The impact of supplementing lambs with algae on growth, meat traits and oxidative status

    The impact of supplementing lambs with algae on growth, meat traits and oxidative status D.L. Hopkins, E.H. Clayton, T.A. Lamb, R.J. van de Ven, G. Refshauge, M.J. Kerr, K. Bailes, P. Lewandowski, E.N. Ponnampalam PII: DOI: Reference:

S0309-1740(14)00137-5 doi: 10.1016/j.meatsci.2014.05.016 MESC 6423

To appear in:

Meat Science

Received date: Revised date: Accepted date:

6 December 2013 27 March 2014 21 May 2014

Please cite this article as: Hopkins, D.L., Clayton, E.H., Lamb, T.A., van de Ven, R.J., Refshauge, G., Kerr, M.J., Bailes, K., Lewandowski, P. & Ponnampalam, E.N., The impact of supplementing lambs with algae on growth, meat traits and oxidative status, Meat Science (2014), doi: 10.1016/j.meatsci.2014.05.016

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ACCEPTED MANUSCRIPT The impact of supplementing lambs with algae on growth, meat traits and oxidative status

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D.L. HopkinsA,*, E.H. ClaytonB, T.A. LambA, R.J. van de VenC, G. RefshaugeA, M.J. KerrA, K. BailesB, P. LewandowskiD, E.N. PonnampalamE A

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NSW Department of Primary Industries, Centre for Red Meat and Sheep Development, PO Box 129, Cowra NSW 2794, Australia

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NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, Pine Gully Rd, PMB, Wagga Wagga NSW 2650, Australia

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NSW Department of Primary Industries, Orange Agricultural Institute, Forest Road, Orange NSW 2800, Australia D

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School of Medicine, Deakin University, 75 Pigdons Road, Waurn Ponds VIC 3216, Australia E Farming Systems Research, Department of Environment & Primary Industries, Werribee, VIC 3030, Australia

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Corresponding author (phone: +61-2-6349-9722; fax: +61-2-6342 4543; e-mail: [email protected] )

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ACCEPTED MANUSCRIPT Abstract The current study examined the effect of supplementing lambs with algae. Forty, three month old lambs were allocated to receive a control ration based on oats and lupins (n = 20) or the

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control ration with DHA-Gold™ algae (~2% of the ration, n = 20). These lambs came from

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dams previously fed a ration based on either silage (high in omega-3) or oats and cottonseed

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meal (OCSM: high in omega-6) at joining (dam nutrition, DN). Lamb performance, carcase weight and GR fat content was not affected by treatment diet (control vs algae) or DN (silage vs OSCM). Health claimable omega-3 fatty acids (EPA + DHA) were significantly greater in the LL of lambs fed algae (125 ± 6 mg/100 g meat) compared to those not fed algae (43 ± 6

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mg/100 g meat) and this effect was mediated by DN. Supplementing with algae high in DHA,

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provides a means of improving an aspect of the health status of lamb meat.

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Keywords: Lamb, growth, meat quality, colour stability, fatty acids, algae

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ACCEPTED MANUSCRIPT 1.

Introduction The concentrations and sources of variation of health claimable long chain omega-3 fatty

acid content in Australian lamb meat have been measured in Sheep CRC Information Nucleus

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(IN) lambs produced at 8 sites across Australia (Ponnampalam et al., 2014a, b). These lambs

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were finished on a range of feed sources including pasture, supplements with pasture or full fatty acids (n-3

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concentrate diets. Levels of health claimable omega-3 polyunsaturated

PUFA) including eicosapentaenoic acid, EPA + docosahexaenoic acid, DHA were significantly affected by site and time of slaughter (Ponnampalam et al., 2014a). In general, EPA+DHA was greater when lambs were mostly fed quality green pasture and lower when

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pellets, grain or dry pasture had become a large portion of the diets prior to slaughter (Ponnampalam et al., 2014a). As a result, lamb from several sites would not meet the

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requirements to be claimed as a ‘source’ of long-chain omega-3 where a level of 30 mg of EPA+DHA per standard serving size of 135 g is required (FSANZ, 2012). Replacing grain based silage or concentrate (pellets) diets rich in linoleic acid (18:2n-6)

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with pasture rich in alpha-linolenic acid (18:3n-3) improves the fat composition of muscle in

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cattle (Wachira, Sinclair, Wilkinson, Enser, Wood, & Fisher 2002; Nuernberg et al., 2005) and sheep (Aurousseau, Bauchart, Calichon, Micol, & Priolo 2004; Aurousseau et al., 2007)

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by increasing long-chain omega-3 or reducing long-chain omega-6 fatty acid concentration. A greater omega-3 concentration and lower ratio of omega-6:omega-3 is considered beneficial for human health (Palmquist, 2009). Inclusion of oat grain at 175-245 g per day in the diet of

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lambs fed supplements significantly reduced total long-chain omega-3 in meat compared with lambs grazing perennial pasture (Ponnampalam, Burnett, Norng, Warner, & Jacobs 2012c), with the results from the IN lambs being consistent with these observations. The results from recent studies indicate that concentrations of EPA and DHA in longissimus muscle (Scerra et al., 2011) and red blood cells (Clayton, Gulliver, Meyer, Piltz, & Friend, 2011) are significantly lower when sheep are fed concentrates compared with pasture. EPA and DHA may be significantly depleted and the ratio of omega-6:omega-3 can be significantly increased with as little as 14 days of concentrate feeding (Scerra et al., 2011) indicating that depletion rates can be rapid. As a consequence, producers require the ability to supplement lambs so as to ensure adequate levels of health claimable omega-3’s.

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concentration of health claimable omega-3 in lamb muscle was significantly increased following the addition of algae high in long-chain omega-3 as a supplement to low quality ryegrass based roughage diet (Ponnampalam, Burnett, Ji, Dunshea, & Jacobs, 2012a). The

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ACCEPTED MANUSCRIPT effect of the addition of algae to the diet on carcase composition and other aspects of meat quality are, however, largely unknown. Therefore, the aim of the current study was to examine the impact of an algae supplement on the growth, carcase and meat quality of lambs

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fed a grain-based diet.

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2.1. Animal background, feeding and slaughter

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2. Materials and Methods

The Poll Dorset x Border Leicester x Merino wether lambs (n = 40) used in the current study were produced as part of a study to examine the effect of ewe nutrition at joining on the

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sex ratio of progeny. The dams of the lambs were fed a ration based on either silage (high in omega-3) or oats and cottonseed meal (OCSM: high in omega-6 fatty acids) for six weeks

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prior to and, three weeks following, joining, using similar methods to those described previously (Gulliver et al., 2013). Of the lambs used in the current study, 12 were single born and 28 were a twin, but each lamb came from a separate dam. At the commencement of an

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introductory feeding period to the base ration, the lambs weighed on average 34.8 ± 2.5 kg

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and were 3 months of age. The introductory period lasted for 14 days over which time the proportion of grain was gradually increased until the proportions of the components, oat

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grain, lupin grain, chopped lucerne, salt and lime were 62.8, 15.7, 19.6, 1.0 and 1.0% respectively of the fed amount. The lambs were then allocated to two treatment groups of 20 based on the nutrition of their mothers at joining (Dam nutrition) and their liveweight. Within

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each treatment group the lambs were grouped into replicates of 5 lambs and then the four replicates per treatment group were allocated at random to 8 pens (1 replicate per pen) (Table 1), with water provided by troughs and shade cloth provided for sun protection. Lambs were fed one of two treatment rations (n = 20 per treatment group) consisting of a basal (control) ration or the basal ration with DHA-Gold™ algae (Martek Biosciences Corporation, Maryland, USA) included at 1.92% dry matter (DM) (algae, Table 2). The metabolisable energy (ME, 11.5 versus 11.6 MJ/kg DM) and crude protein (CP%, 18.0 versus 17.9 %DM) did not differ between the control and algae treatment rations, respectively. The concentration of Vitamin E in the treatment rations was analysed using methods described previously (McMurray & Blanchflower, 1979) and the concentration of Vitamin E in both treatment rations was 6.82 mg/kg DM. The lambs were fed daily and refusals weighed each day before feeding. The level of feeding was increased over the 6 weeks of the experiment, based on a target growth rate of

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ACCEPTED MANUSCRIPT 200 g/hd/day and an energy: protein intake ratio of 0.65. The lambs were weighed at regular intervals and a blood sample was collected at the conclusion of the feeding period. Blood was collected from the jugular vein into a 5 mL VacutainerTM containing lithium heparin.

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Following centrifugation at 1500 x g for 10 min (Centra 7R Refrigerated Centrifuge, IEC,

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USA), plasma was decanted. To 1.5 ml of plasma samples, 15 µl of 20 µM Butylated hydroxytoluene was added and the samples held at -80C. Total F2-isoprostanes (form 8)

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concentration was analysed in plasma using an enzyme immunoassay (EIA) kit (Caymen Chemical Company, USA) following the manufacturers instructions. Prior to analysis plasma samples were hydrolysed by addition of 25 µl 2 M NaOH to each 100 µl plasma sample. The

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samples were incubated at 45°C for 2 hours. Following this, 25 µl 10M HCl acid was added and the samples were centrifuged for 5 minutes at 12,000 g. The supernatant was removed

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and used for the determination of total 8-isoprostane using the EIA kit. This assay is based on the competition between 8-isoprostane and an 8-isoprostane acetylcholinesterase (AChE) conjugate for a limited number of 8-isoprostane-specific rabbit anti-serum binding sites.

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Values were expressed as pg/ml of plasma.

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Throughout the feeding period one lamb exhibited slow growth and at the end of the feeding period was subjected to a post-mortem examination by a veterinarian, which revealed

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pneumonia. The management of the lambs in this study was under the animal ethics approval ORA 12/15/013 issued by the Orange Animal Ethics Committee of NSW Department of Primary Industries.

The lambs were weighed the day before slaughter and transported to a commercial

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abattoir (200 km), where they were held in lairage overnight and slaughtered the following day. The lambs were slaughtered after head only stunning. All carcases were electrically stimulated pre-dressing with a mid-voltage unit (2000 milliamperes with variable voltage to maintain a constant current, for 25 seconds at 15 pulses/s, 500 microsecond pulse width, unipolar waveform) (Toohey, Hopkins, Stanley, & Nielsen, 2008). Carcasses were chilled at a mean temperature of 3°C over a 24 h period. All carcasses were trimmed according to AUS-MEAT specifications (Anoymous 2005) weighed hot (hot carcase weight, HCW) and the depth of tissue at the GR site (the depth of muscle and fat tissue from the surface of the carcase to the lateral surface of the twelfth rib 110-mm from the midline) was measured using a GR knife (GR).

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ACCEPTED MANUSCRIPT 2.2. Measurements After the commencement of chilling, pH and temperature were measured in the left-hand m. longissimus thoracis et lumborum (LL) at the caudal end over the lumbar-sacral junction

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and then subsequently three times to ensure the range in temperature from ~35C to less than

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18C was covered. A section of subcutaneous fat and the m. gluteus medius were cut away to expose the LL and after measurement the area was resealed with the overlaying tissue. pH

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was measured using meters with temperature compensation (WP-80, TPS Pty Ltd, Brisbane, Australia) and a polypropylene spear-type gel electrode (Ionode IJ 44), calibrated at ambient temperature. pH of the LL at 24 h post-mortem (LL24pH) was measured at the 12th/13th rib

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site after calibrating the meter at chiller temperatures.

At 24 h post-mortem the LL muscle was removed (Product identification number HAM

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4910; Anonymous 2005) from the left side loin, between the 6th lumbar vertebrae and the 12th rib. Measures of subcutaneous fat depth (Fat C) and muscle depth and width (EMD and EMW; LL)) were taken at the 12th rib by experienced personnel using a metal ruler and these

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values multiplied by 0.008 to give a cross sectional area estimate (EMA) as applied

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previously (Hopkins, Gilbert, Pirlot, & Roberts, 1992). The cut surface of the LL was exposed to the air at ambient temperature for 30-40 min and the meat colour measured using a Minolta

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Chroma meter (Model CR-400) set on the L*, a*, b* system (where L* measures relative lightness, a* relative redness and b* relative yellowness), with D65 illuminant and 2 standard observer. The chromameter was calibrated with a white tile (Y = 92.8, x = 0.3160, y

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= 03323). Three replicate measurements were taken at different positions and an average value used for analysis. The boned LL section was transported (at 4C) in a portable chiller to the Centre for Red Meat and Sheep Development. A 3 cm length of muscle was cut from the cranial end of the LL, vacuum packed and held at 4C for 4 days. Prior to measurement of colour a fresh surface was cut and the samples were placed on black styrofoam trays (one per tray) and overwrapped with 15μ polyvinyl chloride (PVC) film. Samples on trays were allowed to bloom for 30-40 minutes at a temperature of 3-4oC before making initial colour measurements. Samples were then displayed for 3 days under fluorescent lights set at ~1000 Lux. Each black tray contained one sample of meat only. A Hunter Lab Mini Scan(tm) XE Plus (Cat. No. 6352, model No. 45/0L, reading head diameter of 37 mm) was used to measure light reflectance. The light source was set at “D65” with the 10 standard observer. The instrument was calibrated on a black glass then a white enamel tile following the manufacturer's specifications. At each reading the

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ACCEPTED MANUSCRIPT measurement was replicated after rotating the spectrophotometer 90° in the horizontal plane. An estimate of the oxy/met ratio, was calculated by dividing the percentage of light reflectance at wavelength 630 nm, by the percentage of light reflectance at wavelength 580

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nm.

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From the medial section of the LL a sample (mean 65 g) was removed and vacuum sealed in gas impermeable plastic bags and held at 4C for 4 days after which the samples were frozen at

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-22C. The samples were cooked from frozen for 35 min in plastic bags at 71°C in a water bath (Hopkins, Toohey, Warner, Kerr, & van de Ven 2010). These samples were weighed prior to and after cooking to determine cooking loss (CL).

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A 25 g sample of LL was also taken at day 1 and kept frozen at -20C for later measurement of Vitamin E content as previously described. A 40 g sample of diced LL was

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collected for the determination of intramuscular fat concentration and fatty acid profile. Freeze dried LL was ground using a FOSS Knifetech™ 1095 sample mill (FOSS Pacific, Unit 2, 112-118 Talavera Road, North Ryde, NSW, 2113) prior to fatty acid analysis using the

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one-step procedure of Lepage & Roy (1986) with slight modifications as described previously

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(Clayton et al., 2012). In brief, 10 mg of ground, freeze dried meat was added to an 8.0 mL glass culture tube and exactly 2.0 mL of methanol:toluene (4:1 v/v) containing C19:0 (0.02

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mg/mL) internal standard was added. Fatty acids were methylated (FAME) by adding 0.2 ml acetyl chloride drop-wise while vortexing, followed by heating to 100°C for 1 h. After cooling, the reaction was stopped by slowly adding 5 mL of 6 % K2CO3 while mixing by

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vortex. The sample was centrifuged at 1500 x g for 10 min to facilitate the separation of the layers. The upper toluene layer containing the FAME was transferred to a 2 mL glass vial and sealed with a Teflon-lined screw-cap for subsequent analysis by gas chromatography as described previously (Clayton et al., 2012). Total fatty acids in feed samples were extracted and methylated directly from a freeze-dried sample using the one-step procedure of Lepage & Roy (1986) as modified by Outen et al. (1976). The major fatty acids found in DHA-Gold algae are presented in Table 3. The crude fat level for the algae was determined using the FOSS Soxtec 2050. A 3 gm sample of ground freeze dried meat was weighed into a thimble and extracted in a tin containing 85 ml of hexane for approximately 60 min. Hexane was then evaporated off for a further 20 min before the tin containing the recovered fat was removed from the Soxtec (FOSS, 2003). The tin was then dried for 30 min at 105°C to remove any residual solvent and weighed to allow determination of the crude fat content of the sample. At the conclusion of the period of retail

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ACCEPTED MANUSCRIPT display a 25 g sample of LL was also taken and kept frozen at -20C for later measurement of lipid oxidation. The lipid oxidation in meat was assessed by the thiobarbituric acid reactive substances (TBARS) procedure, expressed in mg of malondialdehyde (MDA) per kg of muscle.

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A 1g sample of fresh meat was ground and suspended in 5ml normal saline before analysis

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using the OXItek TBARS assay kit. Supernatant of standards and samples was read on a

sample concentration of MDA (Zeptomatrix, 2006).

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2.3. Statistical analysis

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spectrophotometer at 540 nm with standard results used to plot a linear regression to determine

A repeated measures analysis of the live weights over time was performed. The full model allowed for birth type (single or twin) effects; included birth weight as a covariate; modelled

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the average trend across lambs within each level of dam nutrition (DN) × Treatment group (Treatment = Algae vs. control) using separate spline models; included random pen effects;

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and allowed random regression deviations from the average trend for individual lambs. Feed efficiency was calculated for each lamb as its average daily growth rate (kg/day) divided by

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the average daily feed intake (kg/day) for all lambs within the pen housing the lamb. Average daily feed intake per pen is used since individual lamb consumption was not available. Feed

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efficiency, So calculated, compared with calculation based on individual lamb consumption, will not bias treatment comparisons, but will reduce statistical power for testing treatment differences given the increased variability in estimating lamb feed efficiency. Feed efficiency

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was analysed using a linear model which included DN, treatment, DN x treatment, birth type and birth weight as fixed effects and pen as a random effect. This same model was applied to the analysis of HCW, GR, Fat C and EMA. The trend for pH with temperature decline across the carcases within each group was modelled using the approach described by van de Ven, Pearce, & Hopkins (2013). This included an average trend for pH versus temperature that was modelled using a spline. This average trend was allowed to differ across the two DN groups, across the two treatment groups, and across all four combinations of DN × Treatment. Spline models were also used to model random deviations from the average trend for individual carcasses. The other random sources of variation included in the model were pen and measurement error. From this modelling, the temperature at pH 6 (Temp@pH6) and the pH at 18C (pH@Temp18) were estimated for each carcase.

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ACCEPTED MANUSCRIPT LL24pH was analysed using a linear model which included DN, Treatment, DN x Treatment, birth type and birth weight as fixed effects and pen as a random effect and fresh colour measures (L*, a*, b*) analysed with the same model, but including LL24pH,

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Temp@pH6 and pH@Temp18 as covariates. Of the colour traits for meat under display, only

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the R630/R580 ratio, and redness (a*) values were analysed as these have been related to consumer acceptance (Khliji et al., 2010). The repeated measures data for R630/R580 ratio

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and a* were analysed using Linear Mixed Model (LMM) analysis. The fixed effects included linear regression on day of display, possibly differing across the four DN × treatment combinations. Possible deviations from linearity over the four days were also included as

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fixed effects, with these deviations also allowed to differ across the four DN × treatment combinations if required. The covariate LL24pH was included, with its effect allowed to

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possibly differ across the four days. Random effects included pen effects; random deviations associated with each carcass (modelled as linear over days); and finally random error. Stepwise regression was used to remove non-significant (P > 0.05) terms in models.

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Shear force of 5 day loin and cooking loss were analysed using a LMM analysis. The full

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model included DN, treatment, DN × treatment, cooking batch and LL24pH as fixed effects (the latter as a covariate) and pen as random effect. A similar model (DN, treatment, DN ×

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treatment, as fixed effects, with pen as random effect) was used for IMF, Vitamin E levels in the muscle, isoprostane levels and TBARS data. The effect of Vitamin E as a covariate for isoprostane levels was also tested as was the effect of isoprostane level on TBARS. Models were fitted in R (R Development Core Team, 2010) using the package asreml (Butler, 2009).

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Of the 40 lambs, two were observed to have particularly slow growth rates and reduced intake, one in the last week of feeding (DN OCSM, control). The other lamb (DN Silage, algae) grew at only an average of 67 g/hd/day (see Fig. 1) during the experimental period. Both lambs were found to have a pneumonia infection and, therefore, post death data from these animals was excluded prior to analysis

3. Results 3.1. Growth and feed intake Average growth rate was not significantly affected by DN, dietary treatment, birth type or birth weight (P > 0.05). There was a non-linear pattern over time on average (as shown in Fig. 1) and significant linear deviations (P < 0.001) from the average pattern across lambs. There was no significant effect of any model terms on feed efficiency.

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ACCEPTED MANUSCRIPT 3.2. Carcase data Hot carcase weight (HCW) was not significantly affected by DN, dietary treatment, birth type or birth weight. GR was, however, significantly (P < 0.05) affected by HCW, birth type and

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birth weight (Table 4) and GR increased as HCW increased (coefficient 0.63 ± 0.25 mm/kg),

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decreased as birth weight increased (coefficient -1.15 ± 0.54 mm/kg) and twin born lambs had a lower GR (3.3 ± 0.97) than singles, where here, and below, the quantified effects are

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conditional of the other terms in the model being fixed. For Fat C there was an effect (P < 0.05) of HCW and birth type, such that Fat C increased as HCW increased (coefficient 0.55 ± 0.20 mm/kg) and was less for twin born lambs (2.0 ± 0.8) than singles. There was no effect of

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DN or treatment on GR or Fat C (Table 4). For EMA there was an effect of HCW, birth type, DN (each at P < 0.001) and treatment (P < 0.05). EMA increased with increasing HCW

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(coefficient 0.59 ± 0.12 cm2 /kg); was 1.7 ± 0.5 cm2 larger for twin born lambs than singles; was 1.0 ± 0.4 cm2 larger for lambs from ewes fed OCSM rather than silage; and 1.4 ± 0.3 cm2 larger for control lambs than lambs fed algae (Table 4).

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The decline in pH was not significantly effected by DN, but there was a uniform

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difference in pH across the two levels of treatment at each temperature (significant at the P = 0.05). The pH for control lambs was estimated to be (0.07 ± 0.03) units less than for algae

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supplemented lambs with the pattern of decline shown in Fig. 2.

3.3. Meat quality data

The LL24pH (overall mean = 5.81 ± 0.02) was not significantly affected by DN or

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treatment (Table 5), nor birth type or birth weight. The lightness (L*) of the loin varied significantly with TemppH6 (P < 0.05), LL24pH (P < 0.001), and across the two levels of DN (P < 0.001). L* increased by 0.08 ± 0.03 units for each increase of 1°C in TemppH6; increased by 16.2 ± 4.1 units for each increase of 1 unit in LL24pH; and was 1.7 ± 0.5 units greater for lambs from ewes fed silage rather than OCSM. The predicted mean L* for lambs having a TemppH6 equal to 22.6°C and LL24pH equal to 5.8 is 34.1 ± 0.3 when the dam was fed OCSM and 35.8 ± 0.3 when the dam was fed silage. By contrast the redness (a*) of the loin was 1.3 ± 0.4 greater for lambs from ewes fed on OCSM than for lambs from ewes fed silage (P < 0.01), and a* decreased by 10.7 ± 3.8 units for each unit increase in LL24pH (P = 0.01). The predicted means for a* for lambs from dams fed OCSM compared with silage, at LL24pH equal to 5.8, were 15.0 ± 0.3 and 13.7 ± 0.3 units respectively. There was no effect of birth type or birth weight on colour parameters.

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ACCEPTED MANUSCRIPT The R630/R580 ratio and a* during display were not significantly effected by DN or treatment, however, and the former measure significantly (P < 0.001) declined with time on display (Table 6). Both R630/R580 ratio and a* declined with increasing LL24pH (P < 0.05)

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with the rates of decline 0.7 ± 0.2 and 1.5 ± 0.4 units, respectively, for each 0.1 unit increase

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in LL24pH. The concentration of vitamin E in the LL was significantly (P < 0.001) lower when lambs were offered the algae ration (1.27 ± 0.05 mg/kg) compared with the Control The concentration of isoprostane in lamb plasma was

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ration (1.64 ± 0.05 mg/kg).

significantly (P < 0.05) higher when dams were previously fed silage (2.06 ± 0.13 pg/ml) compared with OCSM (1.79 ± 0.12 pg/ml). The mean concentration of intramuscular fat (2.5

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± 0.1%) was not effected by treatment diet or DN.

There were no effects of DN, treatment, cooking batch or LL24pH on shear force values

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(Table 5), however, cooking loss was significantly (P < 0.05) greater when lambs were offered the algae ration (28.3 ± 0.5) compared with the control ration (25.6 ± 0.5). 3.4

Fatty acid and TBARS data

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The concentration of the omega-3 fatty acids EPA and DHA and the omega-6 fatty acid

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DPA (n-6) was significantly greater when lambs received the algae treatment ration compared with the control ration (Table 7). The concentration of DHA was highest when the dams of

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the lambs had previously been fed silage compared with OCSM at the time of joining (Table 7). As a result of feeding algae, the level of omega -3 polyunsaturated fatty acids (PUFA n-3) was significantly greater relative to the of level of PUFA n-6, and there was a significant (P < 0.001) effect on the n-6:n-3 ratio. The concentration of TBARS was significantly (P < 0.05)

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greater when lambs received the algae ration (5.2 ± 0.5 mg MDA/kg meat) compared with the control ration (1.5 ± 0.5 mg MDA/kg meat). If isoprostane concentration was included as a covariate for TBARS results it was significant (P < 0.05) such that as isoprostane level increased TBARS values increased for lambs fed algae (1.7 ± 0.7 pg/ml/ mg MDA/kg meat) and decreased for control fed lambs (2.4 ± 0.9 pg/ml/mg MDA/kg meat). This was contingent on removal of one outlier value for TBARS (14.1 mg MDA/kg meat), but even with the value retained the effect on relationship was still significant for the algae fed lambs.

4. Discussion 4.1. Growth rate and carcase effects There was no effect of the algae (DHA-Gold™) supplement on growth rate, which may be expected given the diets were balanced for energy and protein. Although not detected as

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ACCEPTED MANUSCRIPT significant, the lambs fed the algae supplement did refuse some feed, which was observed to be the lucerne component of the diet. In the study of Burnett, Norng, Seymour, Jacobs, & Ponnanpalam (2012), where the same algae supplement was fed there did appear to be a

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depression in feed intake and growth rate of lambs compared to those fed a control diet, but

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because the diets varied in that study between treatments for energy and protein it is difficult to assert whether this depression was related in any way to the algae supplement per se.

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Further study of the impact of the supplement on growth would be of value if conducted under an individual animal feeding program as pen studies are less informative with respect to individual variation in animal performance.

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Given there was no impact of the algae supplement on growth rate, the minimal differences for carcase traits is, as expected. However, the significantly lower level of insulin

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found in lambs fed the same supplement by Ponnampalam et al. (2012a) suggested that a difference in fat deposition may have been expected, given the effect of this hormone on lipase and the uptake of triacylglycerols (Berg et al., 2002). Indeed, the smaller EMA in

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supplement fed lambs in the current study would be consistent with this effect, however, a

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change in insulin concentration cannot be confirmed, as the insulin concentration was not determined. The other effects of birth weight and birth type on measures of fatness have been

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reported extensively previously (e.g. Hall, Gilmour, Fogarty, Holst, & Hopkins, 2001; Hopkins, Stanley, Martin, Ponnampalam, & van de Ven 2007).

4.2. Effects on meat quality

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The lack of difference in ultimate pH between treatments indicates that muscle glycogen levels were similar, with the overall absolute value being on the high side for good keeping quality (Egan & Shay, 1988). This outcome does suggest indirectly that prior to slaughter there was unlikely to be a difference in insulin levels as glycogenesis does not appear to have been affected. Of particular interest was the effect of dam nutrition on fresh meat colour with lambs from ewes fed silage having lighter meat, which was also less red than that of lambs from ewes fed oats and cottonseed meal. The mechanism for such a delayed effect is unclear, particularly as the previous treatment differences were applied through their dams. Potentially linked to this outcome, however, was the finding that the level of 8-isoprostanes was greater in the plasma of lambs from ewes fed the silage diet (higher in EPA and DHA). Isoprostanes are prostaglandin F-like compounds (formed via actions of cyclooxygenases) which are produced by direct free radical development of lipid oxidation and other disorders, and their formation is not dependent on the activity of cyclooxygenases (Roberts & Milne, 12

ACCEPTED MANUSCRIPT 2009). As such they can be used as indicators of oxidative stress (Bekhit, Hopkins, Fahri, & Ponnampalam, 2013) and generally as the antioxidants status increases there will be a decline in isoprostanes. In the present study however, possibly due to the range in the level of

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Vitamin E in the muscle there was no relationship between isoprostanes and Vitamin E. A

(e.g. Hopkins and Fogarty, 1998; Warner et al., 2010).

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high pH in the LL at 24 h reduced redness an effect reported extensively in fresh lamb meat

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During the display period colour stability declined as reflected by the R630/R580 ratio which is consistent with previous studies (for example, see Ledward, Dickinson, Powell, & Shorthose, 1968; Khiliji et al. 2010). In a recent study, the R630/R580 ratio of LL samples (n

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= 3389) did not exhibit an early decline, but increased between the first measurement and the second at 24 h and then declined (Jacob et al., 2011), however, this result was not be

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replicated in a subsequent study (Hopkins, Lamb, Kerr, van de Ven, & Ponnampalam, 2013). The R630/R580 ratio decline was indicative of a change in redness in the current study, but

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this decline was not significantly affected by treatment diet.

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4.3. Effects on fatty acids and lipid oxidation The elevation of the n-3 PUFA EPA and DHA in the LL of the algae fed lambs was

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expected given previous research (Ponnampalam et al., 2012a). Enrichment of PUFA levels in meat may lead to negative flavour/aroma development due to lipid oxidation (Diaz, et al., 2011; Jiang, Busboom, Nelson, & Mengarelli, 2011), however, lipid oxidation in aged muscle (up to at least 4 days post-mortem) by greater PUFA levels is not generally considered a

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problem if vitamin E concentrations are above a critical level of 2.95 mg/kg meat (Ponnampalam et al., 2012b). The colour stability of lamb can also be improved by the addition of Vitamin E to a diet (Wulf, Morgan, Sanders, Tatum, Smith, & Williams, 1995). Vitamin E is a lipid soluble compound that has been shown to protect lipids such as PUFA and myoglobin from oxidation (Faustman, Chan, Schefer, & Havens, 1998). In the current study, there was a detrimental effect of the increased n-3 PUFA on lipid oxidation with the TBAR concentration significantly greater when lambs were fed the algae ration and the level of Vitamin E was not high enough to prevent this oxidation. The higher concentration of n-3 PUFA and the lower concentration of Vitamin E in the muscle of lambs fed algae in the current study may have lead to an increased lipid oxidation. To gain the maximum benefit from algae supplementation, therefore, diets need to contain sufficient Vitamin E.

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ACCEPTED MANUSCRIPT In addition to the observed increase in n-3 PUFA following the consumption of the algae ration, the concentration of DHA was significantly higher in the LL of lambs when dams were previously fed silage at joining. This mediation of n-3 PUFA concentration by DN may point

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to a hormonal mechanism at conception that impacts on the progeny and certainly warrants

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further investigation. In addition, the concentration of isoprostane was also greater in the plasma of lambs from dams fed silage compared with OCSM and greater levels of

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isoprostanes were indicative of greater lipid oxidation. Isoprostane concentration could, therefore, serve as a useful biomarker for increased susceptibility to oxidative stress, but this also requires more investigation

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The concentration of EPA + DHA in meat observed when lambs were fed the control diet in the current study was greater than that observed previously when lambs grazed a low

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quality senesced annual ryegrass pasture (Ponnampalam et al., 2014a). The concentration of these fatty acids in the LL of lambs receiving the algae supplement suggests there may be potential health benefits for consumers of this lamb meat. By having more than 60 mg EPA +

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DHA/135 g serve, lamb meat from algae fed treatments could be claimed as a good source of

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omega-3 according to the Australian nutrient reference standard (FSANZ, 2012). Based on European standards for source and good source of EPA+DHA which are 40 mg and 80

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mg/100 g of meat, respectively (Commission Regulation (EU), 2010) the inclusion of algae as applied in the current study would even allow this standard to be met. The total dose of EPA + DHA in a 135 g serve of meat from lambs offered the algae supplement when their dams were fed silage at joining (189 mg/serve) is similar to the amount found in non-oily fish

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(Williams 2007). 5. Conclusions

The concentration of DHA in the LL was significantly greater when lambs were received a ration containing DHA rich algae at 2% of the dry matter. One of the issues to be resolved is how best to incorporate the algae into rations. The amount of EPA + DHA in the meat following supplementation would mean that the lamb could be considered a “good source” of omega-3. The concentration of vitamin E was reduced and TBARs was increased following supplementation of algae indicating increased lipid peroxidation, however, the colour shelf life of displayed meat was not adversely affected. Supplementing lambs with vitamin E to increase concentrations above the critical threshold of 2.95 mg/kg meat is recommended in order to prevent this lipid peroxidation. The concentration of isoprostane, the colour of meat

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acid metabolism warrants further investigation.

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Acknowledgements

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Staff and resources for this work were provided by NSW Department of Primary Industries. The staff of the cooperating abattoir are thanked for their assistance during sample collection. References

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Zealand Societies of Animal Production Joint Conference, Lincoln University, New Zealand, 2-5 July 2012, p 12.

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polyunsaturated fatty acid and conjugated linoleic acid content of sheepmeat and adipose tissue. British Journal of Nutrition, 88, 697-709. Williams, P. (2007). Nutritional composition of red meat. Nutrition & Dietetics, 64, S113–

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(Revision 2): 1-11.

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Fig. 1. A plot of the predicted trend on average (bold black line) for live weight, including the trends for individual lambs (dotted lines).

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Fig. 2. Plots of the modelled trend of pH versus temperature (C) for each carcass within each treatment level are given above (dashed lines). The solid line is the average trend (averaged over carcasses) within each treatment level.

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Table 1 Allocation of lambs based on the nutrition of their dam (OCSM or Silage) and the feeding treatment in the feedlot (Algae or No algae) Dam nutrition + Treatment Pen OCSM + OCSM + Silage + Silage + Algae Control Algae Control 1 4 1 2 2 3 3 1 4 4 4 1 5 1 4 6 4 1 7 2 3 8 4 1

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Table 2 Nutrient composition of the components of the 2 diets including the n-6:n-3 fatty acid ratio and the proportions of each component fed within each ration. Component ME (MJ/kg) CP (%) n-6:n-3 Base ration Algae ration proportions (%) proportions (%) Oat grain 12.2 15.0 30.77 62.8 61.5 Lupin grain 13.5 35.0 2.13 15.7 15.4 Lucerne 9.0 16.0 0.11 19.6 19.2 DHA-Gold algae 16.6 11.3 0.36 0.0 1.92 Salt 1.0 1.0 Lime 1.0 1.0

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Table 3 Fatty acid profile of DHA Gold™ algae showing only those fatty acids greater than 1% of the total and the ether extract of the algae. Fatty acid % of total fatty acids C14:0 10.13 C16:0 24.52 C20:5n-3 1.54 C22:5n-6 14.85 C22:6n-3 43.81 Ether extract (%) 26.0

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Table 4 Predicted means and standard error (s.e.) for hot GR (GR), subcutaneous fat depth at the C site (Fat C) and eye muscle area (EMA) according to DN grouping. DN (treatment) Birth type GR* (mm) Fat C (mm) EMA (cm2) Silage (control) Single 11.7 (1.2) 6.4 (0.9) 14.1 (0.4) Silage (algae) Single 12.5 (1.2) 8.3 (0.9) 12.7 (0.4) OCSM (control) Single 12.8 (1.1) 6.9 (0.8) 15.0 (0.4) OCSM (algae) Single 11.8 (1.1) 6.7 (0.8) 13.6 (0.4) Silage (control) Twin 8.9 (1.0) 4.4 (0.7) 15.8 (0.3) Silage (algae) Twin 9.6 (1.0) 6.4 (0.8) 14.4 (0.3) OCSM (control) Twin 9.9 (0.9) 5.0 (0.7) 16.7 (0.3) OCSM (algae) Twin 8.9 (0.9) 4.7 (0.7) 15.3 (0.3)

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*First four means are at a birth weight of 6.73 kg and the second four means at 6.35kg.

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Table 5 Predicted means and standard error (s.e.) pH in the loin at 24 h post-mortem (LL24pH), L* (lightness)+, a* (redness)++ and shear force (N) according to DN grouping. DN (treatment) LL24pH L* a* Shear force Silage (control) 5.78 (0.02) 35.6 (0.5) 13.8 (0.5) 54.2 (7.9) Silage (algae) 5.82 (0.02) 36.0 (0.5) 13.4 (0.5) 55.8 (8.3) OCSM (control) 5.80 (0.02) 33.7 (0.5) 15.1 (0.4) 49.6 (7.6) OCSM (algae) 5.85 (0.02) 34.6 (0.5) 14.6 (0.5) 65.5 (7.3) + ++ At a LL24pH of 5.81 and a [email protected] of 22.6°C; At a LL24pH of 5.81

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Table 6 Predicted means and standard error (s.e.) for R630/R580 ratio and redness (a*) of the loin at a LL24pH of 5.81 over the 4 days of simulated retail display. Day of display R630/R580 ratio a* 0 5.38 (0.14) c 16.43 (0.26) a 1 5.19 (0.15) c 19.36 (0.28) d 2 4.40 (0.16) b 18.26 (0.31) c 3 3.96 (0.18) a 17.50 (0.34) b Means for a trait (R630/R580; a*) not having a trailing letter in common are significantly different at P = 0.05.

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Table 7 Predicted means and standard error (s.e.) for fatty acid composition of the intramuscular fat (mg/100g muscle) in the longissimus lumborum of lambs according to treatment diet and previous dam nutrition. Dam Fed Silage Dam Fed OCSM Fatty acids1 Control Algae Control Algae SFA C14:0 164 (19) 169 (21) 191 (18) 199 (18) C16:0 1230 (107) 1333 (114) 1276 (102) 1302 (97) C18:0 917 (74) 818 (77) 979 (70) 806 (67) MUFA C16:1n-7 88 (7) 78 (8) 88 (7) 79 (6) C18:1 n-9 1800 (137)ab 1524 (145)ab 1859 (123)a 1451 (124)b n-6 PUFA C18:2 n-6 297 (16) 305 (17) 290 (16) 295 (15) C20:4 n-6 104 (5) 104 (6) 101 (5) 99 (5) C22:5 n-6 2 (2)a 24 (2)b 2 (2)a 17 (2)c n-3 PUFA C18:3 n-3 39 (3) 39 (3) 41 (3) 37 (3) C20:5 n-3 (EPA) 30 (3)b 48 (3)a 29 (3)b 44 (3)a C22:5 n-3 (DPA) 38 (2) 37 (2) 37 (2) 34 (2) C22:6 n-3 (DHA) 13 (7)c 92 (8)a 12 (7)c 71 (7)b EPA + DHA 44 (9)c 140 (10)a 42 (9)c 115 (8)b SFA 2451 (204) 2479 (216) 2609 (194) 2471 (186) MUFA 1981 (224) 1810 (238) 1904 (213) 1749 (203) n-6 PUFA 433 (23) 467 (24) 425 (22) 449 (21) n-3 PUFA 124 (12)a 220 (13)b 123 (11)a 190 (11)b n-6:n-3 Ratio 3.5 (0.1)a 2.1 (0.1)b 3.5 (0.1)a 2.4 (0.1)b Means for a trait not having a trailing letter in common are significantly different at P = 0.05. 1SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids, n-6:n-3 Ratio = ratio of n-6 PUFA to n-3 PUFA.

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Supplementing lambs with a DHA rich algae diet had no impact on growth, or carcase fatness. DHA in the m. longissimus thoracis et lumborum was significantly increased due to algae feeding Feeding DHA rich algae increased lipid oxidation, but not meat colour stability.

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