Enhanced Omega-3 Polyunsaturated Fatty Acid Contents in Muscle and Edible Organs of Australian Prime Lambs Grazing Lucerne and Cocksfoot Pastures

The enhancement of health-beneficial omega-3 long–chain (≥C20) polyunsaturated fatty acid (n-3 LC-PUFA) contents in the muscle, liver, heart, and kidney of Australian prime lambs through pasture grazing and supplementation with oil infused pellets was investigated. Forty-eight first-cross prime lambs were randomly assigned into a split-plot design with pasture type as the main plot effect and pellet supplementation as a sub-plot effect in a feeding trial that lasted for nine weeks. The n-3 LC-PUFA content in Longissimus dorsi muscle of all lambs was well above the 30 mg threshold for “omega-3 source” nutrition claim under the Australian Food Standards and Guidelines. Pasture type impacted the fatty acid contents in muscle, heart, and kidney of prime lambs. Lambs grazing cocksfoot grass only had high 18:3n-3 (ALA) and n-3 LC-PUFA contents (67.1 mg/100 g and 55.2 mg/100 g, respectively) in the Longissimus dorsi muscle, which was not significantly different (p > 0.8990) from the contents of lambs grazing only lucerne. Supplementation of pellets with or without oil infusion to grazing lambs generally decreased the ALA and n-3 LC-PUFA contents and increased the n-6/n-3 ratio in the Longissimus dorsi muscle. The fatty acid content in the internal organs of grazing lambs was also affected by pellet supplementation. The liver and kidney of grazing lambs were both “good sources” (60 mg/100 g) of omega-3. The cocksfoot grass showed considerable potential for producing healthy, premium quality meat with high contents of n-3 and n-3 LC-PUFA, which may consequently enhance the omega-3 intake of Australian lamb consumers.


Materials and Methods
The study was conducted at the Tasmanian Institute of Agriculture's Cressy Research and Demonstration Station, Burlington Road, Cressy, Tasmania, Australia from October to December, 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).

Animals, Diets, Experimental Design, and Feed Sample Collection
Experimental design: The experiment was a split-plot design with the basal pastures being cocksfoot cv. porto and lucerne. Main plot: Total of 1.5 ha of Cocksfoot cv. porto pasture with three equal 0.5 ha plots. Each 0.5 ha plot was split into four equal 0.125 ha sub-plots. Therefore, cocksfoot cv. porto pasture had 12 equal 0.125 ha sub-plots units in the main plot. Similarly, there were 12 equal 0.125 ha subplots as main plot units for lucerne pasture. These plots were used for rotational grazing.
Sub-plots: Lambs were divided into four groups. Lambs in each group were allocated to one of the following four treatments: (1) cocksfoot cv. porto or lucerne pastures only as the control treatment; (2) cocksfoot cv. porto or lucerne pastures supplemented with: no oil pellets (NOP); (3) canola oil infused pellets (CO); and (4) rice bran oil infused pellets (RBO). Thus, each main plot unit had sub-plots.
Forty-eight White Suffolk x Corriedale first-cross prime lambs with an average liveweight (LWT) of 38.7 ± 0.7 kg weaned at six months were randomly allocated to 12 groups of four lambs balanced by gender. The 12 groups of lambs were allocated to six cocksfoot pasture subplots and six lucerne pasture subplots. The experimental lambs were grazed daily on pastures from 07:00 to 18:00 h and rotated to fresh pasture subplots every 14 days during the trial. Fresh water was available at all times throughout the grazing trial. The lambs in the supplemented treatments were individually offered oil infused pellets at 1 kg/head/day before going to pastures. The feeding trial lasted for nine weeks comprising three weeks of adjustment and six weeks of post-adjustment data collection.
Basal pasture and feed samples: the pasture samples were taken weekly from five area sites 50 cm × 50 cm of each subplot and then homogenized for withdrawing subsamples. The oil infused pellets were sampled from each bag and stored at −20 • C until the end of the trial.

Slaughter Protocol and Fatty Acid Analysis
All lambs were slaughtered at a commercial abattoir (Tasmanian Quality Meats, Cressy, Tasmania) adjacent to the experimental site after staying in the animal house for 12 h without feed and free access to fresh water. The slaughter procedures prescribed by the Meat Standards of Australia guidelines was strictly applied. Samples of liver, heart, and kidney were taken at the abattoir and immediately vacuum-sealed, code-labelled, and stored at −20 • C until FA analysis. Carcasses were chilled for 24 h at 4 • C before transporting to Robinson Meats, Glenorchy, Hobart, Tasmania, Australia. Thereafter, the Longissimus dorsi muscle were sampled at the 12/13 th rib of each carcass as a commercial loin chop (approximately 200 g) for subsequent FA analysis. FA analysis was as described by Malau-Aduli et al. [33]. Briefly, the FA analysis included three processes. 1. A single-phase overnight extraction using CH 2 Cl 2 :MeOH:H 2 O (1:2:0.8 v/v) to extract total lipids from 1 gram of un-homogenised and wet liver, kidney, heart, and muscle tissues and feed samples according to a modified Bligh and Dyer protocol [35]. Phase separation with the addition of CH 2 Cl 2 :saline Milli-Q H 2 O (1:1 v/v) was carried out and followed by rotary evaporation of the lower CH 2 Cl 2 phase at 40 • C to obtain the total lipids. 2. Methylation: An aliquot was taken from each total lipid extract for transmethylation with MeOH:CH 2 Cl 2 :HCl (10:1:1 v/v) for 2 h at 80 • C and Milli-Q H 2 O (1 mL) was then added before the FA methyl esters (FAME) extraction process with hexane:CH 2 Cl 2 (4:1 v/v); extraction was performed three times. 3. Fatty acid quantification: Extracted FAME in glass vials were made up to a volume of 1500 µL with a known concentration of an internal injection standard (19:0). A 7890B gas chromatograph (GC) (Agilent Technologies, Palo Alto, CA, USA) equipped with an EquityTM-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 was employed to analyse the FAME. The GC 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. Peak quantification was performed using Agilent Technologies ChemStation software (Palo Alto, CA, USA). FA identifications were confirmed by GC-mass spectrometric (GC/MS) analysis with a Thermo Scientific 1310 GC coupled with a TSQ triple quadropole (Thermo Fisher Scientific, Milan, Italy) PTV injector and Thermo Scientific XcaliburTM software (Austin, Texas USA). The GC was equipped with a 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 operating conditions was previously described by Miller, et al. [36] and helium served as the carrier gas. FA percentages (FA%) and contents (FA mg/100g) were calculated as follows [37], where 0.916 was the lipid conversion factor as cited by Clayton [38].

Statistical Analysis
FA data were initially transformed into FA contents (mg/100 g). Thereafter, the data were analysed using the split-plot model in General Linear Model procedures (PROC GLM) of the Statistical Analysis System software (SAS Institute, North Carolina, USA) [39]. Pasture types were considered as the main plot effects and supplementation of pellets with or without oil infusion as subplot effects. Non-significant interactions between fixed effects were dropped from the analytical model and treatment differences were declared significant at p ≤ 0.05 using Bonferroni probabilities. Probability values ranging between p ≤ 0.056 and p ≤ 0.059 were deemed as "tending towards significance".

Results
The chemical composition of experimental diets is presented in Table 1. Dry matter (DM) of lucerne and cocksfoot were similar (p > 0.7880), while those of the pelleted supplements were much higher (p < 0.05) and ranged from 89.1% to 91.1%. Crude protein content of the different supplemented Nutrients 2018, 10, 1985 5 of 19 pellets ranged between 13.3% and 15.7%, which was lower (p < 0.05) than that in the basal lucerne feed (18.6%), but higher than in cocksfoot (13.3%). Acid detergent fibre (ADF) and Neutral detergent fibre (NDF) contents of the different supplemented pellets ranged from 6.8% to 8.0% and from 18.3% to 19.9%, respectively, while ADF and NDF content of the basal feed were 35.9% and 43.8%, respectively. In terms of EE content, the level in the supplemented pellets fluctuated between 4.6% and 4.9%, which was at least three-fold higher than the amount in the basal feed (1.8%). ME content of all supplemented pellets was approximately 12 MJ/kg, whilst the basal feed contained 9.5 (MJ/kg) ME.  Table 2 shows the FA composition of supplementary feed and pastures. Cocksfoot cv. porto and lucerne pasture contained high proportions of ALA at 57.6 % and 51.9%, respectively. Supplementary feeds including NOP, CO and RBO had high levels of linoleic acid (LA, 18:2n-6) (50.3%, 32.9%, and 42.2%, respectively). A high relative level of 18:1n-9c was found in the NOP, CO and RBO treatments ranging from 24.5% to 44.5%. Cocksfoot cv. porto and lucerne pasture contained 73.1% and 69.7% of PUFA, respectively, while the PUFA proportion of the supplementary feeds varied from 39.2% to 54.5%. The n-3 PUFA levels of cocksfoot cv. porto and lucerne pastures were 58.0% and 52.2%, respectively, which were considerably higher than the n-3 PUFA levels of the three supplementary feeds. In contrast, NOP, CO, and RBO contained high relative levels of n-6 PUFA, ranging from 33.1% to 50.6%. The n-6/n-3 ratio of NOP and RBO diet treatments was similar and double the ratio of the CO treatment. The cocksfoot cv. porto and lucerne pastures had the lowest n-6/n-3 ratio (0.3) among all treatments.    Table 3, did not affect the total FA, MUFA, and PUFA contents in Longissimus dorsi muscle of grazing lambs. However, supplementation with pellets tended to decrease the ALA and n-3 PUFA contents in Longissimus dorsi muscle of grazing lamb, and the lowest values occurred in the RBO treatment. Lambs grazing on cocksfoot cv. porto or lucerne pastures only had similar ALA content (67.1 mg/100 g and 68.1 mg/100 g, respectively) in the Longissimus dorsi muscle (p > 0.899). Supplementation with NOP and RBO pellets increased the LA content in Longissimus dorsi muscle of lambs grazing on lucerne pasture. Lucerne grazing lambs supplemented with RBO had lower EPA and docosapentaenoic acid (DPA, 22:5n-3) contents in their Longissimus dorsi muscle than lambs grazing on lucerne pasture only. Pellet supplementation tended to decrease the total n-3 LC-PUFA and EPA + DHA + DPA contents (as demonstrated in Figure 1) in Longissimus dorsi muscle of lucerne grazing lambs and the lowest value occurred in the RBO treatment. Supplementation with pellets decreased the 18:0 content in the Longissimus dorsi muscle of lambs grazing cocksfoot cv. porto. The additional access to pellets by grazing lambs increased the n-6/n-3 ratio in the Longissimus dorsi muscle.

FA Composition of Pastures and Supplementary Feeds
Nutrients 2018, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/nutrients supplementation of NOP to lucerne grazing lambs significantly increased the n-6 PUFA, PUFA, and total FA of kidney tissues. Pellet supplementation did not change the EPA + DHA + DPA content in kidney of grazing lambs. Table 7 shows the FA contents of the different internal organs of prime lambs as affected by the two different types of pastures. There was no significant difference in the FA content in liver of lambs grazing on different pasture types. The ALA, EPA, PUFA, n-3 PUFA, and n-6 PUFA contents in Longissimus dorsi muscle of lucerne grazing lambs were higher than that of cocksfoot cv. porto grazing lambs. There was no difference in the n-3 LC-PUFA and EPA + DHA + DPA contents in Longissimus dorsi muscle, liver, heart, and kidney of lambs grazing on cocksfoot cv. porto and lucerne pastures ( Figure 2). The PUFA content in heart of lucerne grazing lambs (676.5 mg/100 g) was greater than that of cocksfoot cv. porto grazing lambs (640.6 mg/100 g). Lucerne grazing lambs had higher 20:3n-6 content in kidney than the cocksfoot cv. porto grazing lambs.  FA of liver: FA content of the liver are shown in Table 4. Pellet supplementation did not affect the SFA, MUFA, PUFA, n-3 PUFA, and n-3 LC-PUFA contents of grazing lambs. Supplementation of cocksfoot cv. porto with NOP and CO to grazing lambs tended to increase the ALA content in the liver. However, supplementation of pellets to lucerne grazing lambs did not change the ALA content in liver. Supplementation of CO to cocksfoot cv. porto grazing lambs resulted in higher EPA, DPA, PUFA, n-3 LC-PUFA, and EPA + DHA + DPA contents in liver in comparison with RBO supplementation. There was no difference in the EPA + DHA + DPA content of liver between grazing lambs with and without pellet supplementation ( Figure 1).

Effect of Pasture Types on the Fatty Acid Contents in Muscle, Liver, Heart, and Kidney
FA of heart: FA contents of the heart are demonstrated in Table 5. Pellet supplementation did not change the FA content of lucerne grazing lambs. Nevertheless, supplementation of NOP and CO significantly decreased the ARA and DPA contents in the heart of cocksfoot cv. porto grazing lambs. Furthermore, NOP supplementation to cocksfoot cv. porto grazing lambs lowered the DHA and PUFA contents in heart tissues. There was no difference in the EPA + DHA + DPA content in heart of grazing lambs.
FA of kidney: Table 6 demonstrates the FA contents of the kidney. The FA contents in kidney of cocksfoot cv. porto grazing lambs were not affected by pellet supplementation. However, supplementation of NOP to lucerne grazing lambs significantly increased the n-6 PUFA, PUFA, and total FA of kidney tissues. Pellet supplementation did not change the EPA + DHA + DPA content in kidney of grazing lambs. Table 3. Effect of pellet supplementation on IMF percentage (g fat/100 g) and fatty acid contents (mg/100 g) in Longissimus dorsi muscle tissue of grazing prime lambs * (LSM ± SE).

Items
Control   Table 5. Effect of pellet supplementation on total lipid percentage (g fat/100 g) and fatty acid contents (mg/100 g) in heart of grazing prime lambs (LSM ± SE) *. 11.5 ± 0.5 a 11.5 ± 0.5 a 9.8 ± 0.5 b 11.5 ± 0.5 a 10.9 ± 0.5 ab 11.7 ± 0.5 a 10.9 ± 0.5 ab 11.2 ± 0.5 ab 20:4n-3 2.  Table 6. Effect of pellet supplementation on total lipid percentage (g fat/100 g) and fatty acid contents (mg/100 g) in kidney of grazing prime lambs (LSM ± SE) *.   3.3. Effect of Pasture Types on the Fatty Acid Contents in Muscle, Liver, Heart, and Kidney Table 7 shows the FA contents of the different internal organs of prime lambs as affected by the two different types of pastures. There was no significant difference in the FA content in liver of lambs grazing on different pasture types. The ALA, EPA, PUFA, n-3 PUFA, and n-6 PUFA contents in Longissimus dorsi muscle of lucerne grazing lambs were higher than that of cocksfoot cv. porto grazing lambs. There was no difference in the n-3 LC-PUFA and EPA + DHA + DPA contents in Longissimus dorsi muscle, liver, heart, and kidney of lambs grazing on cocksfoot cv. porto and lucerne pastures (Figure 2). The PUFA content in heart of lucerne grazing lambs (676.5 mg/100 g) was greater than that of cocksfoot cv. porto grazing lambs (640.6 mg/100 g). Lucerne grazing lambs had higher 20:3n-6 content in kidney than the cocksfoot cv. porto grazing lambs.

FA of Pastures and Supplementary Feeds
The cocksfoot cv. porto and lucerne pastures in this study were abundant in ALA and total n-3 PUFA. Casey et al. [18] reported that cocksfoot pasture had 39.1% of ALA which is considerably lower than the result obtained in this study (57.6%). This could be attributed to the fact that the FA composition of pastures depend on many factors such as cultivar, cutting age, and season. Mel'uchová, et al. [40] found that ALA concentration of pasture plants (mainly lucerne, grass, and herbs) decreased from 62% to 39% (of total FA) from May to August. Garcia et al. [41] also found that cultivar, cutting date, and season significantly influenced the FA composition, the ALA/LA ratio and PUFA. The relative level of ALA of lucerne pasture in this study was 51.9% which was similar to the finding of Wiking et al. [42] (53.5%) and doubled the ALA proportion in lucerne hay (22.1%) as reported by Nguyen et al. [26] in the same region. Glasser et al. [43] also found that the ALA proportion of fresh alfalfa was double that of alfalfa hay. The supplementary feeds used in the current study were rich in LA and total n-6 PUFA. Nguyen et al. [32] also found that 5% canola oil pellet contained high relative levels of LA and n-6 PUFA (26.7% and 27.4%, respectively).

Effect of Supplements on the Fatty Acid Contents in Longissimus dorsi Muscle, Liver, Heart and Kidney
FA of Longissimus dorsi muscle: Supplementation of omega-3 rich feed to lambs in indoor systems can increase the content of health benefit claimable FA in muscle [44]. However, unlike an indoor system, the response of FA content in muscle of grazing ruminants to supplements is not stable, and depends on the quality and quantity of pastures and supplements. Boughalmi and Araba [24] conducted a trial on the Timahdite lamb breed that revealed that lambs raised under pasture only had higher percentages of ALA and n-3 PUFA in the semimembranosus muscle than lambs did under the pasture and concentrate diet. Turner et al. [25] revealed that supplementation with whole cottonseed increased LA and the n-6/n-3 ratio and decreased ALA and n-3 PUFA in Longissimus muscle of Suffolk lambs and Katahdin lambs grazing on a grass-legume pasture. Ponnampalam et al. [45] found that adding oat grain at 245 g or at 175 g with flaxseed or 175 g with flaxmeal per day in the diet of grazing lambs increased the LA content and the n-6/n-3 ratio and did not affect n-3 PUFA and n-3 LC-PUFA content in the Longissimus lumborum, compared with lambs grazing pasture only. In addition, Fruet et al. [46] reported that beef cattle grazing on legume-grass pasture had higher concentrations of ALA in Longissimus thoracis muscle than those grazing on legume-grass pasture supplemented with whole corn grain at 1.4% of body weight.
The results of the current study were in line with previous findings [24,25] that reported supplementation of pellets with or without oil infusion to grazing lambs led to a decrease in ALA and n-3 PUFA contents and increased the n-6/n-3 ratio in Longissimus dorsi muscle. The increase of the LA content in Longissimus dorsi muscle of lucerne grazing lambs supplemented with NOP and RBO pellets could be due to the high n-6 concentration of supplementary diets leading to more n-6 FA being digested, absorbed and finally incorporated in Longissimus dorsi muscle. The decrease of the 18:0 content in Longissimus dorsi muscle of cocksfoot cv. porto grazing lamb with pellet supplementation in this study was in agreement with the findings of Fruet et al. [46], that grass-fed beef had higher concentration of 18:0 when compared to grain-fed animals. The conversion of LA and ALA to their long-chain FA products share several of the elongation and desaturation enzymes and there was competition for incorporation into phospholipids between n-6 and n-3 FA [47]. Thus, the reduction of n-3 LC-PUFA and EPA + DHA + DPA contents in the Longissimus dorsi muscle of lucerne grazing lambs might be due to significant increases in LA content, and therefore, the competition for incorporation of n-6 and n-3 FAs into phospholipids. The lambs grazing cocksfoot cv. porto only had high contents of n-3 LC-PUFA (55.2 mg/100 g) in the Longissimus dorsi muscle, which was similar to that of only lucerne grazing lambs (60.4 mg/100 g) (p > 0.3150). The high content of n-3 LC-PUFA in lamb would be beneficial for meat consumers. According to Nichols et al. [10], the daily requirement per person was 500 mg of the LC omega-3 and a standard serve of red meat was 135 g under Australia and New Zealand regulation [48]. Therefore, consumers having two serves of cocksfoot cv. porto and lucerne grazing lamb meat (= 270 g) each day can meet about 30% of LC omega-3 daily requirement, which could result in a significant increase in LC omega-3 intake to Australians.
FA of liver, heart and kidney: The FA contents of organs (liver, heart, and kidney) can be affected by breeds and nutritional manipulation. Malau-Aduli et al. [33] reported that there were significant sire-breed variations in the FA content of kidney and muscle. Kashani et al. [49] found that Spirulina supplementation to lambs grazing on ryegrass pasture significantly increased the n-3 and n-6 PUFA composition in all organs (liver, heart, and kidney). The results of Nguyen et al. [32] demonstrated that there was no significant difference between liver FA profiles of 5% canola oil pellet-fed and control lambs in an indoor feeding system. This current study clearly demonstrated that supplementation with NOP and CO tended to increase the ALA content in the liver of cocksfoot cv. porto grazing lambs. The provision of NOP and CO supplements to grazing lambs resulted in adding more ALA to the lamb diet, which in turn, could explain the increased ALA content in the liver of cocksfoot cv. porto grazing lambs. In addition, among the supplemented treatments, the cocksfoot cv. porto grazing lambs with RBO supplementation had lower EPA, DPA, n-3 LC-PUFA, PUFA, and EPA + DHA + DPA contents of liver than those lambs in the CO treatment. This is likely due to the large difference in the ALA proportions of the CO (5.7%) and RBO treatments (2.7%). The competition for incorporation of n-6 and n-3 FAs into the phospholipids is a contributing factor, as previously discussed. The competition of incorporation of n-6 and n-3 FAs into the phospholipids also occurred in heart tissue, and could be the reason for the observed lowering of both ARA and DPA contents in heart of cocksfoot cv. porto grazing lambs with RBO and CO supplementation. The increase of the n-6 PUFA, PUFA, and total FA contents in kidney tissues of lucerne grazing lambs with NOP supplementation could also result from the high n-6 proportion (50.3%) of the NOP supplement. The kidney and liver of all lambs in this study contained high n-3 LC-PUFA contents (ranging from 163.2 mg/100 g to 572.6 mg/100 g), equal to and for many species over the n-3 LC-PUFA contents of wild Australian seafood such as fish, shellfish and lobster [10]. In addition, the n-6/n-3 ratio of liver and kidney (from 1.0 to 2.6) were well below the desirable ratio [50]. Therefore, the liver and kidney of grazing lambs could be considered as good sources of omega-3 [51].

Effect of Pasture Types on the Fatty Acid Contents in Muscle, Liver, Heart, and Kidney of Lambs
Pasture type did not affect the FA contents in liver of grazing lambs. However, pasture type impacted the FA contents in the muscle, heart, and kidney tissues. Lambs grazing on lucerne pasture had higher contents of ALA, 20:3n-6, DPA, PUFA, n-3 PUFA, and n-6 PUFA in Longissimus dorsi muscle compared with lambs grazing on the cocksfoot cv. porto pasture. In the present study, the fatty acid composition of cocksfoot cv. porto and lucerne pasture was similar, therefore, the difference in FA content in the Longissimus dorsi muscle of lambs grazing on these two types of pasture could be attributed to the distinctive characteristics of grass and legume pastures in terms of feed intake and the activity of stearoyl CoA desaturase enzyme. The findings of a meta-analysis conducted by Johansen et al. [52] revealed that cows grazing on legume species had 1.3 kg dry matter intake higher than cows grazing on grass species. Wiking et al. [42] found that transcription of stearoyl CoA desaturase in mammary tissue of cows grazing on high proportions of legume (white clover, red clover, and lucerne pasture) was significantly increased in comparison to cows fed maize/grass silage. Fraser et al. [23] also found that lambs finished on legume swards (red clover and lucerne) had significantly higher proportions of ALA in Longissimus dorsi muscle than lambs finished on perennial ryegrass sward. The n-3 LC-PUFA content in Longissimus dorsi muscle of lambs grazing on the lucerne and cocksfoot pastures (50.4 and 55.1 mg/100 g, respectively) were similar and well above the 30 mg cut-off point for "omega-3 source" claim under Australian guidelines [51]. This result could be due to the fact that n-3 LC-PUFA content in Longissimus dorsi muscle of grazing lambs was mainly synthesised from the ALA precursor [53]. It could also be due to the low elongation and desaturation of ALA into n-3 LC-PUFA, and the limited capacity of muscle lipids to incorporate n-3 LC-PUFA as occurs in ruminants [54]. Furthermore, cocksfoot cv. porto and lucerne pastures had similar proportions of ALA (57.6% vs. 51.9%, respectively). Ponnampalam et al. [45] performed a trial with lambs grazing on perennial lucerne and annual phalaris pasture, in which they also found no difference in the n-3 LC-PUFA content in muscle tissue of lambs grazing these two pasture types.

Conclusions
Lambs grazing on lucerne pasture showed higher contents of ALA, 20:3n-6, EPA, PUFA, n-3 PUFA, and n-6 PUFA in Longissimus dorsi muscle in comparison with lambs grazing on the cocksfoot cv. porto pasture. All grazing lambs with or without supplements had high n-3 LC-PUFA content in Longissimus dorsi muscle (50.4 mg/100 g and 55.1 mg/100 g, respectively), which was well over the 30 mg cut-off point for labeling as a source of omega-3. A larger serve size, e.g., 135 or 150 g, as has been used in other studies, would see a good source of omega-3 (60 mg per serve) achieved. Lambs grazing on cocksfoot cv. porto pasture only also achieved high contents of ALA and n-3 LC-PUFA contents (67.1 mg/100 g and 55.2 mg/100 g, respectively), which was the same as those contents of only lucerne grazing lambs, with cocksfoot cv. porto clearly demonstrated to produce premium quality, healthy lamb meat, based on omega-3 PUFA content. Supplementation using pellets with or without oil infusion to grazing lambs generally decreased the ALA and n-3 PUFA contents and increased the n-6/n-3 ratio in Longissimus dorsi muscle. The addition of pellets to grazing lambs decreased the 18:0 content in Longissimus dorsi muscle of cocksfoot cv. porto grazing lambs. NOP and RBO supplementation increased the LA content in Longissimus dorsi muscle of lucerne grazing lambs. Pellet supplementation tended to reduce the EPA + DHA + DPA content in Longissimus dorsi muscle of lucerne grazing lambs. The fatty acid contents of internal organs of grazing lambs were affected by pellet supplementation. The n-3 LC-PUFA contents in the liver and kidney of grazing lambs were equal to the n-3 LC-PUFA contents of wild Australian seafood such as fish, shellfish, and lobster and can be considered and used as a good source of omega-3.