Animals, diets, and experimental design
The feeding trial was carried out at the Xishuixianfuxingmuye Co. Ltd., Guizhou, China (106.198244 E, 28.26403 N). All procedures used in this study were approved by the Guizhou University, Experimental Animal Ethics (Guiyang, China).
The whole experiment stage was 74 d, which from 12 July to 24 September in 2020. The preparation period was 14 d and formal experiment period was 60 d. A total of eighteen Qianbei Ma wether kids (body weight, 21.38 ± 1.61 kg; mean ± standard deviation) were randomly allotted into three groups using a completely randomized design with six duplicates per group. The dietary treatments were: 1) the control group was provided with basal diet; 2) treatment 1 (LA) was provided basal diet with 0.5 g/d PCP; and 3) treatment 2 (HA) was provided basal diet with 1 g/d PCP. The PCP was purchased from Nanjing Herd Source Bio-technology Co., Ltd., Nanjing, China. The level of PCP was used in the research followed by Tian et al. [12]. All experimental kids were housed in clean individual pens and water was taken freely during the experimental period. The equal amount of rations was provided twice daily at 08:30 and 16:30 for ad libitum intake and 10% refusals on an as-fed basis. The nutrient requirement of experimental animals according to the NRC [13], and the diet formulations was listed in Table 1.
Determination of chemical composition
Approximately 100 g of experimental diet was collected once weekly and stored at -20°C. After the end of feeding trials, the basal diet of total mixed ration was dried at 65°C in a vacuum oven for 72 h, then ground and passed through a 1-mm sieve. The dry matter (DM), crude protein (CP), ether extract (EE), Ash, calcium (Ca), and phosphorus (P) were measured as per the method of AOAC [14]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to Van Soest et al. [15] using a FT 122 FibertecTM analyzer (Foss, Hillerød, Denmark). The organic matter (OM) and hemicellulose were obtained by the 100 minus the Ash as well as NDF minus ADF, respectively. Gross energy (GE) was detected using a calorimeter (WGR-WR3, Changsha BENTE Instrument Co., Ltd., China). Each sample was run in triplicate.
Determination of anthocyanin composition
The anthocyanin composition of PCP was extract using 1.5 mole HCl dissolved in 95% ethanol solution (ethanol:HCl = 85:15) was detected by a HPLC-MS/MS according to Tian et al. [11]. The anthocyanin compositions of pelargonidin (Pel), peonidin (Peo), cyanidin (Cya), malvidin (Mal), petunidin (Pet), and delphinidin (Del) were determined in this study. Total anthocyanins (TA) content was calculated from the following formula: TA = Pel + Peo + Cya+ Mal + Pet + Del. The anthocyanin composition of commercial PCP was list in Table 2.
Determination of growth performance
Dry matter intake (DMI) was calculated per day throughout the entire study period. The body weight of each kid was weighted at 1th d and 74th d of the trial period before morning feeding. The average net gain (ANG), average daily gain (ADG) and feed conversion ratio (FCR) were calculated until the end of the experiment. Fecal sample was collected on the last 7 days (2 d of the adaptation period and 5 d of the digestion trial period) and proximate nutrients digestibility was determined by the method of acid-insoluble ash (AIA) [16] according to the following formula: nutrient apparent digestibility (%) =100%-(AIA in diet/AIA in fecal×nutrient in fecal/nutrient in diet) ×100. All fecal samples were oven-dried at 65 °C for 72 h, ground and passed through a 1-mm sieve, and kept at 4 °C until further analysis.
Assessment of meat quality
Six goats per group were slaughtered at the end of the experiment, the experimental kids were slaughtered, and the processing of carcass was described by National agricultural industry standard of the People’s Republic of China [18]. The LD of kid on the left body side was separated for detection of meat quality. The pH of LD was measured at 45 min and 24 h (put into a chiller at 4°C) by a pH-star (Matthäus, Eckelsheim, Germany). The pH value was updated 2 times per second, and until the value was stable and data was recorded. Each sample at per time was run in triplicate. Percentage of water loss was detected by the pressure method [19]. Briefly, 1 cm thick sample was cut using a 2.532-cm diameter with 5 cm2 area circular sampler and weighted the pre-pressure weight. Next, the sample was sandwiched between two layers of gauze, and was placed on a platform with 18 layers of qualitative medium-speed filter paper above and below, and pressurized to 35 kg (pressure gauge measuring range about 138) for 5 min. The platform was removed and immediately peeled meat sample from the gauze to weigh post-pressure weight. Percentage of water loss was calculated using the following formula: Percentage of water loss (%) = [(pre-pressure weight - post-pressure weight)/ pre-pressure weight] ×100. For drip loss, about 10 g and 3 cm length cube of LD was manually trimmed and weighed, and suspended by a fishhook in an inflated plastic bag, sealed and stored for 24 h at 4 °C. The sample was weighted after removing the fishhook and drying on filter paper. Drip loss was calculated as follows: drip loss (%)=[(initial weight−final weight)/initial weight]×100%. For cooked meat rate, about 100 g of LD was weighted without fascia, epimysium or fat. The sample was placed on the steamer and cooked for 30 min, and steamed meat sample was weighted after cooling 4 °C for 2 h. Cooked meat rate was calculated as follows: cooked meat rate (%)= (fresh weight- cooked weight)×100%. Meat color was measured using an Opto-Star equipment (Company MATTHÄUS, KLAUSA). The shear force of sample cores was measured using a digital display tenderness meter (Xielikeji co., ltd., Harbin, China) with a load cell of 15 kg and a 200-mm/min crosshead speed.
Determination of antioxidant status
The procedures of LD homogenate as follow: the sample was washed with pre cooled PBS (0.01 M, pH = 7.4) to remove residual blood, and sample was cut up after weighing. The chopped tissue and PBS were added to the glass homogenizer according to the ratio of 1:9, the mixture was ground thoroughly with ice. The homogenate was broken by ultrasonic using a Bransonic® ultrasonic cleaner (Branson ultrasonics corporation, USA). The homogenate was centrifuged at 4,000 ×g for 10 min at 4°C, and the supernatant was transferred to a 1.5-mL tube and stored at -80°C.
The total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH), catalase (CAT), peroxidase (POD), malondialdehyde (MDA), nitric oxide (NO), superoxide anion (O2-·), and hydroxyl FR (·OH) were determined using commercial available kits from Nanjing Jiangcheng Bioengineering Institute (Nanjing, China; Product Codes were A015-1, A001-3, A005, A006-1-1, A007-1-1, A084-3-1, A084-3-1, A012-1, A052-1-1, A018-1-1, respectively). All measure operation procedures were strictly followed the manufacturers’ protocol.
A FR parameter of 2,2-diphenyl-1-picrylhydrazyl (DPPH; Pcode: 101845869, Sigma-Aldrich, St. Louis, MO) scavenging activity was measured according to spectrophotometric method [17]. Briefly, an aliquot of 20 μL of sample was mixed with 0.6 mL of 0.1 mmol/L DPPH solution in a 1.5 mL tube. The mixture was centrifuged at 4,000 ×g for 10 min at 4°C, then 200 μL of supernatant was immediately transferred to a 96-well plate (TCP011096, JET-BIOFIL®, Beiden Biological Technology Co. Ltd., Nanjing, China) and incubated in the dark at 37°C for 30 min. The absorbance was analyzed at 517 nm via a microplate reader (Epoch, BioTek, Luzern, Switzerland). The equation for calculation was as follows: DPPH scavenging activity (%) = (Ac − As) ×100/Ac, where Ac as the absorbance of the control, As was the absorbance of the sample.
Determination of fatty acid profilesof Longissimus dorsi
A total of 50 mg of LD sample was weighted and was transferred to a 2-mL tube, 1 mL of chloroform methanol solution (2:1) and 2 steel balls were added, and the mixture was shaken vigorously using a tissuelyser with 60 Hz for 1 min, the ultrasonic extraction at room temperature for 30 min, and centrifuged at 12,000 ×g at 4°C for 5 min (H1850R, Hunan Xiangyi Centrifuge Instrument Co., Ltd., China), then supernatant was transferred to a 15-mL centrifuge tube. The sample was esterified in water bath at 80℃ for 30 min after extraction solution was thoroughly mixed with a volume of 2 mL of 1% sulfuric acid methanol solution. After cooling to room temperature, 1 mL n-hexane was added, tube was thoroughly mixed and left to stand for 5 min, then 5 mL distilled water was added and centrifuged at 12,000 ×g at 4°C for 5 min. 100 mg anhydrous sodium sulfate powder was added after a volume of 700 μL of supernatant was transferred to a 2-mL tube with sufficient mixing, then centrifuged at 12,000 ×g at 4°C for 5 min. Next, 300 μL supernatant was transferred to a 2-mL tube, and 15 μL of 500 ppm methyl salicylate was added and mixture was vortexed. The individual FA was detected using a trace 1310-ISQ 7000 gas chromatography-mass spectrometer (GC-MS; Thermo Fisher Scientific, USA). The GC-MS conditions were as follows: separation of FA by a Thermo TG-FAME capillary column (50 m × 0.25 mm × 0.20 µm), the injection volume was 1 µL, split ratio was 8:1; injection port temperature was 250°C, the ionization temperature was 230°C, transmission line temperature was 250°C, Quadrupole temperature was 150°C; temperature program was an initial temperature of 80°C for 1 min, a 20 °C/min rise to 160°C and held for 1.5 min; a 3°C/min rise to 196°C and held for 8.5 min, then a 20°C/min rise to 250°C and held for 3 min; helium gas as carrier, the flow rate of carrier gas was 0.63 mL/min; electron ionization, single ion monitoring, and the energy of ionization was 70 eV.
Statistical analysis
The sample size was calculated by Statistical Analysis System 9.1.3 software (SAS Institute, Cary, NC). Six replications were used in present study, which could make a significance level of 0.05 and a power greater than 0.80. The replicate was considered the experimental unit in all of the statistical analyses. All data analysis were performed through the SAS 9.1.3 according to the method of one-way analysis of variance model: Yij = µ +τi + εij, where Yij means the observation j (j = 1 to 6) in treatment i (i = control, LA, and HA), µ denotes the overall mean, τi describes the effect of treatment (denotes an unknown parameter), and εij represents random error with mean 0 and variance σ2. The relationship between antioxidant status and UFA profiles of LD was analyzed by Pearson correlation coefficients (r) [20]. The significant level was set at P < 0.05.