Effects of dietary energy and crude protein levels on growth performance, blood profiles, and carcass traits in growing-finishing pigs

All experimental procedures involving animals were conducted in accordance with the Animal Experimental Guidelines provided by the Seoul National University Institutional Animal Care and Use Committee (SNUIACUC; SNU-160819-9). A total of 180 crossbred pigs ([Yorkshire × Landrace] × Duroc) with an average body weight (BW) of 30.96 ± 3.068 kg were used for a 12-week feeding trial. Pigs were reared at the Seoul National University experimental farm. Three male pigs and three female pigs were assigned to each pen of a growing facility based on BW, and pigs in each pen were maintained until the end of experiment. Pigs were randomly allotted to their respective treatments by an experimental animal allotment program [19]. Pigs were reared in growing-finishing (2.60 × 2.84 m) facilities for 12 weeks. Feed and water were provided ad libitum during the entire experimental period by a 6-hole stainless feeder and two nipples installed in each pen.

Experimental pigs were allotted to a 2 × 3 factorial arrangement in randomized complete block (RCB) design. The first factor was two levels of dietary metabolizable energy (ME) density (13.40 MJ/kg or 13.82 MJ/kg) and the second factor was three dietary CP levels based on subdivision of growing-finishing phases (high: 18%/16.3%/16.3%/13.2% middle: 17%/15.3%/15.3%/12.2% and low: 16%/14.3%/14.3%/11.2%).

Experimental diets were formulated for four phases: the early growing phase (0–3 weeks), the late growing phase (4–6 weeks), the early finishing phase (7–9 weeks), and the late finishing phase (10–12 weeks). All nutrients in the experimental diets except ME and CP were met or exceeded the nutrient requirements of the NRC [5]. Formula and chemical compositions of the experimental diets are provided in Tables 1, 2, 3, and 4.

Body weight and feed consumption were recorded at 0, 3, 6, 9, and 12 weeks to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F ratio).

Blood samples were taken from the jugular vein of six randomly selected pigs in each treatment to measure blood urea nitrogen (BUN), glucose, and total protein during growing and finishing phages. All blood samples were collected in serum tubes (SST™ II Advance, BD Vacutainer®, Becton Dickinson, Plymouth, UK). Collected blood samples were centrifuged for 15 min at 6,048 × g at 4°C (5810R, Eppendorf, Hamburg, Germany). Serum was carefully transferred to 1.5 mL plastic tubes and stored at −20°C until analysis. BUN (kinetic UV assay, Roche, Germany) and glucose (enzymatic kinetic assay, Roche, Germany) concentrations were analyzed using a blood analyzer. Total protein concentration was measured by a kinetic colorimetry assay using a blood analyzer (Modular analytics, PE, Roche, Germany).

At the end of experiment, four pigs from each treatment group were selected and slaughtered at average 113.42kg ± 1.307 for carcass analysis. Pork samples were collected nearby the 10th rib on the right side of the carcass. After chilling, 30 minutes after slaughter was regarded as the initial time. The pH was measured at 0, 3, 6, 12, and 24 hours and the color of the longissimus muscle was measured at the initial time and 24 hours later. The pork samples were always stored in the freezer (4°C). The pH was measured using a pH meter (Model 720, Thermo Orion, Fullerton, CA, USA) and pork color was measured by CIE color L*, a*, and b* values using a CR300 (Minolta Camera Co., Osaka, Japan). Chemical analysis of pork samples was conducted using the AOAC method [20].

Water holding capacity of pork was measured by the centrifuge method. Longissimus muscles were ground and sampled in a filter tube, then heated in water bath at 80°C for 20 min and centrifuged for 10 min at 2,688 × g at 10°C (5810R, Eppendorf, Hamburg, Germany). To calculate cooking loss, longissimus muscles were packed in a polyethylene bag that was then heated in a water bath until the core temperature reached 72°C and weighed before and after cooking. After heating, samples were cored (0.5 inch in diameter) parallel to the muscle fiber and the cores were used to measure shear force using a salter (Warner Barzler Shear, Norwood, MA, USA). Cooking loss, shear force, and water holding capacity of pork were analyzed by the Animal Origin Food Science laboratory, Seoul National University.

Lipids in pork samples were extracted from duplicate 10 g samples with chloroform: methanol (2.1, v/v) [21] on a shaking incubator (25°C, 8.064 × g) for 24 hours. Extracted lipids were filtered with filter paper (Whatman TM No.4, Buckinghamshire, UK). Twenty-five milliliters of 0.88% NaCl was added to the filtered sample, which was then centrifuged at 2,090 × g for 10 min (Continent 512R, Hanil Co., Ltd., Incheon, Korea). The supernatant was separated and pork lipids were concentrated using N₂ gas at 45°C. After concentrating the lipids, 0.1 g of lipids was transferred to a 15 mL tube with 1 mL of internal standard (1 mg of undecanoic acid in 1 mL of iso-octane) and 1.5 mL of 0.5 N methanolic NaOH. Samples were heated in a water bath at 85°C for 10 min and cooled to room temperature. After cooling, 2 mL of 14% BF₃-methanol was added and then the heating process was repeated one more time. Then, 2 mL of iso-octane and 1 mL saturated NaCl were added, and the sample was centrifuged at 4,200 × g for 3 min (Continent 512R, Hanil Co., Ltd., Incheon, Korea). Upper layer containing fatty acid methyl esters (FAMEs) was dehydrated with anhydrous sodium sulfate and transferred to a vial. The sample in the vial was analyzed using a gas chromatograph (HP 7890, Agilent Technologies, Santa Clara, CA, USA) with a split ratio of 50:1. A capillary column (DB-23, 60 m × 250 μm × 0.25 μm, Agilent, Santa Clara, CA, USA) was used. The injector and detector temperatures were maintained at 250°C and 280°C, respectively. Column oven temperature was varied as follows: 50°C for 1 min, increased to 130°C at 25°C/min, 170°C at 8°C /min, then held at 215°C after increasing to this this temperature at 1.5°C/min. Nitrogen was used as a carrier gas at linear flow of 4 mL/min. Individual FAMEs were identified by comparison of relative retention times of peaks from samples with those of external standards (37 FAME mix and CLA mix, Supelco, Bellefonte, PA, USA) calculated based on the Korean Food Standards Codex (MFDS, 2017).

Data for the various groups were compared by least squares mean comparisons and evaluated using general linear models (GLMs) in SAS (SAS Institute Inc., Cary, NC, USA). Every pen was used as one unit in the feeding trial, and individual pigs were considered experimental units with regard to analysis of blood profiles and carcass qualities. Orthogonal polynomial contrasts were used to detect linear and quadratic responses to CP levels when the significance of the CP effect was evaluated. The treatment effect was considered to be significant if p < 0.01 or p <0.05 while 0.05 < p and p < 0.10 was considered to indicated a trend in the treatment effect.

The effect of dietary energy and CP levels on growth performance are presented in Table 5. Average daily gain and G:F ratio decreased linearly as dietary CP level was decreased (linear, p < 0.05; p < 0.05, respectively) in the early growing phase, and G:F ratio decreased linearly as dietary CP level was decreased (linear, p < 0.05) over the entire growing phase. In the growing period, pigs showed the highest weight gain due to muscle development [16]. Dietary CP and amino acid levels affect protein deposition [16,22]. Several studies have demonstrated that ADG and G:F ratio decreased as CP level increased in the growing phase [10,23]. In this study, dietary energy density did not have any significant effect on growth performance during the growing period, consistent with the study of Kil and colleagues [24].

Dietary energy and protein levels did not differ significantly among groups in the finishing period. Similar to our findings, Kerr et al. [10] represented that a 0.42 MJ decrease in NE/kg and 4% CP in 88.3 –109.7 kg pigs did not significantly affect growth performance. Kil et al. [24] reported that a 1.26 MJ/kg decrease in ME in the finishing pig diet had no effect on growth performance.

However, G:F ratio decreased when dietary ME level was decreased (p = 0.01). Furthermore, BW and ADG showed significant differences over the course of the experiment (p < 0.05), and 13.82 MJ/kg and the high CP level resulted in the highest BW and ADG among the six treatments.

The effects of dietary energy and CP levels on blood profiles are presented in Table 6. There were no significant differences in glucose and total protein concentrations when pigs were fed different dietary ME and CP levels during the growing period, but BUN concentration increased with a decrease in dietary ME (p < 0.01). In the finishing period, total protein concentration decreased when pigs were fed lower dietary ME concentrations (p < 0.05).

Ammonia is produced by the body after eating protein and the liver produces urea as a waste product of protein digestion. Urea nitrogen is a waste product that is released by the liver and then filtered out of the blood by the kidneys [25,26]. Therefore, BUN is used as a standard index for evaluating protein quality or excessive protein intake [27,28]. Fuller et al. [29] demonstrated that feeding animals carbohydrates could increase protein deposition. BUN level is affected by N intake [30], and has a negative correlations with ADG and G:F ratio [26]. In this study, BUN concentration decreased because pigs were fed more energy, which likely improved protein deposition and utilization.

Suboptimal protein intake can decrease the levels of blood total protein [31]. Matthews et al. [32] represented that total protein concentration increased when protein utilization by pigs was improved. In this experiment showed that total protein concentration decreased as ME level in the diet was decreased during the finishing period. This is consistent with the decrease in the G:F ratio with a decrease in dietary ME.

The effect of dietary energy and CP levels on carcass traits are presented in Table 7. In this study, there were no significant differences in moisture, CP, crude fat, or crude ash according to diet. Other studies have reported similar results; Kil et al. [33] showed there was no significant difference in CP and crude fat in pork when pigs were fed diets with different GE (18.34–16.20 MJ/kg). Stewart et al. [34] showed that adding soybean hulls and wheat middling to 30% could improve CP levels in growing-finishing pigs (growing diets: 17.83%–20.30%; finishing diets: 12.44%–15.21%) but this had no significant effects on moisture, CP, crude fat, or crude ash in the resulting pork.

The effect of dietary energy and CP levels on physiochemical properties of the meat are presented in Table 8. Variations in diet had no detrimental effects on cooking loss, shear force, or water holding capacity. These results are consistent with those of Apple et al. [18] who demonstrated that cooking loss was not affected by different levels of ME (14.57–13.82 MJ/kg). Madeira et al. [35] represented that decreasing CP level in the finishing diet (16%–13%) had no effect on cooking loss. Additionally, Widmer et al. [36] reported that decreasing CP in the growing-finishing period by 4% had no effect on cooking loss. Meng et al. [37] demonstrated that decreasing energy (ME: 14.65–14.24 MJ/kg) and CP levels (growing period: 18%–17% and finishing period: 16%–15%) had no effects on water holding capacity or cooking loss. Widmer et al. [36] represented that decreasing energy (growing period: 1.68 MJ/kg of ME and finishing period: 0.82 MJ/kg of ME) and CP level (growing period: 21.3%–15.34%; early finishing period: 18.76%–15.34% and late finishing period: 13.64%–11.24%) had no effects on cooking loss and shear force.

Table 9 shows the effect of energy and CP levels on muscle. TBARS is an index of fat oxidation. Madsen et al. [38] found that the process of fat oxidation could be accelerated in the logissimus by the intake of crude fat. In this study, a decrease in ME of 0.42 MJ/kg and in CP of 2% had no significant effect on muscle TBARS assay. Other studies have also reported that a decrease in ME and CP levels had no effects on TBARS assay [35,37].

Table 10 shows the effects of energy and CP levels on fatty acid composition in growing-finishing pig diets. Saturated fatty acid (SFA) increased (p < 0.01) and polyunsaturated fatty acid (PUFA) decreased (p < 0.05) as ME was decreased by 0.42 MJ/kg in growing-finishing pig diets. In addition, monounsaturated fatty acid (MUFA) content tended to increase when CP level was decreased in growing-finishing pig diets (p = 0.06). European Food Safe Authority (EFSA) recommend that for food to be considered healthy, it should have a low content of SFA [39]. Feed ingredients and nutrition composition can also affect fatty acid composition in pork [40,41]. Nemechek et al. [42] and Davis et al. [43] demonstrated that SFA content could be decreased and PUFA content could be increased by decreasing tallow levels or decreasing ME by 0.42 MJ/kg (13.82–13.40 MJ/kg) in growing-finishing diets. Bessa et al. [44] and Madeira et al. [35] represented no significant differences in MUFA as CP levels were decreased by 3%–4% in growing-finishing pig diets. The discrepancy between this study and previous studies may due to the high CP level in our study. In summary, this experiment showed that 13.82 MJ/kg of ME in a growing-finishing pig diet can improve fatty acids composition of pork.

The change in pH after slaughter is a very important factor affecting the freshness, tenderness, color, texture, and storage of pork. In this study, there were no significant differences in pH at 0 and 24 hours after slaughter (Table 11). This result is in accordance with Apple et al. [18], Widmer et al. [36], Prandini et al. [45], and Madeira et al. [35] who reported that the pH at 0 and 24 hours after slaughter were not affected by ME (14.57–13.82 MJ/kg and 15.49–13.82 J/kg) or CP level (17.7%–14.5% and 16%–13%).

Meat color results are shown in Table 12. There were no significant differences in L*, a*, or b* values as ME level was decreased by 0.42 MJ/kg or CP concentration was decreased by 2%. Apple et al. [18] and Hinson et al. [47] also demonstrated that decreasing energy level in the diet did not affect meat color (14.57–13.82 MJ/kg ME and 14.65–13.40 MJ/kg ME, respectively). In addition, Prandini et al. [45] and Madeira et al. [35] represented that decreasing CP level in diet did not affect meat color (17.7%–14.5% and 16%–13%, respectively).