Effects of dietary lysine levels on growth performance, nutrient digestibility, serum metabolites, and carcase and meat quality of Yacha pigs

Abstract The objective was to evaluate the effect of different standardised ileal digestible lysine (SID-Lys) levels on performance, nutrient digestibility, serum metabolites, and carcase and meat traits of Yacha pigs. A total of 50 pigs were blocked by body weight and randomly assigned to five dietary treatments with six replicate pens (2 pigs/pen) per treatment using a randomised complete block design. Five diets in mash form were formulated to contain SID-Lys at 0.57%, 0.62%, 0.67%, 0.72%, 0.77% of diet in phase 1 (15 ∼ 30 kg), at 0.40%, 0.48%, 0.56%, 0.64%, 0.72% of diet in phase 2 (30 ∼ 50 kg), and at 0.30%, 0.39%, 0.48%, 0.57%, 0.66% of diet in phase 3 (50 ∼ 90 kg), respectively. The results showed that the average daily gain and gain to feed ratio increased with increasing dietary Lys levels in phases 1 and 2 (p ≤ 0.05). Increasing dietary Lys levels increased linearly (p ≤ 0.05) apparent total-tract digestibility of dry matter, gross energy, crude protein, and phosphorus in Phase 3 and also increased (p ≤ 0.05) concentration of serum glucose and tended to increase (p < 0.10) serum total protein in Phase 2. Compared with the Lys deficiently treatment, the adequate Lys treatment had increased carcase weight, lean meat percentage, and shear force and decreased cooking loss (p ≤ 0.05). Moreover, the optimal dietary SID-Lys levels preliminarily determined for 15 ∼ 30 kg, 30 ∼ 50 kg, and 50 ∼ 90 kg Yacha pigs were 0.760%, 0.628%, and 0.553% of the diet, respectively. HIGHLIGHTS Dietary appropriate SID-Lys levels can improve performance and feed utilisation in Yacha pigs. Dietary appropriate SID-Lys levels contributed to enhancing carcase lean meat yield of Yacha pigs.


Introduction
With the increase in people's living standards, there is a growing requirement for the quality of food, including pork products (Tous et al. 2014). Chinese indigenous pig breeds are a crucial component of the world's pig genetic resources and also a critical source of Chinese premium quality pork production (Liu et al. 2017). Yacha pig, as a native excellent pig breed resource, is mainly distributed in Southwest China and has the advantage of indigenous adaptation, exceptional prolificacy, resistance to roughage, high intramuscular fat percentage, and desirable meat quality (Yang 2009). However, compared with improved commercial pigs, the Yacha pig has a much lower lean meat percentage and relatively slower growth rate, which means a lower production efficiency that marred its use for commercial production (Yang 2009). Previous studies have stated that indigenous pigs have a slower growth rate and greater propensity to deposit fat are the result coactively of genetics and essential amino acid (AA) deprived diets (Chen et al. 2021). Therefore, providing optimal nutritional strategies for growing-finishing Yacha pig plays a pivotal role in determining the achievement of these targets. Although the AA requirements of indigenous black pig breeds have been conducted in some studies, it is a wide range given the pig's differences in genotypes and growth periods (Yang 2002;Chang 2012;Yuan et al. 2015). Those studies, in addition, predominantly considered the contents of total AA in diets, whereas there is little information about the requirement of standardised ileal digestible (SID) AA. Currently, diets for the Yacha pig in large-scale piggery are mainly formulated based on the nutrition recommendation tables of the China Nutrition Requirement of Swine (NY/T65-2004) or National Research Council (NRC 2012) owing to a dearth of information about proper nutrient requirements of this indigenous pigs (Chen et al. 2021;Hu et al. 2022). Obviously, the same diet is not appropriate for precise nutrition due to the unique genotype characteristics of the Yacha pig. Unreasonable amino acid supply not only affects pig growth performance and pork quality but also directly causes economic losses and environmental pollution due to increased nitrogen emissions (Hu et al. 2022). Therefore, it is necessary to find the optimal nutritional requirements of the Yacha pig for the improvement of its production performance and the promotion of indigenous pig breed resource development.
Lysine (Lys) is typically the first limiting AA in cornsoybean meal-based swine diets (Lee et al. 2021). It is, therefore, considered as a reference AA for the relative amounts required for the other AA (National Research Council [NRC] 2012). At present, accounting for over 95% of the world's total Lys production is used as a feed additive to improve the utilisation rate of crude protein. The NRC (2012) suggested that the dietary SID-Lys requirements for 25 $ 50 kg, 50 $ 75 kg, and 75 $ 100 kg pigs were 0.98%, 0.85%, and 0.73%, respectively. In animals, dietary Lys is mainly used for protein synthesis, and the efficiency of available Lys for protein deposition is about 72% in growing and finishing pigs (Mohn et al. 2000;Zhang et al. 2011). Katsumata et al. (2002) pointed out that Lys can affect nutrient absorption and metabolism by adjusting the absorption of some functional AA or by regulating endocrine hormone release. In growing and finishing pigs, Lys deprived or excessive diets can reduce or increase nitrogen retention and organism protein turnover, thereby affecting its growth performance, nutrient digestibility, serum parameters, and carcase characteristics (Zeng et al. 2013). The supply of Lys to meet the requirement of different growth stages can improve the utilisation of dietary AA and feed conversion efficiency by reducing nitrogen emission (Strathe et al. 2020). Therefore, the objective of this study was to determine the SID-Lys requirements of Yacha pigs with growth performance, apparent nutrient digestibility, and serum metabolites measured during the growing-finishing period, while carcase and meat traits were measured at the end of the finishing period. Meanwhile, providing scientific and objective data for reference to the optimal nutritional strategies of Chinese indigenous pig breeds.

Materials and methods
The study was conducted according to the guidelines of the Chinese Guidelines for Animal Welfare and approved by the Animal Care and Use Committee of Animal Nutrition Institute, Sichuan Agricultural University (Ethics Approval Code: SCAUAC201806-6).
The study was carried out at the Teaching and Research Base of the Sichuan Agricultural University, Animal Nutrition Institute (Ya'an, Sichuan Province). A total of 50 (male, from 10 purebred sows) castrated Yacha pigs were weaned at 48-day old and fed the same commercial prestart diet for one week. The pigs were purchased from the breeding farm of Yacha pig in Gulin County of Chinese Sichuan Province, all of which were judged to be in good health after clinical examination. Then, pigs [15.29 ± 2.96 (SD) kg body weight] were randomly assigned to one of five dietary treatments in a complete randomised block design, with five replicate pens (2 pigs/pen) per treatment. All pigs were housed in the same room and had free access to feed and water. The animal house was disinfected regularly, and the room temperature and ventilation were kept in commercial conditions (Liu et al. 2022). Pigs were examined daily to ensure the record and, if necessary, therapy of pigs suffering from diseases. During the experimental period, pigs were weighed individually at the end of each phase after fasting overnight. The feed intake of each pen was recorded weekly to determine average daily gain (ADG), average daily feed intake (ADFI), and the body gain to feed intake ratio (G/F). The experiment lasted 18 weeks.

Collection of serum, faeces and muscle samples
At the end of Phases 1, 2, and 3, pigs were fasted for 12 h (drank freely) before collecting blood. Blood samples from each pig were collected from the superior vena cava into non-heparinized tubes. Then, samples were centrifuged (3500 Â g, 4 C, 10 min) to separate the serum and stored at À20 C until analysis. In phase 3, before the collection of faecal samples, pigs were all fed the Cr 2 O 3 -containing diets for five days as an adaption period, and then continuously fed these diets for four days during which faecal samples (100 g per pig) were collected from the rectum after pigs free feed intake for 30 min. All faecal samples were fixed with 10% dilute sulphuric acid and toluene, then stored at À20 C until analysis. At the end of the experiment, 12 pigs (6 in each treatment) were selected from the lowest dose Lys level (Treatment 1) and the optimum Lys level (Treatment 4) which showed the highest growth rate to be slaughtered to determine carcase and meat traits (left side of each   1) The vitamin premix provides the following per kilogram of diets: VA 17 500 IU, VD 3 3 000 IU, VE 15 mg, VK 3 3 mg, VB 1 1.5 mg, VB 2 5 mg, VB 6 3 mg, VB 12 20 lg, d-panthothenic acid 12.5 mg, folic acid 0.75, niacin 25 mg, d-biotin 0.1 mg.

Chemical analysis
At the beginning of each experiment, feed samples were collected and ground to pass through a 1.0-mm screen Wstyler mill. Analyses for dry matter (DM), calcium (Ca), and total phosphorus (P) were conducted according to the Association of Analytical Chemists methods (AOAC 2007). Crude protein (CP) was measured by an Auto Kjeldahl Analysis Equipment (Kjeltec TM 8400, FOSS, Hiller, Denmark). The gross energy (GE) was measured using an Automatic Adiabatic Oxygen Bomb Calorimeter (Parr 6400, Parr Inc., Moline, IL, USA). The amino acids of samples were analysed using an Amino Acid Analyser (L-8900, Hitachi High-Technologies, Tokyo, Japan), referring to the methods of AOAC. The 500 lL serum samples was centrifuged (3000 Â g, 4 C, 3 min), and then, the serum metabolites were determined by an automatic biochemical analyser (7020, Hitachi Ltd., Tokyo, Japan). All faecal samples were dried in a fan-forced oven at 60 C for 72 hours, then ground to pass a 0.45-mm sieve. Representative samples of diets and faeces were taken and analysed in duplicate for chromium (Cr) and nutrients. Cr in digesta and diets were analysed by atomic absorption spectrophotometry (ContrAAV R 700, Analytik Jena AG, Germany). Based on the results of the chemical analyses, apparent total-tract digestibility was calculated using the Cr 2 O 3 concentration in feed and digesta samples as previously described (Zeng et al. 2013). The daily SID-Lys requirement and SID-Lys to metabolisable energy ratio were calculated by the following formula: Where ADFI, dietary GE, and GE digestibility are from the optimal treatment (T4) group in that phase.
Hot carcase weight (dressing percentage ¼ carcase/ body weight), lean meat percentage, tenth rib backfat thickness, and loin eye area (0.7 Â loin eye width Â depth, cm 2 ) were measured after slaughter. The pH of the longissimus was measured with a pH metre (pH-start, Matthaus, Germany) 45 min after slaughter and then stored at 4 C, measured again after 24 hours. The meat colour was determined with a Minolta Chromameter (CR 400, Konica Minolta Inc., Tokyo, Japan). The marbling score (45 min, 24 h) was estimated with pork quality standards (Determination of Livestock and Poultry Meat Quality (NYT1333-2007) 2007). The shear force was measured using a Stable Micro Systems Texture Analyser (TA-XT Plus, Stable Micro Systems, Surrey, UK). Water-holding capacity indicators of the psoas were evaluated by cooking loss (%) and drip loss (%). Intramuscular fat content was measured with a fat metre (Soxtec TM 8000, FOSS, Hiller, Denmark).

Statistical analysis
The growth performance data was statistically analysed using the General Linear Model (GLM) procedure covariance analysis of SAS, version 9.4 (SAS Institute, Inc.2014). The data of nutrient digestibility and serum metabolites were statistically analysed by the GLM procedure variance analysis of SAS. Pre-planned linear and quadratic orthogonal contrast were built using coefficients for equally spaced treatment and used to determine the main effects of increasing Lys levels. Data of carcase characteristics and meat traits were analysed by paired sample T-Test according to the weight of the swine. The SID Lys requirements were estimated for quadratic broken-line models by the NLMIXED procedure of SAS based on the studies by Lee et al. (2021). Results are presented as least squares means for each response variable, and the experimental unit was the pen. Statistical significance was declared at p 0.05, and a trend was expressed when 0.05 < p 0.10.

Growth performance and estimation of lysine requirements
The effects of dietary SID-Lys levels on the growth performance of Yacha pigs are shown in Table 3. The ADG and G/F significantly increased in Phase 1 (p 0.05, Linear) and 2 (p 0.05, Linear or Quadratic) with increasing dietary SID-Lys levels, while ADG and G/F were not significantly affected in Phase 3. The addition of dietary SID-Lys contents tended increased ADFI in Phase 2 (0.05 < p < 0.1, Linear), whereas ADFI did not differ among treatments during the other periods. The body weight at the end of each feeding period was not different among treatments, but it's worth noting that the body weight and ADG in each growth period were maximum in all T4 groups. The fitting quadratic curve analysis of ADG and SID-Lys levels in different treatments in the whole period was carried out and the equations were obtained (Figures 1-3). The optimum SID-Lys requirement of 15 $ 30 kg, 30 $ 50 kg, and 50 $ 90 kg Yacha pigs were estimated to be 0.760%, 0.628%, and 0.553% by the quadratic curve analysis, respectively. When expressed on a g/day or g/MJ ME basis, the optimal daily SID-Lys requirement and SID-Lys to metabolisable energy ratio were 8.60 g/d (0.469 g/MJ ME), 11.85 g/d (0.411 g/MJ ME), and 16.21 g/d (0.361 g/MJ ME), respectively.

Apparent faecal energy and nutrient digestibility
The effects of dietary SID-Lys levels on the apparent total-tract digestibility of energy and nutrient of Yacha pigs (50 $ 90 kg) are presented in Table 4. The apparent total-tract digestibility of DM, GE, CP, and P were significantly increased (p 0.05, Linear) with dietary SID-Lys levels increased. The apparent digestibility of Ca was markedly changed (P ¼ 0.021) by an increasing level of SID-Lys in the diet.

Serum metabolites
The effects of dietary SID-Lys levels on serum metabolites of Yacha pigs are shown in Table 5. Serum glucose (GLU) was significantly altered (p 0.05) with the increase of dietary SID-Lys levels in Phase 1, while GLU was unaltered in Phase 2 and 3. Total protein (TP) of the T3 group tended to be higher (p < 0.10, Quadratic) than that of the other treatments in Phase 2, whereas TP was unaltered in Phase 1 and 3. Increasing dietary SID-Lys levels had no effect (p > 0.10) on urea, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC).

Carcase and meat traits
The effects of dietary SID-Lys levels (T1 representing deficient vs T4 representing adequate) on the carcase and meat traits of Yacha pigs are presented in Tables  6 and 7. Carcase weight, lean meat percentage, and shear force of T4 were significantly higher (p 0.05) than those of T1. Cooking loss of T4 was significantly lower (p 0.05) than that of T1. But increasing dietary SID-Lys levels did not affect other carcase and meat traits (p > 0.10).

Discussion
The optimal nutritional levels are considered to be essential for improving the growth performance and production efficiency of livestock. However, according to the literature data, the effects of dietary Lys levels on growth performance may be varied and associated with the pig genotype, weight range, and dietary type (Asche et al. 1985;Llata et al. 2002). The growth performance of finishing pigs was not improved with the increase of dietary total Lys from 0.55% to 0.68% in studies by Corino et al. (2008), whereas Loughmiller et al. (1998) reported that increased linearly both ADG and G/F in pigs of 91 and 113 kg when dietary total Lys varied from 0.40% to 0.60% and not significant   SEM 1 : Standard error of the mean (n ¼ 6). T1: 0.57%, 0.40% and 0.30% of dietary SID Lys levels in phases 1, 2 and 3, respectively; T2: 0.62%, 0.48% and 0.39% of dietary SID Lys levels in phases 1, 2 and 3, respectively; T3: 0.67%, 0.56% and 0.48% of dietary SID Lys levels in phases 1, 2 and 3, respectively; T4: 0.72%, 0.64% and 0.57% of dietary SID Lys levels in phases 1, 2 and 3, respectively; T5: 0.77%, 0.72% and 0.66% of dietary SID Lys levels in phases 1, 2 and 3, respectively.
differences when the level of dietary total Lys increased from 0.60% to 0.90%. Suarez et al. (2015) observed no effect of dietary Lys levels varied from 0.32% to 0.63% on the growth performance of crossbred pigs during the growing period (30 $ 90 kg BW), whereas found linearly increased ADG, ADFI, and G/F when SID Lys levels increased during the finishing period (90 $ 130 kg BW). In the present study, the ADG and G/F increased linearly in Yacha pigs were observed during the growing period, but not significantly affected in the finishing period. Similarly, Rodriguez et al. (2011) found a linear increase in ADG and ADFI as the total dietary Lys was increased from 0.60% to 0.70% of diet from 100 to 128 kg body weight, and the ADG was not different between sexes. In contrast, Apple et al. (2004) reported that both ADG and G/F of pigs increased linearly as the total Lys to ME ratio increased from 1.7 to 3.1 g/Mcal. Therefore, as a native pig breed with a specific genetic background, whereas the Lys in the diet is mostly used for organism protein accretion and the dietary Lys concentration is directly related to the performance of pigs. It is important to correctly estimate the Lys requirement of Yacha pigs at different growth stages. In the current studies, the quadratic curve regressions estimated the optimum SID-Lys requirements for 15 $ 30 kg, and 30 $ 50 kg, 50 $ 90 kg Yacha pigs to be 0.760%, 0.628%, and 0.553% based on ADG, respectively, which is close to the optimum SID Lys for indigenous growing pigs estimated by a meta-analysis (Yang 2002;Chang 2012). However, the estimated values of optimal SID Lys in this study were slightly lower than the ideal SID values of 11 to 100 kg pigs by the NRC (2012). Normally, Small-physique or fat-type pig breed pigs have lower Lys requirements than largephysique or high lean species at the same growth phase (Chen et al. 2021). In addition, the Lys requirement of pigs decreased with an increase in age and body weight when the Lys was expressed by dietary ratio basis, which was due to the increase in feed intake and fat deposition rate.
It is well known that nutrient and energy digestibility are the important factors affecting growth performance. Previous studies have reported that increasing dietary Lys levels increased DM and CP digestibility of weaned pigs (Kim et al. 2011;Wang et al. 2012). In the present study, the apparent digestibility of DM, GE, CP, and P were linearly increased with increasing Lys levels. Jin et al. (2010) found a decrease in CP digestibility in 58 $ 100 kg pigs with restriction of dietary Lys levels (2.32, 1.92, 1.62 g/Mcal ME). Yang et al. (2008) showed that restriction of Lys resulted in a significant decrease in DM and GE digestibility but an increase in CP digestibility of 35 $ 55 kg pig, whereas showed no effect on the digestibility during the 55 $ 115 kg period. Zeng et al. (2013) also elucidated that the lowest Lys treatment (0.65%, or 0.48 g/MJ DE) showed a significant lower apparent digestibility of GE, DM, CP, and P than the 0.95% (0.70 g/MJ DE) and 1.25% (0.93 g/MJ DE) Lys treatments for 20 kg pigs. These studies showed that when dietary Lys levels of pigs in different periods reached the optimal level, the nutrient digestibility could improve effectively. The reason for this result may be the appropriate level of Lys in the diet can promote the development of animal digestive organs (He et al. 2013;Wang et al. 2012). Some studies indicate that Lys can stimulate the secretion of pepsin and gastric acid, thereby improving protein digestion and increasing appetite (Wu 2010). Previous studies have stated that Lys could chelate with calcium, iron and other mineral elements to form soluble small molecular monomers, which promote the absorption of these mineral elements (Civitelli et al. 1992). In the present study, dietary supplementation with Lys increased the digestibility of calcium and phosphorus also were observed.
It can be considered that the compensation reaction of growth performance and body composition is the result of metabolic changes and physiological activities in response to dietary manipulations, which can be reflected in the serum metabolite profile (Elnesr et al. 2019;. For instance, serum TP level is an indicator of protein utilisation and metabolism in livestock, and the high content of TP indicates that the deposition of organism protein is at a high level (Shen et al. 2019;. However, the effects of dietary Lys on serum metabolites are variable due to different breeds, growth stages, and dietary AA concentrations of pigs (Fernandez et al. 2007;Zeng et al. 2013;Regmi et al. 2018). In the present study, increased Lys levels decreased GLU of pigs in 15 $ 30 kg and increased TP in 30 $ 50 kg. The increase of TP in serum further strengthened the reliability of growth performance results. These results are in agreement with Yang et al. (2008) and Fernandez et al. (2007), who reported that the increase in Lys levels increased TP and albumin but decreased urea and GLU content in the grower period. The effects of dietary Lys levels on TP and GLU in serum are different, which may be related to the utilisation of AAs. Furthermore, It is widely supported that the serum urea content can estimate the extent of AA breakdown and is negatively related to the utilisation of dietary protein (Strathe et al. 2020). Some studies stated that serum urea nitrogen content decreased as the equilibrium ratio of non-limiting AAs relative to Lys increased (Fernandez et al. 2007;Regmi et al. 2018). As expected, the current study indicated that Lys supplementation did not influence the serum urea concentration of Yacha pigs. It was also explained why the concentration of TP increased but not significantly in this study and indicated that increasing dietary Lys levels could increase the AA utilisation of Yacha pigs, but the effect was not significant.
In the previous literature, the effects of dietary Lys on carcase characteristics have been shown to be variable (Bidner et al. 2004). Generally, increasing dietary Lys content increased carcase weight, backfat thickness, lean meat percentage, and loin area in the body. This phenomenon is mainly because optimum Lys results in maximum protein deposition (Witte et al. 2000). In addition to participating in protein biosynthesis as a substrate. Lysine also acts as an endocrine hormone release regulator to promote the synthesis and secretion of growth hormone, insulin, and insulinlike growth factor I. And it acts as a signalling molecule to regulate the activity of translation initiation factors (Eukaryotic initiation factor-2), ultimately affecting the process of protein biosynthesis (Wu 2010). Asche et al. (1985) illustrated that supplementation of dietary Lys increased hot carcase weight, longissimus muscle area, length, and percentage of lean, decreased backfat thickness in the growing and finishing period. In other studies, greater dressing percentage, lean percentage, and wider loin eye area have been observed in pigs fed diets with optimum Lys than in those fed Lys deficient (Witte et al. 2000;Bidner et al. 2004). In the present study, both increased carcase weight and lean percentage were observed in treatment 4 as compared to the negative control, which was consistent with the result of Zhang et al. (2008) and Suarez et al. (2015). Nevertheless, the increased dietary Lys levels have no significant effect on the backfat thickness and loin area of Yacha pigs, which indicated that dietary supplementation of Llysine at an appropriate level might promote the utilisation of AAs, and the subcutaneous fat tissue was unaffected. However, this may be beneficial to improve the meat quality. On the contrary, Llata et al. (2002) reported that there was little effect on carcase characteristics with dietary Lys increased from 0.05 to 0.3% during growing-finishing pigs, which might be explained by the Lys levels of the diet did not reach the dose that affects carcase traits.
Meat quality can be assessed objectively by some traits, including colour, nutrients, water-holding capacity, tenderness, flavour, and intramuscular fat. In general, colour denotes its primary acceptability to consumers, while tenderness is rated as the most critical palatability trait for cooked meat, followed by flavour and juiciness (Chen et al. 2021). Some authors have reported that the gilts fed high Lys diets had increased shear force of semitendinosus muscle and decreased water loss rates (Bidner et al. 2004). Witte et al. (2000) indicated that decreasing dietary Lys levels from 0.64% (1.88 g/Mcal ME) to 0.48% (1.41 g/Mcal ME) of diet did not affect meat colour for 75 $ 105 kg pigs, and the visual score was consistent with the objective measurement results. Wang et al. (2015) also denoted that the visual scores of loin colour and loin marbling were not affected by increasing dietary Lys levels from 0.43% (0.283 kJ/kg GE) to 0.98% (0.659 kJ/ kg GE) of diet for 94 $ 140 kg pigs. In the current study, the Lys supplementation did not affect colour indexes (lightness, redness, yellowness), marbling score, and pH (45 min, 24 h) but significantly reduced cooking loss and increased shear force, which was consistent with the result of Sirtori et al. (2014) and Szabo et al. (2001).

Animal welfare statement
The experiment was conducted in accordance with the Chinese Guidelines for Animal Welfare and the experimental procedures were approved by the Animal Care and Use Committee of Animal Nutrition Institute, Sichuan Agricultural University (Ethics Approval Code: SCAUAC201806-6).

Data availability statement
The datasets analysed in the current study are available from the corresponding author on reasonable request.