Pig stress study maps long noncoding RNA across blood, saliva and brain

A pilot experiment in 36 pigs has shown that the long noncoding RNA Neat1 can be measured in blood, saliva and the ventromedial hypothalamus, offering a foundation for studying molecular responses to psychosocial stress in livestock. The researchers also detected changes associated with sampling time and acute restraint, but the results do not establish Neat1 as a validated biomarker of stress, fear or animal welfare.

The study focused on Nuclear Paraspeckle Assembly Transcript 1, or NEAT1, a mammalian long noncoding RNA involved in stress responses, metabolism, inflammation and transcriptional regulation. It produces two isoforms: the shorter NEAT1_1 and the longer NEAT1_2. NEAT1_2 has an important scaffolding role in paraspeckles, dynamic nuclear condensates associated with cellular stress, while NEAT1_1 has been linked to metabolic and inflammatory regulation. Most previous evidence has come from cultured cells, laboratory mice or human patients, leaving limited information about how Neat1 behaves in large farm animals exposed to social and psychological challenges.

For the pilot, investigators used 18 male and 18 female pigs that were 12 weeks old at the beginning of the experiment. These animals came from a larger study of 144 pigs housed in six mixed-sex groups and assigned to three environmental conditions for four weeks. The neutral condition represented conventional husbandry. The positive condition added regular stockperson interaction, new toys each week and fresh hay each day. The negative condition involved regularly changing pen mates, an approach intended to disrupt social relationships and create conflict and fear. At the end of this phase, half of the 36 sampled pigs were randomly selected for a five-minute snout-snare restraint; the others received no snare. The investigators then collected saliva and blood one hour later before euthanizing the pigs and dissecting the ventromedial hypothalamus, a brain region involved in emotional processing.

Because the pig genome did not then contain a fully annotated sequence for the gene, the team designed primers by aligning the human NEAT1 sequence with the pig genome and identifying a candidate region with approximately 60% similarity to human NEAT1. Conserved portions were used in reverse-transcription quantitative PCR, or RT-qPCR, assays for total Neat1 and the longer Neat1_2 isoform. Amplicon sequencing, melt-curve analysis and amplification efficiency helped select the final primers. Two reference genes were used for each sample type: Rpl4 and Gapdh for hypothalamus, Ppia and Gapdh for saliva, and Ppia and Rpl4 for blood. A later study annotated the pig ss_Neat1 gene and confirmed the region identified by the researchers, according to the paper.

Neat1 was readily detectable in all three biological materials. The ventromedial hypothalamus produced the greatest average RNA yield, 34.36 ± 14.35 micrograms, compared with 6.10 ± 2.39 micrograms from whole blood and 1.86 ± 0.84 micrograms from saliva. However, neither the four-week environmental treatment nor the acute snout snare significantly changed total Neat1 or Neat1_2 expression in the hypothalamus. The proportion of expression represented by Neat1_2 also did not differ by either type of treatment. These null results mean that the experiment found no evidence that its particular housing and restraint exposures altered the measured transcript in this brain region, rather than demonstrating that Neat1 is unrelated to stress.

Saliva produced the clearest acute-treatment signal, but only for total Neat1. Pigs subjected to the snout snare had significantly lower total Neat1 expression than pigs that did not receive the acute challenge, with a reported p value of 0.01. The four-week environmental treatment did not affect total Neat1 in saliva. Neither environmental nor acute treatment significantly altered Neat1_2 expression or the percentage of total expression attributable to that isoform. The finding is notable because saliva can be collected without invasive procedures, but it remains a single result from a small pilot and does not by itself show that salivary NEAT1 reports psychological stress.

Blood measurements revealed a difference between the first and final sampling points rather than a clear response to the experimental treatments. Neat1_2 expression was higher at the first sample, recorded when pigs were transferred to the experimental facility, than at the end of the four-week period. Average relative expression declined from 1.40 ± 1.09 to 0.32 ± 0.24, with a significant time effect at p = 0.023. The environmental and acute treatments did not significantly affect changes in total Neat1, Neat1_2 expression or the Neat1_2 percentage. Before treatment, females had higher blood Neat1_2 expression than males, and both sexes showed lower values at final sampling. The researchers could not rule out age-related change, although the four-week interval represented a modest developmental difference in pigs aged from 12 to 16 weeks and no age-related markers were measured.

Plasma cortisol, a conventional measure of endocrine stress, increased sharply by the final sampling. Its mean concentration rose from 21.4 ± 16.1 nanograms per milliliter at the first sample to 107.3 ± 33.0 nanograms per milliliter at the second. The acute snout snare itself did not significantly affect cortisol, and environmental treatment did not produce a significant difference at the end of the study. The researchers suggested that moving all pigs from communal pens, collecting blood and performing the subsequent procedures likely stressed every animal at the final sampling, potentially obscuring differences between the long-term environmental and acute snare groups. This uniform final stress response is a major limitation when interpreting the molecular results. Neat1 expression also did not correlate significantly with cortisol in blood, saliva or hypothalamus.

There were likewise no significant relationships between Neat1 measurements in the three sample types within individual pigs. Blood and hypothalamic total Neat1, Neat1_2 and the Neat1_2 percentage did not correlate significantly, nor did saliva measurements track those in the hypothalamus or blood. The cellular origins of the measured RNA were not established because the investigators did not isolate specific blood-cell populations. The paper notes that peripheral blood mononuclear cells are a plausible source in blood and can also occur in saliva, while earlier mouse work found especially high Neat1 expression in glia, including astrocytes and oligodendrocytes, rather than neurons. The absence of cross-tissue correlation may therefore reflect tissue-specific regulation, but the study cannot distinguish that explanation from other biological or methodological factors.

Overall, the research provides a technical proof of concept and a set of hypotheses rather than a practical stress test. The team argues that future work should separate acute and chronic challenges, control the intensity and context of stress, and collect samples at several times, including before and after restraint and during the weeks preceding final sampling. The one-hour post-restraint collection may also have been too early to capture a delayed Neat1 response: cell-culture studies cited by the authors detected increases in Neat1_2 roughly four to 12 hours after stress and sometimes as late as 24 hours. More potent, clearly characterized challenges, such as isolation, handling changes, auditory overstimulation or disruption of social bonds, could help determine whether Neat1 tracks specific emotional states. Additional brain regions, including the prefrontal cortex, would be needed to test whether central NEAT1 expression participates in stress processing. Until such evidence accumulates, the measurable decline in blood Neat1_2 across sampling times should not be described as a biomarker of negative welfare, and the absence of treatment effects should not be taken as proof that the transcript is insensitive to stress.

Subject of Research: Measurement of stress-associated Neat1 expression in blood, saliva and ventromedial hypothalamus of pigs exposed to psychosocial challenges

Article Title: Expression of the long noncoding RNA Neat1 in saliva, blood, and ventromedial hypothalamus of pigs exposed to psychosocial stressors

Article References: Webster, D., Ding, L., Zhang, S., D'Souza, D., Tilbrook, A., Tomas, K., Babington, S., Plush, K., Blache, D., & Fox, A. (2026). Expression of the long noncoding RNA Neat1 in saliva, blood, and ventromedial hypothalamus of pigs exposed to psychosocial stressors. Physiological Reports, 14(20), Article e71130. https://doi.org/10.14814/phy2.71130

Image Credits: AI Generated

DOI: 10.14814/phy2.71130

Keywords: pigs, psychosocial stress, Neat1 lncRNA, long noncoding RNA, saliva, blood biomarkers, hypothalamus, animal welfare, paraspeckles, cortisol, Expression, long