to each cow at the afternoon milking. If no residue was left after the morning milking, the cows received 2 kg of the commercial pellet in the afternoon. Residue after afternoon milking was discarded and daily supplement intake was calculated. The milk and fatty acid composition on the first day of feeding were considered to be the control. Variations from Day 1 values in milk and fat composition were considered the treatment effects resulting from PEO supplementation. In addition, milk samples were obtained from the bulk tank to provide the comparative nutrient composition and fatty acid profile of milk from the non-supplemented cows from that dairy farm. Each cow was milked into individually labelled buckets at each milking and milk volume recorded. At both the AM and PM milking, an aliquot of milk was collected into a 50 ml plastic bottle for analysis of milk composition (i.e., crude protein, fat, lactose and solids-not-fat). Another aliquot was collected into a 10 ml plastic tube for analysis of FA composition of milk fat. Both sets of samples were stored in a freezer at −20 ◦ C on the farm until the feeding experiment was completed. On the day they were to be analysed they were thawed at room temperature (i.e., 21 ◦ C) before analysis.

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S.M. Kitessa, P. Young / Animal Feed Science and Technology 170 (2011) 35–44 500

Oil amount (g/d)

Intake 400

Offer

300

200

100

0 1

2

3

4

5

6

7

8

9

10

Day fed PEO Fig. 1. Apparent intake of molasses coated ruminally protected Echium oil supplement (PEO) by cows as the amount of oil in the supplement increased from 100 to 440 g/cow/d.

2.3. Lipid analysis Milk composition was determined using a mid-infra red machine (Milko-Scan 130 Series, Foss Electric, HillerØd, Denmark). Samples from each day’s AM and PM milking for each cow were analysed separately and the milk fat, crude protein, lactose and solids-not-fat contents for each day by cow were determined as a weighted average using milk volume of the two values. Fatty acid methyl esters (FAME) were prepared according to Kitessa et al. (2004). Briefly, total fat from a 5 ml sample was extracted using 1 ml NH4 OH solution, 5 M-ethanol and 12.5 ml diethyl ether. Methylation used a sodium methoxide-diethyl ether; (1:1, v/v) solution, and FAME were extracted using 2.5 ml diethyl ether. The gas chromatograph was a Perkin-Elmer Autosystem GC (PerkinElmer Life and Analytical Sciences Pty Ltd, Melbourne, VIC, Australia) fitted with a flame ionising detector and a split injector, and BPX70 column (SGE Analytical Science Pty Ltd, Melbourne, VIC, Australia). We created a 110 m capillary column by connecting 60 m and 50 m BPX70 columns, both with internal diameters (ID) of 0.32 mm and film thicknesses of 0.25 ␮m. The carrier gas was He and injector and detector temperatures were 210 and 250 ◦ C, respectively. The initial oven temperature was 150 ◦ C and it was ramped at 1 ◦ C/min to 223 ◦ C, when the rate was changed to 45 ◦ C/min to 250 ◦ C which was held for 13.96 min. The internal standard used during extraction, and the standard FAME mixture used for peak identifications were from Sigma–Aldrich (Sigma–Aldrich Pty Ltd, Sydney, NSW, Australia). The FAME standards were a C4 /C24 mix (Supelco Product # 18919), methyl all-cis-7,10,13,16,19-docosapentanoate (Sigma Product # D5679) and methyl stearidonate (Fluka Product # 43959). The internal standard used with each extraction was tridecanoic acid (Sigma product # T0502). FA yield/cup (i.e., 250 ml/d) was calculated by multiplying this volume by the average fat proportion and the mean of the proportion of total FA recorded for each FA for that day. 2.4. Statistical analysis For all fatty acids, differences in their concentrations in milk fat between Day 1 and Day 10 of supplementation were analysed using Analysis of Variance (ANOVA) in the statistical software Genstat® (Lawes Agricultural Trust, 2000). Detailed analysis of the pattern of change over the 10 d period in individual fatty acids was done for the FA in the n−3 biosynthetic pathway using regression equations with “days on supplement” as the independent variable and ALA, SDA, EPA, DPA and total n−3 PUFA as dependent variables. The choice between a linear or quadratic model was determined by comparing the model R2 and errors of prediction. 3. Results 3.1. Supplement intake, milk yield and milk composition There was an immediate linear increase in intake of ruminally protected PEO when the amount offered increased from 100 to 400 g/d (Fig. 1). All cows consumed virtually all the supplements on Days 1–5. A further increase of the daily PEO offer to 440 g/d did not result in increased intake of PEO (Fig. 1). Except for the decrease on Day 9, the average oil intake was 0.72 of PEO on offer. On Day 9, the intake of PEO was only 43% of the 440 g offered. There was a change in daily milk yield during the feeding period with yield remaining at pre-supplementation levels during the first 3 d of supplementation, but dropping below pre-supplementation levels on Day 4 and thereafter (Fig. 2). After PEO supplement withdrawal, milk yield increased and had returned to pre-supplementation levels by Day 30 postsupplementation. Gross milk composition was relatively constant over the PEO supplementation period. The respective

S.M. Kitessa, P. Young / Animal Feed Science and Technology 170 (2011) 35–44

39

35 30

d) Milk yield (L/d

25 20 15 10 5 0 1

2

3

4

5

6

7

8

9

Days fed PEO

10

1

2

30

Post-supplementation

Fig. 2. Average (n = 5) milk yield/d during protected Echium oil (PEO) supplementation and during post supplementation. Table 2 Concentrations (mg/l)a of fatty acidsb in whole milk on the initial and final day of supplementation with ruminally protected Echium oil over a 10 d period. Fatty acid C10:0 C12:0 C14:0 C14:1 n−9 C16:0 C16:1 n−9 C17:0 C17:1 n−9 C18:0 C18:1 n−9, Oleic C18:2 n−6, LA C18:2 CLA, c9-t11 C18:3 n−3, ALA C18:3 n−6, GLA C18:4 n−3, SDA C20:0 C20:3 n−6, DGLA C20:4 n−6, ARA C20:5 n−3, EPA C22:5 n−3, DPA Total n−3 Total n−6 Total SFA Total MUFA a b

Initial (Day 1) 1730 1373 4959 531 11,158 298 285 36 5212 8880 600 246 463 11 38 53 0 12 13 45 559 622 25,321 9897

Final (Day 10) 1610 1269 4624 534 10,771 505 363 114 6195 10,582 746 229 877 72 144 76 31 37 76 65 1162 886 25,518 12,019

SED

P

133.1 106.8 330.1 42.4 746.8 67.2 28.2 17.2 411.5 818.3 82.8 21.6 67.4 12.1 14.5 8.9 8.5 9.1 10.5 6.0 89.2 99.2 1630.2 900.3

0.37 0.34 0.32 0.93 0.61 0.004 0.009 <0.001 0.022 0.045 0.09 0.42 <0.001 <0.001 <0.001 0.012 <0.001 0.010 <0.001 0.002 <0.001 0.012 0.90 0.024

Mean values for both the initial and final day of supplementation were based on 10 data points: 5 AM and 5 PM milk samples (n = 10). Abbreviations as in Table 1.

average milk CP contents for Day 1 and Day 10 were 32.9 ± 0.31 and 33.1 ± 0.42 g/kg. Similarly, there were no changes in milk fat, lactose and solids-not fat contents of milk. 3.2. Fatty acid composition of milk fat 3.2.1. Initial (Day 1) and final (Day 10) fatty acid composition The proportion of saturated fatty acids with ≤16 carbon atoms, which formed the bulk of total saturated fatty acids, did not change between Day 1 and Day 10 (Table 2) but there were increases (P<0.05) in C17:0, C18:0 and C20:0 concentrations between Day 1 and Day 10, although these had no impact on total saturated fatty acid content which remained unchanged at 25.3 versus 25.5 g/l for Day 1 and Day 10, respectively. Of the monounsaturated fatty acids measured, C16:1 n−9 (P=0.004), C17:1 n−9 (P<0.001) and C18:1 n−9 (P=0.045) increased between the initial and final day of supplementation, but C14:1 n−9 did not. Total MUFA content of milk increased from 9.90 ± 0.507 to 12.02 ± 0.782 g/l (P=0.024). Changes in n−6 fatty acids in milk relative to treatment varied among individual fatty acids (Table 2). Concentrations of linoleic acid (C18:2 n−6), which were the bulk of n−6 PUFA, did not change between the initial and final day of supplementation. However, there were changes in the n−6 fatty acids GLA, DGLA and ARA. Overall, total n−6 fatty acid content of milk increased from 622 ± 42.6 to 886 ± 93.1 mg/l between the first and last day of supplementation (P=0.012). In contrast, the

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S.M. Kitessa, P. Young / Animal Feed Science and Technology 170 (2011) 35–44

n−3 fatty acids ALA, SDA and EPA increased by a factor of two or more between Day 1 and Day 10. Milk DPA concentration increased by 44% between Day 1 and Day 10. DHA was not detected in milk at any time. The overall milk n−3 PUFA content doubled between Day 1 and Day 10. 3.2.2. Pattern of change in individual n−3 fatty acids over the 10 Day period Over the PEO supplementation period of 10 d, there were linear increases (P<0.001) in the ALA, SDA and total n−3 PUFA concentration of milk (Fig. 3a–c). The R2 for the linear models fitted to ALA, SDA and total n−3 PUFA were 0.54, 0.44 and 0.59, respectively. For these fatty acids, ‘Days on supplement’ explained about half of their daily variations over the 10 d PEO supplementation. Change in milk EPA was also linear but the fitted regression line was below most of the data points due to samples with undetectable levels of EPA (Fig. 3d). A quadratic curve was not a better fit. “Days on supplement” explained 40% of the variation in milk EPA content during PEO supplementation. In contrast, DPA concentrations in milk during PEO supplementation were best described by a quadratic curve, with a 3–5 d of delay before change in the concentration of this fatty acid was detected (Fig. 3e). The quadratic curve explained 25% of the variation in DPA concentration in milk. In all cases, treatment effects on each n−3 fatty acid were highly significant (P<0.001). There were more missing values for EPA and DPA than other n−3 fatty acids. 4. Discussion We have clearly shown that Echium oil can be used to enrich cows’ milk with some of the LC n−3 PUFA important to human health. The lack of response in milk DHA to PEO supplement was consistent with our previous observation with supplementation of Echium oil to broiler chicken (Kitessa and Young, 2009). Although the monogastric and ruminant, as well as milk versus muscle contrasts across the two studies makes direct comparison difficult, in both cases supplying SDA was only effective in enhancing the EPA and, to a lesser extent DPA but not DHA. In terms of creating milk with enhanced LC n−3 PUFA which enables health claim for human consumers, the change in LC n−3 PUFA achieved was limited to change in milk EPA, as DHA was below the detection limit. Peak EPA yield/cup of fresh whole milk, at about 13.9 mg, was nearly half of the 30 mg cut-off point for ‘source’ claim under the Food Standards Australia and New Zealand (FSANZ) guidelines (FSANZ, 2003). We are not are aware of any published study where dairy cows were supplemented with SDA from Echium oil. The only other published study where enrichment of milk with LC n−3 PUFA using an SDA supplement was considered is that published by Bernal-Santos et al. (2010). In their study, SDA from genetically modified soybean oil was infused into the rumen or abomasum of lactating dairy cows. Abomasal infusion of SDA soybean oil increased milk fat SDA from undetectable levels to a plateau of about 1.8 g/100 g of total fatty acids in milk that had 44 g/kg fat. Based on the SDA concentration and milk fat content reported, this was equivalent to 792 mg SDA/l milk or more than 5 fold the 144 mg/l in our study. In contrast, abomasal infusion of SDA soybean oil increased the EPA level from about 0.05 at the start of supplementation to 0.18 g/100 g total fatty acids, which was equivalent to 79.2 mg/l, and almost identical to the 76 mg EPA/l in our study. Hence, the high SDA content of SDA soybean oil resulted in more than quadruple the milk SDA content which we achieved using Echium oil, but did not lead to a difference in milk EPA content per litre. The similarity in EPA content of milk between the two studies, despite a 5 fold difference in SDA concentration of milk fat, suggests that metabolic processes other than the -6 desaturation step in n−3 PUFA biosynthesis need to be optimised to substantively enrich livestock products with EPA and DHA. Further studies are needed to elucidate these limiting factors. The SDA soybean oil study showed milk fat from ruminally infused cows and control cows had similar ALA, SDA and EPA levels; clearly indicating the need for ruminal protection of the precursor fatty acids ALA and SDA. Comparing the relative efficacy of SDA oils in enriching milk with EPA to that of oils that contain pre-formed EPA (e.g., tuna oil) provides an interesting insight, as at the same dietary oil inclusion rate, the peak EPA concentration reached using PEO was only a third of that achieved using ruminally protected tuna oil (PTO) (Fig. 4). This can largely be explained by a combination of two factors: (1) studies have shown that supplying EPA and DHA in the diet is more effective in altering the fatty acid concentrations of tissues than using their precursors (James et al., 2003), and (2) because EPA and DHA constitute 25–27 g/100 g of total fatty acids in tuna oil, while SDA is only 10–15 g/100 g of total fatty acids in Echium oil. Thus, comparison of the studies needs to be tempered by differences in the dose rate of the individual FA. The 5–6 d delay before a shift occurred in EPA concentration in milk fat under PEO supplementation (Fig. 4) provides a contrast to PTO supplementation in that the delay in response, and the lower peak concentrations achieved, lends support to our earlier suggestion that including EPA and/or DHA in the diet is a more effective way of enriching animal products with that LC n−3 PUFA than using precursor fatty acids. Still, use of PEO has resulted in an increase in total LC n−3 PUFA in milk and abomasal infusion data from Bernal-Santos et al. (2010) showed less delay in change in EPA levels (i.e., peaking by Day 3) but with a similar limitation of lower peak concentration compared to including EPA in the diet. The lack of response in DHA in milk fat is congruent with observations in other species. In chickens, we showed that SDA supplementation was only effective in changing EPA and DPA, but not DHA (Kitessa and Young, 2009). SDA supplementation in humans also caused a doubling of plasma EPA levels without effect on DPA or DHA (James et al., 2003). It is possible that the lack of response in DHA might not be limited to biosynthetic inefficiency. For example Cunnane (2003) reported an increase in ␤-oxidation of C18 fatty acids with each increase in the number of double bonds from C18:0 (stearic) to C18:3 (ALA), and suggested that the fourth double bond in SDA might further enhance ␤-oxidation of SDA. Hence, some of the advantages

S.M. Kitessa, P. Young / Animal Feed Science and Technology 170 (2011) 35–44

(b)

3.50

0.70

3.00

Stearidonic aciid, g/100 g tota S al fatty acids

Alpha lin nolenic acid, g/100g total fatty g acids

(a)

2.50 2.00 1.50 1.00 y = 1.011±0.0767 + 0.130±0.0125x R² = 0 0.54; 54 P<0.00 P<0 001 1

0.50 0.00 0

2

4

6

10

8

0.60 0.50 0.40 0.30 0 30 0.20 y = 0.087±0.0242 + 0.0337±0.0040x R² = 0.44; P<0.001

0.10 0.00 -0.10 0

12

2

4

6

10

8

12

Day fed PEO

Day fed PEO

(d)

(c)

0.35

4.5

Eicosapentaenoic acid, g/ 00 g total fatt acids

Total n-3 PUFA, g/100 g total fatty aciids

41

4.0 3.5 3.0 2.5 2.0 1.5 10 1.0

y = 1.156±0.1001 + 0.191±0.0165x R² = 0.59; P<0.001

0.5 0.0 0

2

4

6

8

10

12

y = - 0.033±0.0165 + 0.021±0.0027x R² = 0.40; P<0.001

0.30 0.25 0.20 20 0 0.15 0.10 0.05 0.00 -0.05

0

2

4

Day fed PEO

6

8

10

12

Day fed PEO

(e) Docosap pentaenoic acid, g/100 g tota al fatty acid ds

0.25

0.20

0.15

0.10

0.05 y = 0.143±0.0229 - 0.020±0.0032x +0.002±0.0004x2 R² = 0.25; P<0.001 0.00

-0.05

0

2

4

6

8

10

12

Day ay fed ed PEO Fig. 3. Observed (scatter plot) and fitted values (line) for concentrations of (a) ALA, (b) SDA, (c) total n−3 PUFA, (d) EPA and (e) DPA in milk over 10 d of supplementation of dairy cows with ruminally protected Echium oil.

of bypassing the -6 desaturase step required to convert ALA to SDA may be countered by this enhanced ␤-oxidation. The importance of this finding for SDA metabolism needs to be confirmed using isotope studies. The pattern of change in milk SDA concentration as days on PEO supplementation increased was similar to changes in milk EPA achieved through increasing days on dietary tuna oil in our previous study (Fig. 5). In both studies, milk concentrations of dietary fatty acids plateaued from Day 7 onwards. The difference in the peak concentration achieved was most likely a reflection of the nearly two fold difference in the concentration of EPA + DHA in tuna oil (25–27 g/100 g) versus SDA in Echium

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Eicosapentaenoic acid, g/100 g total fatty acids

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0.8 PEO, this study

0.7

PTO, Kitessa et al. 2004

0.6 0.5

Peak concentration difference

0.4 0.3 0.2 Respose delay

0.1 0.0 -0.1

1

2

3

4

5

6

7

8

9

10

Days fed supplement

Fig. 4. Change in EPA concentration in milk fat of cows supplemented (30 g/kg dry matter intake) with ruminally-protected tuna oil (PTO, Kitessa et al., 2004) or ruminally-protected Echium oil (PEO, this study).

g/100 g total fatty acids

0.8 0.7

SDA, this study

0.6

EPA, Kitessa et al. 2004

0.5 0.4 0.3 0.2 0.1 0.0 -0.1

1

2

3

4

5

6

7

8

9

10

Days fed supplement Fig. 5. Change in milk SDA and EPA concentration when cows were supplemented (30 g/kg dry matter intake) with ruminally protected tuna oil (Kitessa et al., 2004) or ruminally-protected Echium oil (this study).

oil (12–14 g/100 g). The importance of this increase in SDA content in milk fat from a human health perspective remains to be determined. Peak yield of SDA/cup of fresh whole milk, at 40 mg, was a third of that reported by Kitessa and Young (2009) for a serving of 100 g of thigh muscle from SDA supplemented broiler chicken (141 mg). At present, there is no health claim for SDA other than that related to its metabolic surrogate status for EPA (Whelan, 2009). Although none of the changes amounted to nutritional significance in terms of product differentiation (i.e., high MUFA milk), supplementation of SDA had a positive impact on total MUFA in milk. That is, SDA supplementation increased total milk MUFA content, but did not create high MUFA milk, at least at our rate of inclusion. Nevertheless, this was consistent with our previous observation, where grazing dairy cows were supplemented with ruminally protected tuna oil, of modest changes in total SFA and MUFA in milk (Kitessa et al., 2004). The marked fluctuations in supplement intake from Days 5 to 10 and the accompanying reduction in milk yield were unexpected. In the past, we were able to supplement dairy cows (Kitessa et al., 2004), dairy sheep (Kitessa et al., 2003) and dairy goats (Kitessa et al., 2001) with ruminally protected tuna oil without effects on intake and milk yield. It was only when unprotected tuna oil was fed to lactating dairy goats that 49.8% reduction in intake and 28.2% reduction in milk yield occurred (Kitessa et al., 2001). In the current study with PEO, milk yield over Days 5–10 was 16.9% lower than that over Days 1–4 of PEO supplementation, which suggests that the decrease in milk yield may be due to inadequacy in ruminal protection of the Echium oil rather than use of Echium oil per se. However, further work with in vitro incubation of ruminally protected Echium oil for different lengths of time is needed to determine whether the reduction in intake is related to the proficiency of our ruminal protection method, or secondary compounds in the oil. As the Echium oil used in this study was human food grade oil, the former appears more likely than the latter. In available natural oils containing SDA, it is a small component of their total FA, usually less than 10 g/100 g of total FA. Even the richest natural source, Echium oil, has SDA at only 10–15 g/100 g of total FA. This means the only avenue to increase its intake by livestock is to increase the amount of source oil in the diet, and this has implications in its use in ruminant diets to increase LC n−3 PUFA in milk and tissues. Richer dietary source of SDA have now been produced using plant biotechnology. Indeed, using -15 desaturase genes, Ursin (2003) and Eckert et al. (2006) reported SDA concentrations of 16–23 g/100 g of total fatty acids in canola oil and 30 g/100 g of total fatty acids in soybean oil, respectively; up to triple the concentration of SDA used in our study. However, as stated earlier results from the study by Bernal-Santos et al. (2010),

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where the abomasally infused oil contained 27 g/100 g SDA, double the amount in our Echium oil, did not result in greater enrichment with EPA than observed in our study. Another limitation of using current SDA containing oils is that, even before feed formulation and ruminal protection costs are considered, the high price for the oil (at more than $52,000 US dollars/t), is a considerable disincentive. SDA from genetically modified plants has the potential to provide more SDA/unit volume of oil, as well as (or more importantly) a less expensive unit price which may make use of SDA in livestock feed a way of increasing n−3 PUFA in milk and meat commercially feasible. A substantiated human health claim for SDA, rather than as a surrogate for EPA, would also substantially improve the commercial prospects of using SDA containing oils to enhance the human health benefits of animal derived foods. 5. Conclusions Supplementation of dairy cows grazing irrigated ryegrass white clover pasture with SDA containing oils was partially successful in raising the n−3 long chain PUFA content of their milk fat, but the EPA content of milk fat 4 fold to 13.9 mg/250 ml. DHA was undetectable in milk fat before, during or after Echium oil supplementation. Overcoming the -6 desaturation step did not seem to yield higher milk enrichment with EPA and DHA at a level which would be expected based on overcoming a rate limiting step. In order to optimise the role of land based n−3 precursors in animal nutrition, there is a need to examine further the role of processes other than the -6 desaturation step in n−3 metabolism in livestock. With respect to Echium oil, further studies with more cows/treatment over a longer period (e.g., whole lactation) are recommended to elucidate the optimal inclusion rate and commercial feasibility of using SDA oils to raise the claimable EPA and DHA content of their milk fat. Such studies will also need to consider shelf stability and organoleptic integrity of the enriched milk products. Acknowledgements We are very grateful to the Maughan family in Harvey, Western Australia, for providing their dairy facility for this experiment. The manager of the farm, Mr. Stuart Maughan, played a crucial role in accessing the dairy facility. We would like to thank Mr. Albert Shultink who helped us with milking. This project was wholly funded by the CSIRO Food Futures Flagship. Laboratory facilities were provided by CSIRO Livestock Industries. Both authors have no conflict of interest to disclose. References Bernal-Santos, G., O’Donnell, A.M., Vicini, J.L., Hartnell, G.F., Bauman, D.E., 2010. 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