Water scarcity is a real and pressing concern worldwide. Only approximately 3% of the world’s water is freshwater, and only 0.5% is usable [1]. Importantly, agriculture uses over 70% of freshwater resources [2]. As climate change and global population expansion will further tax an already strained resource, it is estimated that two thirds of the global population, or 4 billion people, will experience severe water shortages for at least one month a year [3]. Indeed, rising temperatures coupled with fluctuation in distribution and amounts of rainfall will dramatically change water resources and create uncertain availability. Although tackling water scarcity necessitates action at every level of use, agriculture is uniquely situated in that any reductions in water use must also maintain animal production and welfare standards to meet the needs of the growing population.

It is estimated that broiler chickens consume 1.6–2.0 grams of water for each gram of feed [4]. Considering an average market age of 47 days for US broilers in 2023, and an average feed intake [5], this equals 8–10 kg (2.2–2.7 gallons) of water consumed per bird during production. The US alone produced 9.16 billion broilers in 2023, which equates to over 20 billion gallons of drinking water consumed by these birds. Additionally, during times of elevated environmental temperatures, productivity, in terms of growth, feed efficiency, meat yields, and livability, is decreased and the volume of water consumed can increase significantly [6], further impacting water usage. Even a small improvement in water efficiency in broilers could save billions of gallons of water annually, allowing that water to be diverted for other uses. Recently, our research group has published on 2 lines of chickens divergently selected for water efficiency (ratio of water consumed to body weight gain). We have shown an 18% reduction in water intake in the high water efficient (HWE) as compared to their low water efficient (LWE) counterparts, while maintaining body weight gain (BWG) and improving in feed conversion ratio (FCR) [7]. Additionally, the HWE line performed better under chronic cyclic heat stress (HS) conditions, despite consuming less water and having a higher core body temperature [7]. In that study, however, the cumulative, and possibly adaptive, effects of 20 days of HS were examined. As older and heavier broilers are more susceptible to the negative effects of HS [8], we undertook this study to determine the effect of acute (3 h) HS on 49 d-old LWE and HWE chickens. In particular, increased heat load has been shown to induce oxidative stress and the inflammatory response through alteration of cytokines, chemokines, and other inflammatory markers [9, 10, 11, 12], likely contributing to growth declines and HS-productivity losses seen in poultry [13]. What is unknown, however, is how selection for water efficiency may impact these parameters. Determining the profile of these factors would be of significant interest to the poultry industry, as robustness (defined as maintaining a high level of production while being adaptable to a wide variety of conditions [14]) is a key goal of breeding and selection programs. Here, in order to further characterize these divergently selected lines under HS conditions, we report the gene expression and relative levels of circulating chemokines, cytokines, and other inflammatory markers.

Water availability is already a concern for roughly a quarter of the world’s cities, which experience perennial water shortages [20]. As climate change and global warming will likely lead to more frequent and intense heat waves [21], the need for water, which is already limited, will increase. The poultry industry is particularly susceptible to HS [22], as modern chickens have high metabolic activity, increased body heat production, and decreased thermotolerance [6]. Therefore, the combined impact of these environmental challenges must be addressed in order to not only maintain, but improve productivity of a critical, efficient, and affordable protein source for a growing world population.

It is imperative to understand potential associated changes, whether positive or negative, that can arise during the selection process in broilers. Indeed, intensive genetic selection for improved growth rate and feed efficiency for more than 80 years has given rise to unintended negative traits that are now concerns for the industry [23]. Here, overall, we show that the impact of 4 generations of selection for improved water efficiency had minimal impact on circulating inflammatory markers in acute heat-stressed broilers. As we measured gene expression in whole blood, it encompasses a heterogeneous mix of cell types, including white blood cells, the traditional players in the immune and inflammatory response, as well as red blood cells. This is of importance, as in chickens, red blood cells are nucleated, and therefore are transcriptionally active and have protein synthesis abilities, and likely substiantially impact the immune response, through both sending and receiving cellular signals [24]. Additionaly, obtaining a blood sample is minimally invasive, and can allow for monitoring of health status or response to specific stressors (such as HS) within a breeding program.

Some of the spotted effects of HS might be associated with dysregulation of protein homeostasis-induced by protein misfolding and aggregation. To counter these detrimental effects, the cell activates the survival pathways through systematization of highly conserved responses. Contingent on the stress type, severity, intensity, amplitude, and duration, cells can activate a rapid and efficient stress response and protein quality control systems (refolding, degradation, etc.) to ensure their survival or start off the cell-death pathway [25]. At the cellular and molecular levels, a well known standard rapid response to HS is induced synthesis of heat shock proteins (HSPs). Based on their known functions, elevated HSP expression due to acute HS was not unexpected, and has been previously shown in multiple tissues in poultry [11, 26, 27]. The differences seen in HSP70 gene expression between the lines, however, are more intriguing. Lower blood HSP70 expression in the HWE line, regardless of environmental temperature, may indicate an improved state of intracellular homeostasis in these birds as compared to the LWE line, as HSP70 is a highly inducible molecular chaperone that catalyzes proper folding of nascent proteins under normal conditions and assist in refolding and clearing damaged proteins under stress conditions [28]. Further studies are needed to define the role and mechanisms of HSP70 in protein homeostasis, cell survival, and thermotolerance in HWE birds.

Unsurprisingly, HS increased gene expression of the majority of cytokines and chemokines as has been previously reported [9, 10, 29], and alteration of the innate cytokine and chemokine response by stressors is well noted [29, 30]. The fact that the induction of these factors was to a similar extent in both HWE and LWE lines indicates that there were neither detriments nor improvements to this response associated with selection for water efficiency. However, IL-10, IL-4, and CCL4 were differentially expressed between the two lines. IL-10 is increased in circulation in response to inflammatory challenges, such as viruses, bacteria, fungi, protozoa and helminths [31]. Studies have shown it to exhibit immunosuppressive properties, through inhibition of cytokines such as IFN-$\gamma$ [32], IL-8 [33], IL-12 [34], and TNF$\alpha$ [35]. IL-4 plays a key role in the promotion of T-helper 2 cell differentiation and can suppress synthesis or function of many proinflammatory cytokines [36]. Similarly, CCL4 helps orchestrate the immune response to infection or inflammation, as it is a chemoattractant for monocytes/macrophages, T-lymphocytes, natural killer cells and dendritic cells [37]. As IL-4 and CCL4 gene expression were upregulated in HWE chicken during HS, but not in LWE birds, it may indicate a more robust response to the HS challenge and promote anti-inflammatory properties, which may help explain the improved performance of this line during HS [7]. However, these cytokines and chemokines are not well studied in avian species; therefore, research targeting the mechanism by which they may regulate growth and production during HS stress challenges is necessary.

The inflammasomes are a group of intracellular multimeric protein complexes that influence inflammation and the protective immune response [38]. Multiple members of the NLR family exist, with differences in stimuli and activated pathways, and both inflammatory and anti-inflammatory properties [39, 40]. Here, the mRNA abundances of the NLRP3, NLRC5, and NLRX1 inflammasomes were all upregulated in circulation by HS, without any differences between the water efficiency broiler lines. NLRP3 is the most well studied inflammasome, and is generally considered pro-inflammatory. Although NLRC5 and NLRX1 are less well understood, NLRC5 has been shown to have inflammatory properties via association with NLRP3 [41] and through IFN production, but may also be able to suppress IFN and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-$\kappa$B) signaling [42], depending on the cell-type and experimental conditions. Here, its role is still unclear. Gene expression patterns between NLRP3 and NRLC5 are similar; however, circulating IFN$\gamma$ is decreased during HS in the HWE lines. Interestingly, NLRC3 gene expression was lower in the blood of HWE as compared to the LWE line. It had been shown that the NLRC3 inflammasome has an anti-inflammatory role via inhibition of the NF-$\kappa$B pathway [43], and can inhibit pyroptosis through blocking the formation of NLRP3 inflammasomes [44]. As these inflammasomes are not well defined in chickens, further exploration to their roles is merited.

The brain is a critical regulator and protector of homeostasis via neuroendocrine responses which guard set-points of body water concentration and volume [45]. AVP is a key hormone in fluid balance, as it is released from the posterior pituitary in response to increased plasma osmolarity, and acts to increase water retention at the kidney [46]. There are several studies in human medicine examining the difference between individuals with low and high habitual fluid intake [47]. In both intake groups, plasma osmolarity and sodium levels remain within normal ranges; however, one of the hallmarks is higher levels of circulating AVP in those that consume less [47]. Here, we found no differences in circulating AVP protein, although gene expression was lower in the HWE as compared to the LWE line. Other research has shown AVP expression in peripheral tissues [48, 49], therefore it is plausible that AVP produced by circulating cells may have autocrine effects separate from, or in addition to, centrally produced AVP. Studying these cells in isolation could help define the role of this locally produced AVP. Overall, our data suggests that although HWE birds are drinking less water, their blood inflammasome and chemocytokine expression profile seemed to not be altered. It is plausible that the HWE birds have more efficient metabolic water production and water (re)absorption in the gut, allowing overall homeostasis to be maintained in the body. Further research is warranted to examine other physiological, cellular and molecular pathways in the HWE birds with more selection generations to make sure that there are no undesirable changes occur.

In conclusion, water scarcity is a global concern that must be addressed at all levels of water use, but modifications in agricultural systems must also maintain optimal production levels. Herein, to the best of our knowledge, we are the first to report that broilers selected for high or low water efficiency displayed an overall similar expression pattern of circulating inflammatory markers in response to acute HS. This likely indicates that selection for improved water efficiency has not negatively impacted inflammation status in these birds.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

SD designed the research study. ESG, TT, WGB, SO, and SD performed the research. ESG analyzed the data and wrote the manuscript. SD edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

This study was conducted in accordance with the National Institutes of Health recommendations guide for laboratory animal use and care. All the procedures in this study were approved by the University of Arkansas Animal Care and Use Committee under protocol #23015.

Authors would like to thank the University of Arkansas Poultry Research farm and the University of Arkansas feed mill for their assistance.

This study was supported by a grant from USDA NIFA Sustainable Agriculture Systems (#2019 69012-29905) to S.D.

The authors declare no conflict of interest.