Dietary Linseed Supplementation Improves Meat Quality of Sheep by Altering Muscle Fiber Characteristics and Antioxidative Capacity


 Background: In ruminants, due to the hydrogenation of the rumen, muscles contain a large amount of saturated fatty acids (SFA), which have a negative effect on meat quality. Linseed as a common oil crop which is rich in polyunsaturated fatty acid (PUFA), it affected the deposition of PUFA. Unfortunately, PUFA can exert a negative influence on the oxidative stability of meat. Fortunately, linseed is not only rich in PUFA, but also rich in phenols, which are a good source of antioxidants. Therefore, linseed may be can serve as an effective feed additive to improve meat quality of sheep. The aim of this investigation was to establish the effects of dietary linseed supplementation on carcass traits, meat quality, muscle fiber characteristics and antioxidative capacity of sheep. Results: Results of this study indicated that linseed supplementation significantly increased the loin-eye area, crude protein and ash. Reduced pH24h, L* and shear force. Moreover, linseed supplementation affected the relative content and enriched the kinds of volatile flavor substances. Increased mRNA expression of MyHC I and MyHC IIx, and a decrease in cross-sectional area (CSA) and muscle fiber diameter was also observed. Additional changes included enhanced activity of succinic dehydrogenase (SDH), decreased activity of lactate dehydrogenase (LDH), increased total antioxidative capacity (T-AOC) activity. The mRNA expression of glutathione peroxidase (GSH-PX) and catalase (CAT) were increased while malondialdehyde (MDA) decreased. Conclusions: The results suggest that linseed is an effective feed additive in improving meat quality. The underlying mechanism(s) for its effectiveness may be partly due to a change in muscle fiber characteristics and antioxidative capacity.


Background
As consumers' requirements for meat quality increase, it becomes a major problem for livestock producers to provide safer, healthier, and more delicious meat. Compared with pasture feeding, sheep maintained in feed lots do exhibit improved carcass weight but unfortunately also exhibit a decrease in meat quality [1,2]. Therefore, improving the meat quality of feed lots sheep has attracted worldwide attention. Previous research has reported that diet nutrition had an effect on meat quality of sheep [3][4][5][6].
For example, dietary rumen-protected betaine supplementation was shown to increase the average daily gain and antioxidant status of muscle in male Hu sheep [7]. Also, according to PE Simitzis, MA Charismiadou, M Goliomytis, A Charalambous, I Ntetska, E Giamouri and SG Deligeorgis [5], incorporation of avonoids in sheep diets improved plasma and meat antioxidant capacity. Growth performance and carcass trait were also affected by dietary inulin supplementation [8]. These results indicate that improvement of meat quality with dietary supplementation is possible.
As a common oil crop, linseed is rich in polyunsaturated fatty acid (PUFA), such as α-linolenic acid and linoleic acid. In ruminants, due to the hydrogenation of the rumen, muscles contain a large amount of saturated fatty acids (SFA), which is not conducive to the improvement of meat quality. According to HV Le, DV Nguyen, QV Nguyen, BS Malau-Aduli, PD Nichols and AEO Malau-Aduli [9], n-3 LC-PUFA contents were increased in lambs by dietary axseed supplementation. Furthermore, when linseed was added to the diets of bulls [10], pigs [11], and chickens [12], it affected the deposition of PUFA. Unfortunately, PUFA can exert a negative in uence on the oxidative stability of meat. Fortunately, linseed is not only rich in PUFA, but also rich in phenols, which are a good source of antioxidants [13]. Improving antioxidation can prevent the occurrence of peroxidation reactions thereby reducing the generation of offensive avors in meat.
Previous research has indicated potential associations between muscle ber types and meat quality. It is now well established from various studies that muscle ber characteristics are related with meat color, tenderness, postmortem pH, etc. [14][15][16]. In this respect no investigations on the effect of linseed supplementation on meat quality of sheep have been performed.
Therefore, the aim of this investigation was to assess the effect of linseed supplementation into the diet of sheep with respect to on carcass traits, meat quality, muscle ber characteristics and antioxidative capacity. A total of twenty-four Sunit sheep with an average body weight of 16.5 ± 1.6 kg from Inner Mongolia, China were weaned for 90 d and then randomly allocated into control (C) and linseed supplemented (L) groups. Three replications of four sheep per replicate were used for each group. The C group was fed a basic diet, which consisted of whole plant silage and sun ower cake supplemented with commercial fattening feed. The L group diet consisted of a similar diet but supplemented with 8% linseed. The nutrient contents of the diets are shown in Table 1. Water was provided ad libitum throughout the experiment. The feeding trials lasted for 90 d; a feed intake and weight gain were recorded throughout. Meat quality LL muscle from the 10th and 12th ribs were divided into four samples. The rst sample was stored at − 20 °C and used to determine avor and nutritional quality. The second sample was used to measured pH. Third sample and forth samples were used to measure meat color, cooking loss and shear force respectively.

Animals and diets
Volatile avors were determined using solid phase microextraction (SPME) and gas chromatography mass spectrometry (GC-MS) as described by V Vasta, G Luciano, C Dimauro, F Rohrle, A Priolo, FJ Monahan and AP Moloney [17] with some modi cations. Samples (5 g) were grounded with a grinder and placed into a 15 mL vials capped with PTFE septa.
The SPME bre (DVB/CAR/PDMS 50/30 mm; 57328-U; Supelco, Bellefonte, USA) was exposed to each sample and placed in vial for 40 min at 60 °C. After adsorption, the bre was inserted into the injection port at 250 °C for 3 min. For the GC (TRACE 1300, Thermo Fisher Scienti c, USA) analysis: the injector operated in splitless mode at 250 °C. The initial temperature was 40 °C, held for 5 min, heated up to 200 °C at 5 °C / min, held for 5 min, then heated up to 250 °C at 20 °C / min, held for 5 min. Helium was used as the carrier gas and the ow rate is 1.0 ml/min. The mass spectrum was acquired at 70 eV, and the scan mass range was m/z 30 ~ 400 m/z. Volatile compounds were identi ed by comparison with the mass spectra from the library database (NIST MS Search 2.0). The results of volatiles compound were expressed as the percentage (%) of each compound in total identi ed compounds.
The protein content was analyzed by Kjeldahl (GB 5009.5-2016) and intramuscular fat content was analyzed by Soxhlet (GB 5009.6-2016). The moisture was determined by drying to a constant weight in an oven (105 °C), and the ash on the sample residue was measured after drying at 550 °C for 12 hours in a mu e furnace.
The pH was determined at 45 min and 24 h post-mortem using a pH meter (pH-Star; Ingenieurbüro R. Matthäus, Ebenried). Meat color was evaluated at 45 min post-mortem by measuring the lightness (L), red (a *), and yellow (b *) values using a chromatic meter (CR-410, Konica Minolta, Japan). Samples were subsequently chilled at 4 °C for 24 h. Each sample was weighed (W1, g), then heated at 85 °C for 40 min in sealed plastic bag using a water bath. After cooling, absorb surface moisture with lter paper and weigh sample again (W2, g), and cooking loss (%) was calculated as (W1 − W2)/W1 × 100. To determine the shear force, samples were chilled at 4 °C for 24 h, heated in a sealed plastic bag using a water bath at 75 °C for 45 min. After cooling, any surface moisture was absorbed with lter paper. Rectangular cores (3 cm × 1 cm × 1 cm), parallel to the longitudinal orientation of the muscle bers, were taken and analyzed. Measurements were performed at least 8 times using a tenderness meter (C-LM3B, Northeast Agricultural University, Harbin, China).

Muscle ber characteristics
Transverse serial muscle ber sections (10 µm), obtained using a cryostat microtome (MEV, SLEE, Germany) at − 25 °C, were incubated using myo brillar adenosine triphosphatase (mATPase) staining methods (pH 4.60) to classify the muscle ber types [19]. Approximately 1500 bers per sample, which randomly selected from no tissue disruption and freeze damage, were detected and de ned as ber type I, ber type IIA, ber type IIB. All samples were analyzed by image analysis program (Laica QWin V3 Processing-Analysis Software, Leica, Germany). The cross-sectional area (CSA) of the muscle ber was determined as the ratio of the total measured area to the total number of bers count. The muscle ber density was expressed as the mean number of bers/mm 2 . The total number of muscle bers measured as the loin-eye area multiplied by the muscle ber density.
RNA extraction, cDNA synthesis and real-time PCR according to the instructions of the manufacturer. The purity and concentration of total RNA was detected by a microspectrophotometer (ND-1000, gene, China). Ratios of 260 nm and 280 nm absorption (A260/A280) between 1.8 and 2.0 was considered good quality of extracted RNA. The integrity of extracted total RNA was detected using 1% agarose gel electrophoresis. Total RNA was reversed transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China) according to manufacturer's instructions. The mRNA expression levels of all genes were examined by real-time quantitative polymerase chain reaction (RT-PCR) using TB Green™ Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, Dalian, China) and RT-PCR detection system (Roche, China). All primers were synthesized commercially by Sangon Biotech (Shanghai) and shown in Table 2.

Statistical analysis
All analyses were carried out using IBM SPSS Statistics 19.0. The Shapiro Wilk test and Levene test were used to test the normality of data distribution and homogeneity of variance. All data were analyzed using one-way ANOVA; each result was reported as the average ± standard error. P < 0.05 or P < 0.01 signi ed signi cance between means.

Results And Discussion
Carcass characteristics and meat quality Linseed supplementation had no effect (P > 0.05) on carcass weight, average daily food intake, average daily gain, dressing loss, backfat thickness, however, it did increase (P < 0.05) loin-eye area which is mainly used to evaluate carcass meat production (Table 3). It was also observed that linseed supplementation signi cantly reduced pH 24h (P < 0.01), L* (P < 0.05) and shear force (P < 0.01), but had no effect (P > 0.05) on pH 45min , a*, b* and cook loss (Table 4). Additionally, crude protein and ash signi cantly increased (P < 0.01) in the linseed group (Table 5).   [20][21][22][23][24]. In the present study, however, linseed supplementation did exert a signi cant effect on carcass traits, meat quality and chemical composition. Con icting results might be related to the level of linseed supplementation and the duration of the feeding trial [25]. For example, A Guerrero, C Sañudo, MM Campo, JL Olleta, E Muela, RMG Macedo and FAF Macedo [26] reported that a diet supplemented with 10% linseed and fattening for either 30 or 50 d could improve the fatty acid composition, productive, carcass characteristics and meat quality of cull ewes. According to R Marino, M Caroprese, G Annicchiarico, F Ciampi, MG Ciliberti, Ad Malva, A Santillo, A Sevi and M Albenzio [4], dietary linseed supplementation improved the meat tenderness and agrees with our current observations, which showed that dietary supplementation with linseed exhibited a lower shear force. The pH of muscle after slaughter is an important indicator of glycolytic activity; and was the basis for identifying potential PSE and DFD. In agreement with CE Devine, AE Graafhui, PD Muir and BB Chrystall [27], the pH 45min and pH 24h fell within the normal range. The pH 24h in the linseed group was lower than that of the control group. This difference in pH may account for the difference in muscle ber composition between the two groups since muscle ber type is related to the ultimate pH [28,29]. In the case of meat color, supplementation with linseed only reduced L* values, which is contrary to previous [4,30].
Major volatile avors in LL muscle including alcohols, aldehydes, ketones and hydrocarbons are given in Table 6. Aldehydes were the most, prominent followed by alcohols. A total of 47 volatile compounds were detected in current study, of which 33 volatiles were detected in the control group. In contrast, the linseed group contained 38 volatiles. The relative content of 1-pentanol, hexanal, nonanal, 2,3-octanedione and allyl 2-ethyl butyrate in the control group were signi cantly higher than that in the linseed group (P < 0.05).
Dietary linseed supplementation signi cantly increased the relative content of Z-10-Pentadecen-1-ol, pentanal, 2-Octenal, (E)-, decanal, butane and 2-heptanone (P < 0.05). Aldehydes, which had a low threshold and were mostly derived from fat oxidation and degradation, moreover, it played an important role in the formation of mutton avor [31]. Linseed signi cantly increased the relative content of pentanal and enhanced the fruity odors of mutton [32]. However, the relative content of hexanal was signi cantly reduced in linseed group, indicating that the control group had stronger grass like odors [32]. Hexanal was derived from the oxidation of linoleic acid and arachidonic acid. The reason why the relative content of hexanal in the control group was higher than that in the linseed group may be related to the antioxidant capacity of meat. The increase of antioxidant capacity can reduce the degree of oxidation of unsaturated fatty acids, thereby reducing the content of hexanal, the product of linoleic acid oxidation [3,33]. Linseed signi cantly increased the relative content of 2-octenal, (E)-,which provided a stronger meat and fatty and green odors [32]. Relative content of nonanal in the control group was signi cantly higher than that in the linseed group. Nonanal is associated with various odors in mutton including fat, oral and citrus [34]. 2,4-Decadienal, (E,E)-was a product of linoleic acid, which had a meaty and grilled odors, and was a unique avor substance in the linseed group [33]. The relative content of decanal in the linseed group was signi cantly higher in the control group (P < 0.05), contributing to stronger odors of soap, orange peel and tallow in mutton [32]. Undecanal were the special avor substances in the control group, which exhibited fat, wax and soapy odors [34]. Dodecanal was endemic to the linseed group and has been reported to have an onion and yeast odor [34].
The formation of meat avors is generally related to low thresholds of volatile compounds. For example, aldehyde compounds, produced by the oxidation of fat were reported by [35] to have low thresholds with respect to smell. Compared with glycolytic muscle ber, oxidized muscle bers had higher a content of phospholipid [36] which were one of the main substances affecting meat. avor Free amino acids are also vital avor precursors in meat [37]. D Mashima, Y Oka, T Gotoh, S Tomonaga, S Sawano, M Nakamura, R Tatsumi and W Mizunoya [38] reported that there was a positive correlation between the MyHC and total free amino acid concentrations. These results revealed that muscle ber type had a strong effect on the avor of meat. In addition, PUFA in meat were prone to oxidation and were important precursors of volatile avor substances. But excessive oxidation could cause bad smell. According to ND Cameron and MB Enser [39], PUFA in meat was easily oxidized which was inversely related to avor.
Therefore, the difference in avor may be related to the antioxidant capacity. The antioxidant system and the oxidizing system restrict each other to prevent the occurrence of lipid peroxidation.

Muscle ber characteristics and MyHC isoforms mRNA expression level
Representative photomicrographs of control and linseed fed groups are shown in Fig. 1. Muscle bers were classi ed into types: I (black), IIA (white) and IIB (brown) using myosin ATPase staining. Muscle ber characteristics are shown in the Table 7. Linseed supplementation decreased CSA (P < 0.01), increased total ber number and density (P < 0.01). The ber diameters of types I, IIA and IIB were observed to decrease (P < 0.01) with linseed supplementation, but had no effect on muscle ber numbers and area composition (P > 0.1). According to the polymorphism of the myosin heavy chain (MyHC), muscle bers can be divided into four types: MyHC I (slow-twitch oxidative), MyHC IIa (fast-twitch oxidative), MyHC IIx (fast-twitch oxidative-glycolytic) and MyHC IIb (fast-twitch glycolytic) [40]and serve as a fast, reliable and accurate method to identify muscle ber types. As shown in Fig. 2, linseed supplementation increased the mRNA expression of MyHC and MyHC x (P < 0.01), but had no effect on the mRNA expression level of MyHC a and MyHC b. These results suggest that the number of oxidized muscle bers were higher in the linseed group. Muscle from the linseed group contained increased (P < 0.01) activity of SDH which is a key enzyme involved in aerobic metabolism. In addition, a decrease (P < 0.01) in LDH activity, major enzymes involved in anaerobic glycolysis, were observed (Fig. 3). These results suggest that a diet supplemented with linseed increased the oxidative metabolism of muscle. Several reports have shown that the of number muscle bers contributed to no signi cant changes in postnatal mammalian muscles [41,42]. Therefore, muscle ber hypertrophy in postnatal lies with the total number of muscle bers within a muscle [42]. This nding was consistent with that of AJ Fahey, JM Brameld, T Parr and PJ Buttery [43] who found that maintenance, nutrition, and mobility of lambs during the postnatal period had no effect on the muscle bers numbers and ber types However, nutrition did have a signi cant effect on muscle ber diameter. Fewer number of muscle bers may lead to an increase in the muscle ber diameter [44]. This nding was also reported by F Gondret, L Lefaucheur, H Juin, I Louveau and B Lebret [45]. In current study, linseed supplementation resulted in higher total number of muscle ber and lower muscle ber diameters. In the control group lower numbers of muscle ber and higher muscle ber diameters were consistent with previous research results. A relationship between the number and size of muscle bers and muscle mass has been reported [15,46]. Several reports have shown that mean CSA bers [47] and muscle bers diameter [48] were negatively related to tenderness. Moreover, a previous study has shown that shear force was negatively related to tenderness [49]. All in all, as the muscle ber diameter and CSA increased, the shear force increased, and the tenderness decreased. In our study, dietary linseed supplementation decreased CSA and the diameter of muscle bers, meanwhile the lower shear force value was observed in linseed group. These results t well

Muscle antioxidative capacity
Dietary supplementation with linseed had no effect (P > 0.10) on muscle CAT and GSH-PX enzyme activity but did result in greater (P < 0.01) T-AOC activity (Table 8) and the mRNA level of GSH-PX (P < 0.01) and CAT (P < 0.05) (Fig. 4), and decreased T-SOD enzyme activity and mRNA level ( Table 9, Fig. 4).
Moreover, MDA content was decreased (P < 0.01) by dietary supplementation with linseed. Lipid peroxidation had a negative effect on meat quality [52]. Therefore, it is common knowledge that inhibit oxidation and reduce lipid peroxidation by dietary natural antioxidants were effective for improving meat quality. Previous studies had shown that axseeds are rich in phenolic substances which can directly participate in anti-free radicals active [13]. I Moñino, C Martínez, JA Sotomayor, A Lafuente and MJ Jordán [53] reported that sheep which are rich in polyphenols in the diet, free radical scavenging ability will also be enhanced. Because of MDA was a secondary product of lipid oxidatirron, MDA content could re ect the degree of lipid peroxidation in the tissue [54]. Therefore, MDA may indicate an antioxidant capacity. In current study, T-AOC activity and the mRNA level of GSH-PX and CAT were increased, MDA was decreased in linseed group, which results indicated that dietary linseed supplementation can enhance antioxidant capacity of sheep. This nding was consistent with that of LB Pouzo, AM Descalzo, NE Zaritzky, L Rossetti and E Pavan [55] who found that by supplementing with low levels of axseed, the antioxidant capacity of beef can be improved. SOD was the rst enzyme to ght oxidative stress, the content of axseed oil and fat were as high as 30-45%, and the oil and fat may act as a pro-oxidant to inhibit the activity of SOD, which may be the reason for the decreased SOD activity of linseed group [56,57]. Moreover, at the same time point, aldehyde compounds which was considered to be a characteristic product of the oxidation process [35] had changed was observed in linseed group, which may be related to increased antioxidant capacity. In general, we speculate that the improvement of meat quality and the change of avor substances may be attributed to the enhancement of antioxidant capacity.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests. Cross-sections of LL muscle stained for myosin ATPase after pre-incubation in pH 4.60-4.65.

Figure 2
Effect of linseed supplementation on MyHC isoforms of LL muscle ** Signi cantly different (P<0.01) from control.

Figure 3
Effect of linseed supplementation on SDH and LDH activities of LL muscle ** Signi cantly different (P<0.01) from the control.

Figure 4
Effect of linseed supplementation on the mRNA expression level of antioxidant genes of LL muscle * Signi cantly different (P<0.05), ** signi cantly different (P<0.01) from the control.