Gene Study Reveals How Stunning Methods Shape Stress Responses in Pigs

Every year, billions of pigs pass through slaughterhouses worldwide, and the final minutes before slaughter can leave molecular fingerprints that ripple all the way to the dinner plate. A team of South Korean researchers has now peered into those minutes at an unprecedented level of detail, using whole-blood RNA sequencing to compare how finishing pigs respond biologically to two of the most widely used stunning methods: electrical stunning and carbon dioxide controlled atmosphere stunning. Their findings, published in Food Science of Animal Resources, reveal that the two techniques trigger strikingly different gene expression programs, one associated with tissue damage and inflammation, the other with oxidative stress buffering and tissue protection.

The stakes of this research extend well beyond animal welfare, although that alone would justify the effort. Stress at slaughter is a well-established driver of pale, soft and exudative meat, a quality defect that costs the pork industry dearly. When pigs experience acute stress, muscle glycogen is depleted, lactic acid accumulates, pH drops rapidly and proteins denature, degrading color, tenderness and moisture retention. Because pork is the most consumed meat in the world, even modest improvements in slaughter handling could translate into substantial gains in quality and consumer trust, particularly as welfare-certified products gain market share in countries such as South Korea, which introduced a pig farm welfare certification system in 2013.

Electrical stunning, the most common method in commercial slaughterhouses, works by passing a current through the brain to render the animal instantly unconscious. In Korea, the recommended protocol applies at least 1.25 amps at 240 volts to the brain for two to four seconds. Yet the method has known drawbacks: high voltage can induce PSE meat and rupture capillaries, and the sudden muscular force can cause fractures, including pelvic fractures. Carbon dioxide stunning takes a different route, exposing pigs to 80 to 90 percent CO2 for roughly three minutes. As CO2 dissolves into the blood, breathing quickens, blood pH falls, cerebrospinal fluid acidifies and consciousness is lost. Previous studies have linked gas stunning to a lower incidence of PSE meat and a reduced likelihood of animals regaining consciousness during exsanguination.

What remained largely unknown, however, was what happens at the molecular level. To find out, the researchers, led by Seok-Won Lim and corresponding authors Jung Hwan Jeon and Jun-Mo Kim, studied twenty crossbred finishing pigs of about 106 kilograms raised on a farm in the Chungnam region. Ten pigs received electrical stunning and ten received CO2 stunning, with all pre-stunning conditions matched so that the stunning method was the only major variable. Whole blood was collected in RNA-stabilizing tubes before and after stunning, and high-quality RNA was sequenced on an Illumina NovaSeq6000 platform using 151-base-pair paired-end reads, with globin depletion to improve detection of the transcriptome.

The sequencing produced an average of roughly 20.9 million raw reads per sample, with more than 95 percent of trimmed reads mapping to the pig reference genome. After quality control and removal of one outlier sample, the team identified 104 differentially expressed genes in the electrical stunning group, 67 upregulated and 37 downregulated, and 56 in the CO2 group, 39 upregulated and 17 downregulated. Thirteen genes were upregulated under both methods, including FASLG, PRF1, KLRK1, IL12RB2, CCL4, GZMA and IFNG, a shared core pointing to a universal cytotoxic immune response to acute tissue stress regardless of how consciousness is lost.

Functional annotation and gene set enrichment analysis showed that both methods activated natural killer cell-mediated cytotoxicity, cytokine-cytokine receptor interactions and pathways associated with graft-versus-host disease, allograft rejection and type I diabetes, all driven by genes such as IFNG, PRF1 and FASLG that mobilize cytotoxic T cells and NK cells to remove damaged cells. But the similarities ended there. Under electrical stunning, the chemokines CXCL10, CX3CR1 and CCL5 were recruited, suggesting a broader influx of immune cells into tissues, while the gene DCN, which encodes decorin, a proteoglycan that stabilizes the extracellular matrix, was sharply downregulated. The authors interpret this as evidence of extracellular matrix destabilization and potential inflammatory reactions triggered by the abrupt sympathetic-adrenal surge that electrical stimulation provokes.

The CO2-stunned pigs told a different molecular story. Albumin, the most abundant plasma protein, was the most strongly upregulated gene in this group, consistent with a systemic defense against the oxidative and hypoxic stress that CO2 exposure creates. At the same time, the matrix metalloproteinases MMP1 and MMP9 were downregulated, pointing to preserved extracellular matrix integrity and limited immune cell infiltration. Crucially, the CO2 group also engaged inhibitory receptors: KLRD1, which recognizes MHC class I molecules and restrains NK cells from attacking the body’s own tissues, and CD244, which dampens cytotoxic responses to prevent immune overactivation. Together with selective activation of the MHC class I recognition pathway, these findings suggest that gas stunning coaxes the immune system into a more balanced, tissue-protective mode rather than an all-out inflammatory assault.

Perhaps the most intriguing signals emerged from the pathway networks unique to the CO2 group. One network linked oxidative phosphorylation and proteasome activity with neurodegenerative disease pathways, indicating that cells were clearing damaged proteins and restoring mitochondrial ATP production while regulating reactive oxygen species. Another network comprised DNA replication, nucleotide excision repair and mismatch repair, implying that cells were actively repairing oxidative DNA damage rather than succumbing to apoptosis. Meanwhile, the phospholipase C and PI3K-AKT signaling axes were broadly suppressed, a metabolic downshift that the authors suggest may reduce intracellular calcium release, slow the onset of rigor mortis, cut energy consumption and limit ROS generation, an adaptive response that could help explain why CO2 stunning is associated with a lower incidence of PSE meat.

The study is not without limitations. It focused on a single CO2-based protocol, and the authors themselves call for work on alternative gas mixtures such as nitrogen and argon, as well as studies that integrate blood transcriptomics with physiological stress indicators and meat quality traits. Sample sizes were modest, and blood transcriptomes reflect a blend of stunning effects and individual responses to handling and sampling. Still, the research opens a genuinely new window on slaughter stress, demonstrating that the choice of stunning method is written into the transcriptome within minutes. If validated further, the gene signatures identified here, from decorin suppression to albumin induction and inhibitory receptor engagement, could serve as biomarkers for objective welfare assessment, guiding the industry toward methods that minimize suffering while delivering better meat to consumers.

Subject of Research: Comparative blood transcriptome analysis of electrical versus CO2 controlled atmosphere stunning in finishing pigs

Article Title: Comparative transcriptome profiling of electrical and controlled atmosphere stunning in finishing pigs

Article References: Lim, S.-W., Lee, S.-H., Lim, B., Lim, C., Jang, M.-J., Lim, S. J., Cheon, S. N., Jeon, J. H., & Kim, J.-M. (2026). Comparative transcriptome profiling of electrical and controlled atmosphere stunning in finishing pigs. Food Science of Animal Resources, 46(1), Article 53. https://doi.org/10.1007/s44463-026-00058-9

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00058-9

Keywords: pigs, RNA-Seq, transcriptomics, electrical stunning, CO2 stunning, animal welfare, meat quality, stress response, immune response, gene expression, PSE meat, slaughter