Cold resistance refers to the ability to tolerate cold conditions, gradually developed through natural selection during the prolonged adaptation of animals to cold environments. Studies have shown that exposure to cold increases overall energy expenditure and induces changes in systemic metabolism, mainly due to the activation of adaptive thermogenesis [[1], [2], [3], [4]]. Animals usually resist cold through behavioral and physiological changes [5]. Exposure to cold leads to a gradual decline in skin temperature, which varies with the duration and intensity of cold exposure [6]. Observable signs include back arching, shivering, and disrupted behavior. Simultaneously, the body responds through neuroendocrine regulation, increasing the secretion of catecholamines, corticosteroids, and thyroid hormones. These hormones act on skeletal muscle, adipose tissue, and the liver, enhancing tissue metabolism. This response is accompanied by increased lipid droplets (LDs) and mitochondria [[7], [8], [9], [10], [11]], along with an enlargement of mitochondrial volume and increased cristae density, facilitating heat production to counteract cold conditions [11,12]. The evidence suggests that cold exposure is vital for whole-body lipid metabolism in animals. Brown adipose tissue, a major thermogenic organ, can improve lipid metabolism by regulating glucose and free fatty acid (FFA) uptake during cold acclimatization [13,14]. Cold exposure can also lead to the formation of beige fat, also known as browning [15]. Skeletal muscle is a specialized heat-producing organ in mammals, and its metabolism is mainly dependent on the action of mitochondria, which are its main organelles for energy production [16,17]. Moreover, the liver, serving as a central regulator of whole-body energy metabolism, responds to low-temperature stimulation by enhancing FFA oxidation. It meets the body's energy demands under cold conditions by secreting specific factors that stimulate thermogenic activity in other tissues (brown fat) [[18], [19], [20]]. Therefore, the liver is essential in maintaining body temperature and should not be overlooked. Cold resistance in animals is a polygenic trait controlled by intricate regulatory mechanisms. Recent studies reported the role of lipid metabolism in regulating cold resistance. For example, Gui et al. [21] discovered that in yak adipocytes, the transcription factor CCAAT enhancer-binding protein alpha (CEBPα) can directly bind to the promoter region of the fatty acid-binding protein 4 (FABP4) gene, potentially contributing to thermogenesis in yaks adapted to high-altitude environments. Fan et al. [22] found that cold exposure induced the expression of Kruppel-like factor 9 (KLF9) in mice, which promoted white fat browning and brown fat thermogenesis. Liu et al. [23] reported that perilipin 5 (PLIN5) in mouse liver may be involved in regulating liver lipid metabolism to maintain the body's adaptation to cold. Moreover, it has been reported that a low-temperature environment can promote the interaction between LDs and mitochondria, thus accelerating lipid metabolism and participating in cold resistance [24]. The FATP4 protein is crucial in mediating the interaction between LDs and mitochondria [25]. The cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) protein family is localized on the surface of LD-to-LD contacts, facilitating lipid exchange and LD fusion [[26], [27], [28]]. However, the mechanisms regulating cold resistance in pig liver tissues after low-temperature treatment remain poorly understood. Therefore, it is essential to investigate these mechanisms to gain insights into how pigs adapt to cold environments.

Proteomics studies can be used to analyze the regulation of proteins and to discover new biomarkers [29,30]. Compared to the traditional data-dependent acquisition (DDA) method, the advanced data-independent acquisition (DIA) technique divides the entire mass spectrum scanning range into multiple windows based on the mass-to-charge ratio (m/z). Within each window, all parent ions are fragmented and detected, thereby significantly enhancing the accuracy of quantitative analysis and the identification of low-abundance proteins [[31], [32], [33]]. Proteomics techniques (including DIA) have been successfully used to study heat stress in different animals such as chickens [34], pigs [35], dairy cows [36], and fish [31]. However, there are no reports on applying DIA proteomics technology to study cold resistance in pigs. Exposure to low-temperature environments triggers the body's regulatory mechanisms to maintain basic metabolism and essential functions, and proteomics technology is an effective approach to uncover the molecular mechanisms underlying these cold-induced responses [37].

Hezuo pig, a subspecies of the Tibetan pig and a plateau-type local pig species can grow and reproduce normally at temperatures as low as −15 °C in winter without any warming measures, demonstrating a remarkable resistance to cold. DIA proteomics was performed to elucidate the molecular mechanisms underlying cold resistance in Hezuo pig livers and analyze changes in the liver proteome under low-temperature conditions. This approach offers a novel perspective for future research on cold resistance mechanisms in animals.