Carcase traits, meat quality, and lipogenic gene expression in muscle of lambs fed wheat bran feruloyl oligosaccharides

Abstract This study investigated the effects of dietary feruloyl oligosaccharides derived from fermented wheat bran (FOs-FWB) supplementation on carcase traits, meat quality, muscle fatty acid composition, and the expression of lipogenic genes in lambs. In a completely randomised design, 50 Dorper × thin-tailed Han lambs (20.21 ± 3.36 kg) were randomly divided into five treatments with ten replications. The lambs in the control group were fed a basal diet, while those in the experimental groups were fed the basal diet supplemented with 50, 100, 200, and 400 mg/kg FOs-FWB. The shear forces of longissimus dorsi (LD) in lambs fed 50 and 100 mg/kg FOs-FWB were significantly lower than that of the control group (p < .05). Lambs fed with 200 mg/kg FOs-FWB had higher crude protein and lower fat contents in LD muscle (p < .05), the ash content in LD muscle was increased by FOs-FWB supplementation (p < .05). Lambs fed 50 mg/kg FOs-FWB had significantly higher proportions of C18:1n − 9, C20:1, C18:3n − 3, C18:3n − 6 and total n − 3 (p < .05). The proportion of C18:3n − 6 was significantly increased by 100 mg/kg FOs-FWB supplementation (p < .05). Feeding 200 mg/kg FOs-FWB significantly decreased proportions of C14:0 and total saturated fatty acid (SFA), but increased proportions of C18:1n − 9, C18:2n − 6, total monounsaturated fatty acid (MUFA) and unsaturated fatty acid (UFA) to SFA ratio (p < .05). Moreover, increases in the proportion of C22:6n − 3 and total polyunsaturated fatty acid (PUFA) were observed in lambs fed FOs-FWB (p < .05). The relative expression levels of ACC, FAS, and SCD genes in the LD muscle of lambs fed 200 and 400 mg/kg FOs-FWB were higher than the control group (p < .05). Dietary supplementation with 100, 200, 400 mg/kg FOs-FWB significantly increased the relative mRNA expression level of LPL (p < .05). Furthermore, a decrease in the relative mRNA expression level of PPARγ was observed in lambs fed 50 mg/kg FOs-FWB (p < .05). In conclusion, dietary supplementation with FOs-FWB reduced shear force, enhanced intramuscular fat content and PUFAs proportion and stimulated lipogenic gene expression in LD muscle of lambs, especially at a level of 200 mg/kg addition. Highlights Dietary FOs-FWB improved the tenderness of the meat. Dietary supplementation with 200 mg/kg FOs-FWB increased the content of intramuscular fat and PUFAs, as well as the ratio of UFA to SFA. Dietary FOs-FWB affected the expression of genes related to lipid metabolism.


Introduction
Mutton is one of the most popular red meat worldwide, which can supply a range of essential nutrients in the human diet.Both sensory quality characteristics and nutritional value have strong influences on the choice of consumers for mutton products.The two important consumer drivers are linked through intramuscular fat content and composition.However, the excessive pursuit for growth rates of lambs has led to an increase of saturated fatty acid (SFA) contents and induced deterioration of the sensory quality of lamb meat (Ponnampalam et al. 2012).Many studies have reported the possibility of modifying the fatty acid profile and sensory quality of lamb meat by nutritional strategies (Scollan et al. 2017).For example, oilseeds (Aldrich et al. 1997), algae (Clayton et al. 2014), fish oils (Najafi et al. 2012) and plant extracts (Smeti et al. 2018) increased the proportion of PUFAs and sensorial attributes of lamb meat.
Ferulic acid (FA) is the most abundant phenolic acid in the cell wall of wheat bran and has been proven as a potential source of antioxidants (Mathew and Abraham 2004;Wang et al. 2019a).The dietary supplementation of FA in improved hot carcase dressing and Longissimus thoracic (LD) muscle area of lambs (Peña-Torres et al. 2022) and sensory quality and PUFAs content of steers (Gonz alez-R ıos et al. 2016).Feruloyl oligosaccharides (FOs), oligosaccharide ester-linked FA, is the mainly bound forms of FA, which could be produced from feruloyl polysaccharides hydrolysis (Ou and Sun 2014;Malunga et al. 2016).It has been proven that FOs exhibit the biological activity FA and oligosaccharides, and its in vitro antioxidant capacity is stronger than that of free FA (Zhao et al. 2018).In the previous study, we found that dietary supplementation of FOs derived from fermented wheat bran (FOs-FWB) promoted growth performance, modulated rumen fermentation and increased serum antioxidant enzyme activities and GSH concentration of lambs (Wang et al. 2019b).Thus, it was hypothesised that FOs-FWB may be an effective alternative to modify the meat quality and lipid metabolism of lambs.To test the hypothesis, the effects of dietary FOs-FWB supplementation on carcase traits, meat quality, fatty acid composition and lipid metabolism gene expression of lambs were evaluated.The data collected in this study will add to our existing understanding of FOs-FWB and identify a potential feed additive for lamb production.

Experimental design, animal management, and diets
The FOs-FWB preparation and animal management regimen for the present experiment have been reported previously (Wang et al. 2019b).Briefly, fifty crossbred male lambs (Dorper Â thin-tailed Han) at two months old with 20.21 ± 3.36 kg body weight were used.Lambs were randomised into five dietary treatments with ten lambs each.The lambs were fed a basal diet supplemented with 0 (control group), 50, 100, 200, and 400 mg/kg FOs-FWB, respectively.The inclusion level of FOs was chosen based on our preliminary study (Wang et al. 2018).The lambs were housed in individual stalls equipped with feeders and a water source.The experimental period lasted 56 d and was preceded by an adaption period of 14 d.Lambs received the diets twice daily (at 08:00 and 18:00) to ensure 10% refusal, and had free access to fresh water.The ingredient and chemical composition of the basal diet is shown reported in Table 1.The feed was formulated based on the Feeding Standard of Meat-Producing Sheep and Goat of NY/T 816-2004 (Ministry of Agriculture of the People's Republic of China 2004) recommendation as a pelleted mixed diet.

Slaughtering, carcase and meat sampling
At the end of the feeding trial, all lambs were slaughtered in the local abattoir using Halal methods.The lambs were fasted from feed and water for at least 12 h before slaughter.The pre-slaughter live weight and hot carcase weight were recorded, and the dressing percentage was calculated.The loin eye area was measured using a digital planimeter on the cut surface of LD muscle at the interface between the 12th and 13th rib on the left sides of the carcase.The total tissue depth (GR value) between the 12th and 13th rib, 100 mm from the midline of the spine were recorded (Ponnampalam et al. 2007).Three pieces of the left LD muscles were sampled, one piece was immediately used for meat physical characteristics measurement, the second was lyophilised and minced for chemical and fatty acid composition analysis, and the third was snap-frozen in liquid nitrogen for genes expression analysis.

Meat quality analysis
The meat quality analysis was conducted according to methods published by Gonz alez-R ıos et al. (2016).Briefly, the pH value of LD muscle was detected using a pH metre (Russell CD700; Russell pH Limited, Germany) at 45 min after slaughter.Meat colour including lightness (L Ã ), redness (a Ã ) and yellowness (b Ã ) was measured by an automated colorimeter (CR-300, Minolta, Osaka, Japan).Chroma (C Ã ) was calculated by the formula: C Ã ¼ (a Ã þ b Ã ) 1 = 2 and Hue angle (H o ) using the formula: H o ¼ tan À1 (b Ã /a Ã ).For the cooking loss analysis, 120 g of LD muscle sample was cooked in a water bath until the core temperature reached 70 C.After that, the sample was cooled, and cooking loss was calculated as a percentage of weight change before and after cooking.Subsequently, meat was cut into pieces of 1 cm Â 1 cm Â 2.5 cm along the direction of the muscle fibres, and the shear force was determined using a texture analyser (C-LM2, Xieli Technology Development Co., LTD, Qinhuangdao, China).The water loss rate was determined using the filter paper press method (Du et al. 2022).The LD muscle sample 1.0 cm in diameter and 0.5 cm in thickness was taken.The samples were weighed, wrapped in an 18-layer filter paper, pressed using a compression machine (YYW-2, Nanjing Soil Instrument, Nanjing, China) at a force of 35 kg for 5 min and then reweighed.The water loss rate was calculated as a percentage of weight change before and after pressing.

Meat chemical and fatty acid composition analysis
The moisture, crude protein, ether extract and ash contents of LD muscle samples were determined according to the method of the Association of Official Analytical Chemists (AOAC 2007): moisture by Method 930.15; crude protein by Kjeldahl method 984.13; ether extract by the Soxhlet method 963.15 and ash by incineration method 924.05.
The intramuscular fatty acids composition were determined according to the Determination of fatty acids in food of GB 5009.168-2016(State Food and Drug Administration of the People's Republic of China) using the internal standard (methyl nonadecylate, Sigma Chemicals, St. Louis, MO, USA).The lyophilised LD muscle sample (80-100 mg) was mixed with 100 lL of the internal standard.Then, 2 mL of NaOH/CH 3 OH was added.The mixture was homogenised with a vortex mixer, heated for 30 min at 85 C, and then cooled for 6-7 min.Then, 3 mL of trifluoro(methanol)boron was added.The mixture was then shaken and reheated for 30 min at 85 C.After cooling to ambient temperature, the upper extraction layer (n-hexane) was collected.A TG-5MS (Thermo Fisher Scientific, USA) fused-silica capillary column (30 m Â 0.25 mm ID Â 0.25 mm film thickness) was used to separate the methyl esters, which were detected with a flame ionisation detector.The oven temperature program consisted of the following program: 80 C for 1 min, increased to 200 C at 10 C/min, increased to 250 C at 5 C/min, increased to 270 C at 2 C/min, and hold for 3 min.The injector and detector temperatures were maintained at 290 C and 280 C, respectively.The concentrations of individual fatty acids were quantified according to the peak area and are indicated as g/100 g total fatty acid.

Gene expression analysis
Total RNA was extracted from LD muscle using the TRIzolV R Plus RNA Purification Kit (Invitrogen No 12183-555).The concentration and purity of the RNA was assessed using a Bio Tek Epoch2 Microplate Spectrophotometer (Aglient Bio-Tek, USA) at 260/280 nm absorbance.The SuperScript TM III First-Strand Synthesis SuperMix(Invitrogen NO 11752-050) was used for cDNA synthesis.b-actin and glyceraldehyde phosphate dehydrogenase (GAPDH) were analysed to determine the suitable housekeeping gene.b-actin had the best gene expression stability.Therefore, the housekeeping gene b-actin was used as the internal control.Quantitative polymerase chain reaction (qPCR) was performed according to the manufacturer's instructions (Power SYBRV R Green PCR Master Mix, Roche, NO:4913914001).Forty cycles of amplification (comprising 95 C for 15 s and 63 C for 25 s) were performed.Amplification and melting curve analyses were then performed using the Quant Studio O3 software program (Life Technologies, USA).Amplifications were conducted in triplicate, and the relative expression level of each gene was analysed using the 2 ÀDDCt method.Information of primer sequences for peroxisome proliferator-activated receptor c (PPARc), lipoprotein lipase (LPL), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-coenzyme A desaturase (SCD) are listed in Table 2.

Statistical analysis
Individual lambs served as experimental units Data were analysed using one-way ANOVA (SAS 9.2, SAS Inst.Inc., Cary, NC, USA).Mean comparisons were performed using Duncan's multiple tests.The final test results were presented as mean and SEM.Significance was considered when p < .05.

Carcase traits and meat quality
The carcase traits of lambs were not affected by dietary FOs-FWB supplementation (Table 3).The shear force of LD muscle from lambs received 50 and 100 mg/kg FOs-FWB was significantly (p < .05)lower than the control group (Table 4).There was no significant difference in the pH 45 min , meat colour, water loss rate, and cooking loss rate of LD muscle among the groups.

Chemical composition of longissimus dorsi muscle in lambs
Compared with the control group, lambs fed with 200 mg/kg FOs-FWB had significantly lower crude protein content but higher ether extract content in LD muscle (Table 5; p < .05).Furthermore, the content of ash in LD muscle was significantly increased by dietary FOs-FWB supplementation (p < .05).

Lipid metabolism gene expression
The mRNA levels of genes related to lipid metabolism in LD muscle are shown in Figure 1.The relative mRNA expression levels of ACC, FAS and SCD in LD muscle of lambs fed 200 and 400 mg/kg FOs-FWB were higher than that of the control group (p < .05).Feeding 100, 200, 400 mg/kg FOs-FWB significantly increased the relative mRNA expression level of LPL (p < .05).Furthermore, a decrease in the relative mRNA expression level of PPARc was observed in lambs fed 50 mg/kg FOs-FWB (p < .05).

Discussion
It has shown that FOs exhibit the biological activity of FA and oligosaccharides, such as antioxidant, immunomodulatory and probiotic physiological effects (Ou and Sun 2014).In our previous, FOs was obtained by fermentation of wheat bran, and the esterified FA   ,b,c,d Means (n ¼ 10) with different superscripts within the same row differ significantly (p < .05). 1 Lambs received a basal diet (control) or basal diet supplemented with 50, 100, 200 or 400 mg/kg FOs-FWB.content in FOs-FWB was 0.99 mmol/g (Wang et al. 2019b).Furthermore, FOs-FWB can be used as a feed additive in lamb production to improve the growth performance and antioxidant status, thus has application prospects in modifying meat quality and lipid metabolism of lambs.In this study, the effects of FOs supplementation on carcase traits, meat quality, fatty acid composition and lipogenic gene expression of lambs were evaluated for the first time.
The carcase traits of lambs were not affected by dietary supplementation of FOs-FWB.Our results are consistent with previous research that found dietary addition of FA or polysaccharides with antioxidant properties had no influence on carcase characteristics of lambs (Gonz alez-R ıos et al. 2016; Pereira et al. 2020).Carcase traits are directly related to the preslaughter live weight of animals and affected by nutrient consumption.Therefore, the lack of difference might be attributed to the isoproteic and isoenergetic diets used in this study.
Meat colour is a direct factor in judging meat quality and customers' willingness to buy.Meanwhile, the pH value reflecting the rate and intensity of muscle glycogenolysis after slaughter is another important index of meat quality and related to meat colour and water holding capacity (Li, Wang, et al. 2015).Cooking loss and water loss rate can be used to measure the water-holding capacity of meat.It has been proven that feed additives with great antioxidative capacity could delay glycolysis and oxymyoglobin oxidation and reduce oxidative-induced conformational alterations and fragmentation of myofibrillar proteins (Salami et al. 2015).However, in the present study, some traits of the meat quality such as pH 45 min , L Ã , a Ã , b Ã value, water loss rate and cooking loss were not affected by dietary FOs-FWB supplementation.And the observed pH 45 min , L Ã , a Ã and b Ã values of applied treatments and control group maintained within an acceptable range of lamb meat.These data were in accordance with previous research that found that dietary FA supplementation did not affect the muscle pH and colour of lambs (Valadez-Garcia et al. 2021) and pigs (Li, Li, et al. 2015).Furthermore, Peña-Torres et al. (2021) reported no influence of FA supplementation on the water loss rate and cooking loss of heifers.Further studies are required to elucidate the reason why FOs-FWB did not influence the pH, colour, water holding capacity of lambs.
In the current study, lambs fed FOs-FWB had reduced shear force compared to the control group.Similarly, Gonz alez-R ıos et al. ( 2016) found that dietary supplementation of FAs decreased the shear force in crossbred steers.Shear force is used to assess the tenderness of the meat, which is affected by muscle fibre types (Schiaffino and Reggiani 1996).In mice (Chen et al. 2019) and pigs (Wang et al. 2021), FA supplementation triggers a preferential differentiation in muscle from fast glycolytic muscle fibres towards slowly oxidising muscle fibres (type I).Peña et al. (2018) also observed increases in the area and diameter of oxidative fibres response to FA supplementation in lambs.It has been reported that an increase in the proportion of type I fibre reduced the shear force of meat (Choi and Kim 2009).Thus, the positive effects of FOs-FWB on shear force may be due to the altered composition of muscle fibre types.
The content of intramuscular fat (IMF) is closely related to meat quality, such as tenderness and flavour (Joo et al. 2013).To the best of our knowledge, few study has been conducted to examine the effects of FA on the IMF of domestic animals.Gonz alez-R ıos et al. ( 2016) reported there is no difference in IMF content of steer fed with and without FA.Similar results were also found by Peña-Torres et al. ( 2021) that feeding FA did not affect the IMF content of heifers.In contrast, our study shown that the fat content of LD muscle in FOs-FWB groups was higher, which was agreed with the reduction of shear force.The discrepancy in IMF might be caused by the specie, physiological state, oxidative status of animals as well as dosage and supplementation period of FA.
The proportional composition of fatty acids in meat is conventionally used for evaluating the nutritional and healthy values of meat (Cabrera and Saadoun 2014).The levels of unsaturated fatty acids (UFA) in the mutton are known to influence the fat firmness, meat nutritional value and consumers' acceptance.Especially, the percentage of long-chain n-3 PUFA, such as C18:3n À 3 (a-linolenic acid, ALA), C20:5n À 3 (docosapentaenoic acid, DPA), and C22:6n À 3 (docosahexaenoic acid, DHA) are beneficial for preventing atherosclerosis and cardiovascular disease and promoting immune function of human.In this study, we found that the C22:6n À 3 and total PUFA proportion in lipids of LD muscle were increased by dietary FOs-FWB supplementation.The modulating effects of FOs-FWB on the fatty acids profile of LD muscle might partly be attributed to FA, which could be released in the rumen (Yue et al. 2009).Consistent with our findings, Gonz alez-R ıos et al. (2016) reported that a diet containing FA could increase the percentage of PUFA in the muscle of steers.Phenolic compounds have been proven to modify the rumen microbiota and reduce the probability of biohydrogenation of UFA in the rumen (Cabiddu et al. 2010;Jayanegara et al. 2011;Jafari et al. 2016).It was confirmed that the PUFA proportion in lipids of mutton was increased by the dietary addition of plant polyphenols (Cimmino et al. 2018;Smeti et al. 2021).Furthermore, in the present study, feeding 200 mg/kg FOs-FWB significantly decreased proportions of C14:0 and total SFA, but increased proportions of C18:2n À 6, total MUFA and UFA to SFA ratio.Thereby our data indicate that the inclusion of 200 mg/kg FOs-FWB is a superior choice.
Furthermore, the mRNA expression of a gene involved in fatty acid metabolism in the LD muscle were analysed in this study.Notably, this is the first study to investigate the effects of FOs-FWB on transcripts of fatty acid metabolic genes in the muscles of lambs.Of these genes, LPL was found to be involved in fatty acid uptake, and FAS and ACC were responsible for de novo fatty acid synthesis (Deng et al. 2018).In this study, along with increased IMF level in LD muscle, mRNA expression of LPL, FAS and ACC was upregulated by dietary supplementation of 200 and 400 mg/kg FO-FWB.Furthermore, D9 desaturase (encoded by the SCD gene) participates in the formation of MUFA, mainly C16:1 and C18:1n À 9, from SFA in ruminants (Daniel et al. 2004).As expected, FOs-FWB supplementation at 200 and 400 mg/kg upregulated mRNA expression of SCD, which is in agreement with the higher C18:1n À 9 proportion in lipids of LD muscle.However, in contrast with our results, Milojevic et al. (2020) did not observe any effect caused by the dietary plant extracts (rich in phenolic compounds) on the above genes expression of ewes.The inconsistent findings may result from factors such as the growth stages of lambs, rearing environment, additives kinds and dietary nutrition level.
PPARc plays an important role in the induction of adipocytes differentiation and adipogenesis of mature adipocytes (Kersten et al. 2000;Perera et al. 2006), thus acts as an important regulator of IMF content in animals (Yang et al. 2019;Mohammadpour et al. 2020).It has been proven that the transcription of genes involved in lipid metabolism, such as LPL, FAS, and ACC are negatively correlated with the activation of PPARc (Takahashi et al. 2002;Ilavenil et al. 2015).Based on molecular dynamics stimulation studies and stability of binding, FA was predicted to be a potential PPARc inhibitor (Senthil et al. 2021).Yin et al. (2022) suggested that FA could downregulate the expression of PPARc in zebrafish.Similarly, in this study, the expression of PPARc in LD muscle of lambs fed 50 mg/kg FOs-FWB was lower than the control group, and lambs fed 200 mg/kg FOs-FWB had similar PPARc expression level.Therefore, we speculated that the addition of FOs-FWB may achieve the aforementioned effects by activating PPARc signalling pathway.However, additional studies are needed to

Table 1 .
Ingredient and chemical composition of basal diet (%, air-dry basis).

Table 2 .
Information of primer sequences of lipid metabolism genes.

Table 4 .
Effect of dietary feruloyl oligosaccharides derived from fermented wheat bran (FOs-FWB) supplementation on physical characteristics of Longissimus dorsi muscle in lambs.Means (n ¼ 10) with different superscripts within the same row differ significantly (p < .05). 1 Lambs received a basal diet (control) or basal diet supplemented with 50, 100, 200 or 400 mg/kg FOs-FWB.

Table 6 .
Effect of dietary feruloyl oligosaccharides derived from fermented wheat bran (FOs-FWB) supplementation on fatty acid composition of Longissimus dorsi muscle in lambs (% total fatty acids).