As energy prices continue to rise worldwide, an increasing number of pig farmers are adopting measures to reduce heating in winter and implementing low-temperature breeding models to save energy consumption. Pigs are particularly sensitive to temperature fluctuations, with the critical temperature range typically considered to be 26°C–28°C for weaned piglets and 18°C–20°C for finishing pigs [1]. Consequently, lowering the temperature in pig barns not only increases gastrointestinal motility and reduces feed efficiency, affecting growth performance, but also alters muscle metabolism, thereby increasing the risk of inferior meat quality [2]. Therefore, when adopting lower rearing temperatures for energy conservation, reliance on the pigs’ own regulatory mechanisms alone is insufficient. It is essential to optimize nutritional supply to enhance growth performance in cold environments and mitigate negative impacts on meat quality. As an essential nutrient in animal diets, protein significantly contributes to regulating physiological activities, and enhancing muscle protein synthesis and supporting flavor amino acid (FAA) deposition [3,4]. Protein metabolism encompasses both the synthesis pathway mediated by the mammalian target of rapamycin (mTOR) and the degradation pathway mediated by the ubiquitin-proteasome system. mTOR regulates protein synthesis by activating downstream factors, primarily promoting protein synthesis and deposition in animal muscle through the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) [5]. Conversely, MuRF1 and MAFbx as E3 ubiquitin ligases promote degradation together with FOXO [6]. We hypothesize that appropriate protein intake may help pigs alleviate protein metabolic disorders and cold-induced meat quality deterioration via the mTOR pathway, while also improving their growth performance. Currently, few studies have investigated dietary protein supplementation under low-temperature conditions. In this study, feeding trials with graded dietary protein levels under cold conditions in winter to explore the effects of dietary protein concentration on growing-finishing pigs’ growth performance, meat quality, and nutritional quality, as well as their underlying regulatory mechanisms.

In low-temperature winter environments, pigs’ basal metabolic rates increase and protein breakdown intensifies; therefore, adequate protein intake is essential for maintaining growth, development, and productivity. The results of this study showed that, throughout the experimental period, the F/G was significantly lower in T2 compared to T3 and T4, suggesting that excessively high dietary protein levels may increase production costs due to protein wastage. Pigs weighing 25–50 kg are in the growth phase, characterized by rapid muscle and bone development and high protein requirements [9]. However, due to the immature digestive system of pigs at this stage, excessive protein intake will increase intestinal burden, thereby inhibiting ADFI, reducing ADG and elevating F/G [10], which compromises farmer profitability. Similarly, our research found that during this stage, T4 had lower ADFI and ADG, but an increased in F/G, which may be due to insufficient energy intake caused by low ADFI, resulting in slow growth during this stage [11]. As pig body weight increases, the absorption and utilization efficiency of nutrients in the diet also rises [12]. In this study, the ADG of T4 was higher than that of other groups during the 50–75 kg stage, alongside a lower F/G compared to T1 and T3. Therefore, providing a higher dietary protein level during this stage may promote rapid growth in pigs. This may be attributed to the progressive maturation of the digestive system during this stage, allowing greater protein absorption and utilization, thereby reducing the feeding days in this stage. Although the weight gain rate of T4 accelerated at this stage, it still cannot fully compensate for the growth retardation caused by insufficient energy intake in the early stage. The ADFI of T2 during this phase was lower than that of the other three groups, and F/G was also lower than that of T1 and T3. This suggests that T2’s digestibility may have improved [13], sufficient to meet maintenance requirements under low-temperature conditions. During the late finishing phase (100–125 kg), pigs primarily accumulate fat, reducing protein requirements. The ADG of T2 was significantly higher than that of T3 and showed an upward trend compared to T1. This growth advantage was likely the primary reason for the shorter rearing period in T2 compared to the other groups. Analysis of the various growth stages indicated that, although there were no significant differences in overall daily weight gain among the treatment groups, the growth advantage exhibited by T2 at each stage resulted in a shorter total rearing cycle, which has positive implications for reducing labor, management, and heating costs. Furthermore, the F/G of T1 and T2 was lower than that of T3, indicating that appropriately reducing dietary protein levels during this phase not only meets the growth requirements of finishing pigs but also reduces F/G, thereby controlling production costs. Low temperatures increase the maintenance requirements of pigs and elevate their energy needs. Research found that raising the net energy level for growing-finishing pigs by 6% above the NRS recommendation could mitigate the negative effects of low temperatures on pig growth [8]. Building on this, the present study investigated the optimal dietary protein level for growing-finishing pigs under low-temperature conditions and found that under a uniformly elevated net energy level, moderately increasing the protein level improved growth performance and shortened the production cycle. This suggests that the optimal energy-to-protein ratio for pigs under low-temperature may differ from that under normal temperature.

Meat quality as an important indicator of pork consumption quality, evaluated by pH, color, cooking loss, drip loss, and tenderness, influences consumer choices. Previous studies have indicated that under suitable temperature conditions, dietary protein levels have no effect on the pH value of pig muscle [14], but our study found that in low-temperature conditions, the pH_24h of LT in T4 was lower than that in T1, which may negatively impact meat tenderness [15]. Furthermore, this study found that compared to T3, the L* and b* of the LT were reduced in T2 and T4 at 45 min postmortem, and consumers typically prefer meat products with higher a* and lower b* [16], given that no color differences were observed among the groups after 24 h, it is inferred that their impact on actual consumer preferences is likely limited. Higher drip loss indicates poorer sensory quality in meat (such as poor tenderness, flavor intensity, etc.) [17]. In this investigation, T2 had lower drip loss than T1 and T4, we speculate that this may be due to T2 having a more stable muscle cytoskeletal structure, which reduces myofibrillar contraction and retains more water within the muscle [18]. Concurrently, we observed a positive correlation between drip loss and cooking loss in LT, which may also explain the lower cooking loss in T2. These results may indicate that pork from T2 exhibits higher marbling [19]. Moreover, increased F/G correlate with increased shear force and greater cooking losses in pork, resulting in diminished tenderness and water-holding capacity of meat products. This indicates that feed conversion efficiency impacts not only economic returns but also meat quality. Consequently, winter farming should consider the interplay between growth performance and meat quality, aiming to improve pork quality and consumer appeal while optimizing cost efficiency.

With growing health awareness, conventional meat products have become inadequate to satisfy contemporary consumer requirements, they prioritize meat that is nutritionally dense, flavorful, and has a higher energy density, typically featuring elevated levels of protein and AA [20]. Dietary protein typically does not affect muscle moisture [14], but in this study, T2 had lower moisture than T1, possibly indicating increased marbling [21], and enhanced palatability. Animal muscle is a high-quality protein that provides a richer source of nutrients than plant-source proteins. Low temperatures decrease muscle protein synthesis and meat nutritional value, while dietary protein correlates positively with muscle protein deposition [22]. Balanced digestible AA supply enhances AA utilization [23], whereas imbalance may promote protein degradation. In this study, T2 and T4 had higher muscle CP than T1, but T3 showed no difference, possibly due to increased muscle protein degradation. Furthermore, we observed that moisture in LT exhibited a negative correlation with CP and EE. This may be attributable to CP and EE replacing moisture deposits within the tissue [24]. Skeletal muscle is the most significant AA pool in the animals. AAs are not only key factors in determining the quality of pork but also an important basis for assessing its nutritional value [25,26]. More importantly, AAs as flavor precursors that can undergo chemical reactions to produce volatile aromatic compounds, which are key to forming the enticing flavor of meat. Meat products that are both highly nutritious and flavorful are precisely what contemporary consumers seek. Low temperatures reduce Val, Leu, and Ile in Yorkshire pig muscle [27], while increased dietary protein raises the concentration of most AAs in pig muscles [28]. Our study showed that T2 and T4 increased AA concentrations in the LT, with T2 showing increased levels of total TAA, EAA, and FAA, likely from reduced AA limitation after moderate protein intake [29]. This study found that the content of TAA and FAA in LT was negatively correlated with drip loss. This may be because AA help maintain myofibrillar protein structure, thereby improving the water-holding capacity of muscle [30]. Consequently, increasing dietary protein can mitigate nutrient loss caused by low temperatures, enhance muscle protein and AA content. Since high-protein foods can meet nutritional needs with smaller portions, consumers are increasingly inclined to choose high-protein products and are willing to pay a premium [31]. Therefore, raising the protein content in pork products helps enhance their appeal to consumers, thereby improving their market competitiveness.

Dietary protein levels significantly influence serum biochemical parameters in pigs. UN reflects protein catabolism: higher UN indicates lower protein utilization, and high-protein diets increase UN. In this experiment, we found that the UN content of T3 was higher than that of T1, which indicated that protein decomposition in the body was accelerated. Interestingly, the UN content of T4 did not show significant difference, which we hypothesized might be due to an imbalance in the ratio of digestible AAs in T3’diet, leading to increased AA deamination and consequently elevated UN content [32]. Correlation analysis indicates a positive relationship between UN and F/G, suggesting that growing-finishing pigs with low feed utilization may exhibit higher protein breakdown, leading to nutrient wastage. Blood ALB and GLOB reflect nutritional and immune status, with a high ALB/GLOB being beneficial [33]. Increasing dietary protein raises ALB/GLOB in pigs [10]. Similarly, our findings showed that T2, T3, and T4 had higher ALB and ALB/GLOB than T1, indicating improved nutritional status under cold conditions. ALT and AST reflect liver health, one characteristic of liver damage is the massive release of AST and ALT. In this experiment, T4 had higher ALT than other groups, but no significant alterations in AST activity were detected, suggesting liver metabolic burden and mild damage. It is noteworthy that serum ALT levels were negatively correlated with ADG. We speculate that the growth retardation observed in T4 may be attributable to the metabolic burden caused by excessively high dietary protein levels. Thus, exceeding T3’s protein level may harm animal health.

Animal protein metabolism balances synthesis and degradation via mTOR-mediated synthesis and ubiquitin-proteasome-mediated degradation. IGF-1, an mTOR upstream regulator, promotes protein synthesis by activating IRS-1. This study found that the mRNA expression levels of IGF-1 were upregulated in both T2 and T4, consistent with the existing knowledge that protein levels influence IGF-1 expression [34]. Similarly, IR binding to insulin activates mTOR by phosphorylating IRS [35]. IR expression in the LT of T2 was significantly higher than in T1, indicating that T2 more effectively activates the mTOR pathway to promote muscle CP deposition. Analysis of phosphorylation levels at key signaling nodes provided evidence for these regulatory mechanisms. T2 and T4 enhanced p-mTOR and its downstream effector p-S6K1 compared with T1, driving translation initiation and ultimately leading to increased CP and AA content in the muscles of T2 and T4. Given that 4E-BP1 plays a critical role in releasing translation initiation inhibition, it is more important than S6K1 in protein synthesis [36]. Consequently, increased p-4E-BP1 levels in T2 likely conferred the strongest protein deposition capacity to this group. This may account for the production of premium meat products with lower moisture content and higher nutritional value in T2. we hypothesize that the lower F/G observed in this group may reflect its ability to convert feed more efficiently into high-quality lean meat. The primary function of MuRF1 in skeletal muscle is to inhibit protein synthesis and promote degradation. T3 had higher MuRF1 expression, possibly due to digestible AA imbalance [37], explaining its unchanged muscle protein. AA serve as a nutritional signal to activate mTOR. In this study, AA transporter SLC38A2 mediates branched-chain AA uptake and exhibits upregulation in both T2 and T4. This finding aligns with prior research indicating that elevated dietary protein levels enhance SLC38A2 expression, thereby increasing cellular AA transport capacity and subsequently activating mTOR via Rag GTPase [38]. Notably, IGF-1, IR, and SLC38A2 exhibited synergistic upregulation trends, this suggests that appropriately increasing dietary protein may promote efficient protein deposition by synergistically activating the mTOR pathway through upregulating protein synthesis-related genes and enhancing cellular nutrient uptake capacity. We further analyzed the association between muscle AA and CP synthesis-related genes. The results showed that the concentrations of TAA, FAA, and EAA in muscle exhibited varying degrees of positive correlation with the mRNA expression levels of IGF-1, IR, and SLC38A2. This suggests that AA deposited within LT may themselves act as a nutritional signal, positively regulating the IGF-1/insulin signaling pathway [39]. Collectively, these findings show that appropriate protein supplementation under cold conditions enhances muscle protein synthesis via the mTOR pathway, improving meat nutritional value and encouraging consumer purchases. Therefore, protein supply levels must be adjusted according to environmental factors in production practice to better meet consumer demand for nutrient-dense, high-quality meat products.

In view of the continuous shortage and rising prices of protein feed resources, simply increasing dietary protein supply is not a cost-effective feeding strategy, and any adjustment of dietary protein content should be comprehensively evaluated based on input costs and economic benefits. Because pigs’ protein requirements change at low temperatures, determining the appropriate protein intake while ensuring production performance is beneficial for promoting swine production in cold regions. The present study focused on exploring the appropriate dietary protein level for growing-finishing pigs exposed to low ambient temperatures. In general, a higher dietary protein level will inevitably lead to increased feed input costs across different growth stages. Nevertheless, moderate protein supplementation under low-temperature conditions still presents favorable economic benefits and practical application value. Appropriately elevated dietary protein can effectively shorten the overall feeding cycle of finishing pigs. Cold seasons require continuous barn temperature maintenance, which brings sustained high fixed expenditures including heating, labor, facility operation and maintenance, electricity, and site occupation. A shortened rearing period can greatly reduce such daily comprehensive management and heating costs, and also decrease the total feed consumption throughout the production cycle. Moreover, reasonable protein improvement can optimize meat quality by reducing drip loss and promoting the deposition of CP and AAs in muscle tissue. With the continuous upgrading of consumer demand, high-quality meat products with superior nutritional properties can further improve market acceptance and consumer preference. Collectively, moderately increasing dietary protein to a proper proportion of the standard recommendation is a feasible nutritional regulation strategy under low-temperature environments, which can comprehensively balance growth performance, production cost control, and meat quality improvement.

The study showed that increasing dietary protein content to 104% of the NRS recommended level under winter low-temperature conditions serves as a viable nutritional strategy to shorten production cycles and enhance pork quality. Furthermore, this approach significantly increased AA and protein content in pork by activating the mTOR/S6K1/4E-BP1 pathway, thereby optimizing flavor characteristics and overall nutritional quality. These findings underscore the value of optimizing protein supplementation strategies under low-temperature farming conditions, providing reference for the production of high-protein pork products.