Fatty acid profiles of muscle, liver, heart and kidney of Australian prime lambs fed different polyunsaturated fatty acids enriched pellets in a feedlot system

We investigated the effect of various dietary polyunsaturated fatty acid (PUFA) sources on the fatty acid profiles of muscle, liver, heart and kidney of Australian prime lambs. Seventy-two White Suffolk x Corriedale first-cross lambs weaned at 6 months of age were randomly allocated to the following six treatments: (1) Control: Lucerne hay only; wheat-based pellets infused with 50 ml/kg dry matter (DM) of oil from (2) rice bran (RBO); (3) canola (CO); (4) rumen-protected (RPO), (5) flaxseed (FSO) and (6) safflower (SO) sources in a completely randomized experimental design. Lambs in CO, FSO, SO and RPO treatments achieved contents of eicosapentaenoic acid (EPA, 22:5n-3) plus docosahexaenoic acid (DHA, 22:6n-3) in the longissimus dorsi muscle ranging from 31.1 to 57.1 mg/135 g, over and above the 30 mg per standard serve (135 g) threshold for “source” claim under the Australian guidelines. There was no difference in n-3 LC-PUFA contents in longissimus dorsi muscle of lambs fed dietary oils of plant origin. The highest 18:3n-3 (ALA) contents achieved with FSO diet in the muscle, liver and heart were 45.6, 128.1 and 51.3 mg/100 g, respectively. Liver and kidney contained high contents of n-3 LC-PUFA (ranging from 306.7 to 598.2 mg/100 g and 134.0 to 300.4 mg/100 g, respectively), with all values readily exceeding the ‘good source’ status (60 mg per serve under Australian guidelines). The liver and kidney of PUFA fed lambs can be labelled as ‘good source’ of n-3 LC-PUFA based on EPA and DHA contents stipulated by the Food Standards of Australia and New Zealand guidelines. Therefore, if lamb consumers consider eating the liver and kidney as their dietary protein sources, they can adequately obtain the associated health benefits of n-3 LC-PUFA.


Results
Fatty acid profiles in basal and supplementary feed. Fatty acid composition (as % total fatty acids) of both basal and supplementary feeds are presented in Table 1. Lucerne hay contained high proportions of PUFA and n-3 PUFA (48.7 and 28.2%, respectively). The major fatty acids in lucerne hay were 18:2n-6 (LA) and 18:3n-3 (ALA), with relatively high levels of 19.0 and 27.5%, respectively. Similarly, flaxseed oil infused pellets (FSO) contained relatively high levels of PUFA and n-3 PUFA (62.2 and 20.7%, respectively) and the major fatty acids in FSO were LA (41.0%), ALA (20.5%) and 18:1n-9 (18.8%). The remaining treatments were rich in PUFA and n-6 PUFA with relatively high levels ranging from 39 to 59 (%) and 34 to 57(%), respectively, in which the major fatty acids were LA and 18:1n-9. The highest n-6 PUFA level was found in safflower oil infused pellets (SO) (mainly LA: 56.5%). The rumen protected oil infused pellets (RPO) had the highest proportions of n-3 LC-PUFA com- Fatty acid profiles in the longissimus dorsi muscle. Fatty acid contents (mg/100 g) of the longissimus dorsi muscle tissue is depicted in Table 2. There was no difference among treatments in total fatty acids (FA), saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA). The PUFA, n-3 LC-PUFA, n-3 PUFA and 18:1n-7t of supplemented treatments were significantly higher than that of control. The RPO treatment had the highest contents of n-3 LC-PUFA (66 mg/100 g); mainly EPA, DPA and DHA. The highest mean of ALA was found in the FSO treatment (45.6 mg/100 g) and the lowest value was in the control. All supplemented treatments, except for RPO, had significantly higher values of LA than that of control. The SO treatment contained the highest contents of n-6 PUFA and n-6/n-3 ratios of all treatments varied from 1.8 to 3.5. Fatty acid profiles in edible organs. Fatty acid profiles in the liver. Table 3 shows the fatty acid contents (mg/100 g) of liver tissue. The means of all fatty acid components in the liver were markedly higher than those in the muscle and significant differences were evident in both saturated and unsaturated FA. The CO treatment had the highest total FA (3775.0 mg/100 g) and MUFA (1018.0 mg/100 g) contents. The lowest SFA and 18:0 contents were found in the RPO group (1086.0 mg/100 g and 595.5 mg/100 g, respectively). Contents of n-6 PUFA and LA in all supplemented treatments, except for RPO, were significantly higher than in the control. RPO had the highest n-3 PUFA and n-3 LC-PUFA which mainly consisted of EPA, DPA and DHA. The ratio of n-6/n-3 in all treatments ranged from 0.6 to 1.9. The highest value of ALA was found in the FSO treatment (128.1 mg/100 g).
Fatty acid profiles in the heart. Table 4 depicts fatty acid contents (mg/100 g) of the heart. There was no difference in total FA, SFA and MUFA; similar to the same trend observed in the longissimus dorsi muscle. PUFA and 18:1n-7t in all supplemented treatments were significantly higher than in the control treatment. The control had the lowest contents of n-6 PUFA and LA. The RPO treatment had the highest n-3 LC-PUFA and n-3 PUFA, whilst the lowest contents of these FA were in the control treatment. FSO had the highest ALA content (51.3 mg/100 g). SO and rice bran infused pellets (RBO) treatments had the highest n-6/n-3 ratio and RPO had the lowest. Generally, the n-6/n-3 ration in all treatments ranged from 2.7 to 6.6.
Fatty acid profiles in the kidney. Table 5 shows the fatty acid contents (mg/100 g) in the kidney. SFA contents did not differ among treatments. All supplemented treatments had significantly higher PUFA and LA contents than the control. RPO treatment had the highest n-3 LC-PUFA and n-3 PUFA contents. The highest total FA, n-6 PUFA and LA contents were in the SO treatment (2305.0; 752.6 and 375.8 mg/100 g, respectively). The observed trend of n-3 LC-PUFA content was similar to the pattern observed in the liver where only FSO treatment had greater n-3 LC-PUFA content than the control. The range in n-6/n-3 ratio in all treatments was from 1.3 to 4.0 in which the SO treatment had the highest ratio.

Discussion
Past studies had used raw oil or oilseeds as supplements in ruminant feeding trials. In order to minimise oxidative rancidity, improve stability and shelf life of both feeds and meat products, we chose to use oil-infused pellets as an innovative means of delivering n-3 LC-PUFA to lambs. Besides, available information on the effect of oil-infused pellets on the fatty acid profile of lamb is scanty in the published literature. Furthermore, the use of oil-infused pellets is less bulky than raw oil or oilseeds, hence our strategy minimises transportation and storage issues in commercial lamb feed production. The fatty acid profile of lucerne hay was similar to that reported by Nguyen et al. 10 . Many past studies had reported that flaxseed oil contained a high concentration of ALA [13][14][15][16] . Our study showed that FSO had the highest concentration of ALA among the supplemented treatments. The high concentration of LA in SO observed in this study was consistent with other previous studies 12,17 . The RPO treatment contained high concentrations of EPA, DPA and DHA because of its fish oil content.
In the longissimus dorsi muscle, the higher contents of n-3 LC-PUFA in the supplemented treatments can be explained by the higher levels of by-pass lipids and protection from excessive lipolysis and extensive biohydrogenation in the rumen 18,19 as observed in previous studies 9, 10,20 . On the other hand, it would seem that the levels and extent of biohydrogenation and conversion of stearate (18:0) into Oleate (18:1n-9) were similar, hence the   observed result where no difference was found in the 18:1n-9 content of the longissimus dorsi muscle between the supplemented and control treatments. This observation could also be indicative of similar delta-nine desaturase enzyme activities among treatment groups since it is the primary enzyme responsible for the conversion of stearate to oleate in the biochemical pathway of in vivo lipid synthesis. One of the biohydrogenation intermediates of ALA and LA is 18:1n-7t 21 . The increased contents of ALA with flaxseed supplementation has also been reported in a previous study by Wachira et al. 22 who reported that supplementation with flaxseed doubled the proportion (x100) of 18:3n-3 in the longissimus dorsi muscle from 1·4 to 3·1. Demirel et al. 23 reported that feeding protected linseed resulted in 3.0-fold higher concentration of ALA in lamb muscles compared with Megalac (calcium soap of palm fatty acid distillate). The observed trend in our current study where the ALA content in the longissimus dorsi muscle of lambs in the FSO treatment was 1.9 times that of the control but similar EPA and DHA contents, could be due to the low elongase enzyme activity for the elongation of C18 to ≥C20 FA and subsequent desaturation of ALA into more potent n-3 LC-PUFA, and the capacity of muscle lipids to incorporate ALA into n-3 LC-PUFA 18 .
In contrast, our finding that the RPO treatment had double EPA and triple DHA contents in longissimus dorsi muscle tissue in comparison with the control was in line with the results of Wachira et al. 22 who reported that feeding fish oil increased the muscle proportion (x100) of EPA from 0·7 to 2·3 and DHA from 0·3 to 0·8. The greatest n-6 to n-3 ratio in SO treatment could be explained by high n-6 PUFA contents of safflower oil (57%) as shown in Table 1. According to Pannier, et al. 24 , a standard serve was 135 g of meat. Food Standards of Australia and New Zealand (FSANZ) regulate that a given food can be claimed as a 'source' of omega-3 fatty acids if the EPA + DHA contents of that food is greater than 30 mg per serve 25 . Lambs in the CO, FSO, SO and RPO treatments had EPA + DHA content in the longissimus dorsi muscle exceeding the 30 mg per serve cut-off point for "source" claim under the FSANZ guidelines 25 (as depicted in Fig. 1) and the n-6/n-3 ratio within the desirable  29 revealed that DPA was related to various improvements in human health and served as a storage depot for EPA and DHA in the human body. Howe, et al. 27 suggested that the importance of DPA in delivering the health benefits associated with n-3 LC-PUFA was equal to that of EPA and DHA. Therefore, if DPA was considered as an omega-3 content claim, a significant amount of n-3 LC-PUFA intake (~39%) would be added to lamb meat consumers as demonstrated in Fig. 1. Edible by-products (organ meats) from cattle, pigs and lambs range from 20 to 30% of the animal's liveweight 30 . The findings of Bester et al. 31 showed that lamb organ meats are nutrient-dense animal source foods that provide protein, zinc and iron to meet the nutritional requirements of humans. Furthermore, the edible internal organs of prime lambs (heart, liver and kidney) are rich in essential fatty acids 32 . Edible internal organs are widely consumed in many countries as special ingredients in traditional foods such as kidney pie in the United Kingdom, cooked and diced delicacies in South America, sheep liver in Iran and paté in many countries 33 . Therefore, knowledge of the fatty acid profiles of lamb edible internal organs could contribute innovations in value-addition of edible meat by-products and generate extra income for lamb producers and slaughterhouses.
The liver is the first organ to receive and metabolise fatty acids after lipid digestion and absorption. As with previously reported studies 11,32 , individual fatty acid content was markedly high in the liver. Our observation of the lowest SFA and 18:0 contents in RPO treatment is supported by the work of Gallardo et al. 34 who found that supplementation of ewes with 3% Ca soap FA in fish oil based diets dramatically reduced 18:0 content in milk. Shingfield et al. 35 also observed that increased fish oil percentage in the diet linearly decreased 18:0 contents in the duodenum. The highest MUFA in CO (mainly 18:1n-9 contents) can be explained by the high MUFA (45.7%) input of CO into the diet. Similarly, the high LA and ALA contents (128.1 mg/100 g) in the liver of FSO fed lambs indicates that despite rumen biohydrogenation, a significant amount of ALA had escaped and was absorbed in the  intestines and metabolised in the liver. Demirel et al. 23 reported that feeding linseed more than doubled the concentration of ALA in the liver compared to Megalac diet. Out of all the PUFA of plant oil origin in this study, only FSO treatment had an n-3 LC-PUFA content that was significantly higher than the control. This could be explained in part, by the high ALA content in FSO being converted to n-3 LC-PUFA. Our result was in line with the findings of Alhazzaa et al. 36 who reported that the gradual increase in ALA concentration led to a proportional increase in EPA in hepatocytes. The liver EPA and DHA contents of PUFA fed lambs ranged from 162.2 to 414.0 mg/100 g which was similar to EPA and DHA contents of wild Australian seafoods such as fish (containing 350 mg/150 g (~233 mg/100 g), shellfish (containing 225 mg/150 g (~150 mg/100 g) (see the review by Nichols et al. 37 ). Furthermore, the n-6/n-3 ratio in the liver is within desirable level 26 . Therefore, the n-3 LC-PUFA content in the liver of PUFA fed lambs can be labelled as reaching the claimable 'good source' based on EPA + DHA threshold of the Food Standards of Australia and New Zealand guidelines 25 . Therefore, if lamb consumers consider the liver as their dietary protein source, they would obtain the associated dietary health benefits. In the heart, Kashani et al. 38 reported that supplementation of lambs with Spirulina at the medium-level increased both n-3 and n-6 PUFA compositions. The results from our study showed that total PUFA, n-3 LC-PUFA and n-6 PUFA contents of heart were all enhanced by supplementation with various PUFA enriched pellets. Furthermore, n-3 LC-PUFA contents in the heart of supplemented lambs ranged from 70.1 to 147.5 mg/100 g; indicating that the heart could be a 'good source' of n-3 LC-PUFA 25 . However, the n-6/n-3 ratio of the heart in all lambs with the exception of RPO, was much higher than the minimum desirable level of 4.0 26 . The kidney samples in the present study had high contents of key health-benefitting n-3 LC-PUFA in agreement with the findings of Malau-Aduli et al. 32 . Specifically, our current findings showed that the contents of n-3 LC-PUFA ranged from 121.4 to 300.4 mg/100 g which is equal to the n-3 LC-PUFA content in Australian wild fish or shellfish (see details in the review by Nichols et al. 37 ). The result of the present study demonstrated that supplementation of lambs with PUFA enriched pellets  substantially increased kidney n-3 LC-PUFA contents. Nguyen et al. 11 also reported that supplementation of lambs with canola and flaxseed oils at 5% enhanced n-3 LC-PUFA contents of the kidney.

Conclusion
Supplementation of lambs with PUFA enhanced pellets in a feedlot system is needed to boost the contents of n-3 LC-PUFA in the longissimus dorsi muscle. Lambs in CO, FSO, SO and RPO treatments had contents of EPA + DHA in longissimus dorsi muscle that were over the claimable 'source' level of omega-3 as stipulated by the Foods Standards of Australia and New Zealand guidelines. Supplementation of lambs with oils of plant origin including canola, rice bran, flaxseed and safflower had similar effects on the contents of n-3 LC-PUFA in lamb longissimus dorsi muscle. FSO had the highest contents of ALA in the muscle, liver and heart tissues. Liver and kidney of PUFA fed lambs can be utilised as omega-3 rich foods on the basis of their high n-3 LC-PUFA contents and desirable n-3/n-6 ratios. In line with the tested hypothesis in this study, it is proven that canola, flaxseed, safflower and rumen protected oil infused pellets can be used in feedlot systems for producing healthy lamb meat as a source of omega-3 food. The cost-efficacy of supplementing lambs with these oil-infused pellets has been presented in detail in a separate stand-alone manuscript (currently under review) and details will not be duplicated herein. Suffice it to say that the feed cost per unit gain in Australian dollars/kg (AU$/kg) for RBO and CO supplements were 3.0 AU$/kg each, 4.1, 4.2 and 6.3 AU$/kg for RPO, FO and SO supplements, respectively.

Materials and Methods
This study was carried out at the Tasmanian Institute of Agriculture's Cressy Research and Demonstration Station, Burlington Road, Cressy, Tasmania, Australia (41° 43′S, 147° 03′E) from April to June, 2016. The use of animals and procedures performed in this study were all approved by the University of Tasmania Animal Ethics Committee (Permit No A0015657).  Table 5. Fatty acid contents (mg/100 g) of the kidney. * Values within the same row bearing different superscripts differ (P < 0.05). All other abbreviations are as defined in Tables 1 and 2 respectively. Each lamb in the supplemented treatments was fed 1 kg pellets/day. The feeding trial lasted 10 weeks including a 3-week adaptation period and followed by 7 weeks of full supplementation. Freshly offered feed was given every day at 9.00 h after residual feed had been collected and weighed. The level of refusal of the concentrate supplement ranged from 0.14-0.18 kg DM/head/day. The chemical composition of the experimental and basal diets is presented in Table 6.
Procedures of feed sample and chemical composition determination. Every week, feed samples were taken from each pellet bag and bale of lucerne hay and bulked. The bulked samples were stored at −20 °C    32 . In short, total lipids in 1 gram of un-homogenised and wet liver, kidney, heart and muscle tissue and feed samples were extracted overnight using a modified Bligh and Dyer 41 . The first step of the process was a single-phase overnight extraction using CHCl 3 :MeOH:H 2 O (1:2:0.8 v/v). The second step involved phase separation with the addition of CHCl 3 :saline Milli-Q H 2 O (1:1 v/v) and followed by rotary evaporation of the lower chloroform phase at 40 °C to obtain total lipids. An aliquot from each total lipid extract was used in transmethylation process with MeOH:CHCl 3 :HCl (10:1:1 v/v) for 2 h at 80 °C and then fatty acid methyl esters (FAME) were extracted three times using hexane:CHCl 3 (4:1 v/v). A known concentration of an internal standard (19:0) was added in 1500 µL vial containing the extracted FAME. The FAME was analysed on a 7890B gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an Equity TM -1 fused 15 m silica capillary column with 0.1 mm internal diameter and 0.1-μm film thickness (Supelco, Bellefonte, PA, USA), a flame ionisation detector, a split/splitless injector and an Agilent Technologies 7683 B Series autosampler. The gas chromatograph conditions were: splitless mode injection; carrier gas He; initial oven temperature 120 °C and then increased to 270 °C at 10 °C/min and to 310 °C at 5 °C/min. The Agilent Technologies ChemStation software (Palo Alto, California USA) was used to quantify fatty acid peaks. The fatty acid identities were confirmed by gas chromatograph-mass spectrometric (GC/MS) analysis using a Finnigan Thermoquest GCQ GC/MS fitted with an on-column injector and Thermoquest Xcalibur software (Austin, Texas USA). The gas chromatograph (GC) was equipped with an HP-5 cross-linked methyl silicone-fused silica capillary column (50 m × 0.32 mm internal diameter) which was of similar polarity to the column described above. The carrier gas was helium and GC conditions were according to the description of Miller, et al. 42  Statistical analysis. Fatty acid data were initially transformed into fatty acid contents (mg/ 100 g). These data were then analysed using the General Linear Model procedure (PROC GLM) of Statistical Analysis System 45 . The fixed effects of treatment and gender and their second order interaction effects on fatty acid contents were tested. The initial full statistical model used for the analysis was: Y = μ + O i + G j + (OG) ij + (OG) 2 ij + (OG) 3 ij + e ijk where Y = dependent variable, μ = overall mean, O i = oil supplementation treatment, G j = gender, brackets and superscripts represent linear and cubic second-order interactions and e ijk = residual error. Linear and cubic orthogonal contrasts indicated that gender was not a significant factor, hence it was removed from the final model that assessed the impact of treatment only. Duncan's multiple range tests were used to determine the differences amongst treatments at a minimum threshold of p < 0.05 level.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.