Heat stress (HS) adversely impacts poultry production by altering several neuroendocrine, molecular, and physiological processes, which are still not completely understood. It poses deleterious effects on the health, production, and welfare of humans and livestock, as such demanding the persistent search for novel and effective strategies that would mitigate its multifaceted impacts on productivity and sustainability. HS influences several physiological responses in the body, and direct assessment of metrics such as rectal temperature (RT), surface temperature, respiratory rate, panting, and heat production, can be used to examine the responsiveness and adjustment during acute or chronic HS (Yahav, 2000; Renaudeau et al., 2012). Transcriptional analysis has shown that HS strongly over-represents several pathways such as the electron transport chain, apoptosis, heat shock response, inflammatory response, and oxidative stress signaling (Kapila et al., 2016). Identification of these transcriptional responses would assist in understanding the responsiveness of organisms to environmental heat load.

Broilers subjected to HS often undergo physiological responses, such as hyperthermia, metabolic disorders, oxidative stress, respiratory alkalosis, systemic inflammation, and mortality (Loyau et al., 2014; Brownstein, 2016). Moreover, thermal and oxidative stress initiates the expression and chaperoning activity of inducible heat shock proteins, which function to protect cells from damage (Zhao et al., 2006). An important physiological response during HS is an increase in blood flow to the body surface or upper respiratory tract (Majdeddin et al., 2020). These perturbations are necessary to provide the fluid needed for heat dissipation during panting which is accompanied by peripheral vasodilation and an increase in body temperature (Yahav et al., 1997). Nitric oxide (NO) is an active effector of vasodilation that directly regulates vascular tone and blood flow in the vascular smooth muscle (Luiking et al., 2010). The primary precursor for the endogenous synthesis of NO by nitric oxide synthase (NOS) is L-arginine. Therefore, increasing arginine availability via L-arginine to L-citrulline (L-Cit) recycling might be beneficial for heat-stressed birds.

L-Citrulline, a non-protein amino acid found mainly in watermelon, is an important metabolite of the urea cycle (Azizi et al., 2020). Recently, L-Cit has been revealed as a nutritional supplement with the ability to lower body temperature and it can afford thermotolerance during thermal challenge in chicks (Chowdhury et al., 2021). It was demonstrated that a single oral dose of L-Cit (15 mmol/10 ml/kg body weight) could induce hypothermia in chicks throughout the 5 h study period, whereas, dual administration of L-Cit was necessary to sustain thermotolerance in chicks (Chowdhury et al., 2017). L-Cit is regarded as a HS biomarker since changes in the plasma L-Cit concentration can reflect the extent and length of thermal challenge in chicks (Chowdhury et al., 2014; Chowdhury, 2019). Plasma L-Cit level is increased during short periods of HS, but exposure to long term HS reduces circulating L-Cit concentration (Chowdhury et al., 2014; Chowdhury, 2019). Kvidera et al. (2016) reported that L-Cit slightly affected thermal response during cyclical HS in gilts by decreasing respiratory rate, with a tendency to decrease the RT. In another study, dietary supplementation of 1% L-Cit decreased the respiration rate during HS but did not influence the RT (Liu et al., 2019). These researches demonstrate the role of L-Cit in thermoregulation, however, there is still paucity of information regarding the molecular responses and mechanisms responsible for these actions. Importantly, L-Cit can be recycled during L-arginine generation and NO synthesis (Bahri et al., 2013). The three NOS isoforms; neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) can catalyze L-arginine, to generate NO and L-Cit (Schwedhelm et al., 2008). Dietary L-Cit supplementation increased the serum amino acid concentration for arginine, citrulline, and ornithine in laying hens (Uyanga et al., 2020). Studies have shown that L-Cit can effectively increase systemic arginine levels (Jegatheesan et al., 2015; Kim et al., 2015), much higher than direct L-arginine supplementation (Lassala et al., 2009; Morita et al., 2014; Agarwal et al., 2017). L-Arginine, an essential amino acid in poultry nutrition, mediates several biological functions during stress conditions (Wideman et al., 1995; Fouad et al., 2012; Varasteh et al., 2018). However, L-arginine deficiency may occur due to increased catabolism, L-Cit unavailability for de novo synthesis, and under certain inflammatory conditions (LeBaron et al., 2020). Thus, augmenting L-arginine availability would provide a measure to promote NO synthesis and bioavailability during stress condition. In addition, the application of L-Cit in animal production is yet an avenue to explore for its direct benefits, as well as its indirect actions via arginine and NO generation (Uyanga et al., 2020).

Nitric oxide is involved in a wide range of physiological functions, including anti-oxidative (Hayashi et al., 2005), anti-inflammatory, appetite regulation (Zendehdel et al., 2017), vasodilation (Flam et al., 2007), and thermoregulation (Coleone et al., 2009; Dantonio et al., 2016). NO is an important modulator of body temperature because it can act on the central nervous system (CNS) and in the periphery to elicit thermoregulatory responses (Simon, 1998; Steiner and Branco, 2001). NO decreases body temperature at sites of thermo-integration, whereas, it acts to increase body temperature at pyrogenic and thermo-perceptive regions within the CNS (Gerstberger, 1999). The hypothalamus functions as the thermoregulatory center, where NO secondary target, soluble guanylate cyclase is abundant (Gerstberger, 1999). Stress-induced hyperthermia and infection-induced fever are two mechanisms that lead to an increase in body temperature via activation of the autonomic nervous system and the immune system respectively (Briese and Cabanac, 1991; Oka et al., 2001; Vinkers et al., 2009). The rise in body temperature induced during stress, is considered relatively similar to fever caused by exogenous pyrogens (such as lipopolysaccharides) in animals (Soszynski, 2001; Vinkers et al., 2009). Stress-induced fever is associated with the upregulation of pro-inflammatory cytokines, suggesting that cytokines play an important role in body temperature regulation (Evans et al., 2015). Pyrogenic cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), can induce fever when released peripherally or centrally within the CNS (Monroy et al., 2001). The activity of these cytokines requires activation of the cyclooxygenase (COX) pathway, for the subsequent release of prostaglandin E2 (PGE2) (Lazarus, 2006; Krall et al., 2010). PGE2 is recognized as a final mediator of fever and is responsible for the upward resetting of the thermoregulatory set point in the preoptic area of the anterior hypothalamus (PO/AH) (Lazarus, 2006; Evans et al., 2015). Since high temperature results in immune dysfunction, inflammation, and in extreme cases, multiple organ disorders (Ahmed, 2005), we investigated the involvement of COX-PG signaling during HS condition. Therefore, this study examined the effects of L-Cit supplementation on thermotolerance, redox balance, heat shock response, nitric oxide regeneration, and inflammatory responses of acute heat-stressed broilers.

The present study was designed to gain an understanding of the acute HS paradigm, and how dietary intervention with L-Cit would mediate acute HS effects in broiler chickens. Similar to previous reports, exposure of broilers to acute HS affected thermotolerance by initiating severe hyperthermia and tachypnea. Body temperature measurements including ear temperature, RT and CBT were elevated by HS during the study. Interestingly, L-Cit reduced the RT and CBT intermittently at certain time points during HS and at TNZ. Also, the mean CBT of broilers was lowered by L-Cit supplementation. Reduction in CBT can occur as a beneficial stress response to a variety of stimuli, including, hypercapnia, hypoxia, and hypoglycemia because it lowers oxygen consumption, and energy requirements, thus improving survival (Almeida and Branco, 2001). Although the molecular mechanisms behind this determinant are yet to be elucidated, it is understood that L-Cit may act on the central control of body temperature to achieve hypothermia, but whether this effect is temperature dependent or independent is yet to be fully ascertained. Plasma levels of T₃ can be considered as a reliable indicator of HS in chickens due to its involvement in regulating heat production, and metabolic rate to achieve thermotolerance (Yahav et al., 1996). As a physiological adjustment to HS, reduction in T₃ concentration allows for a lowered rate of heat production, metabolism, and energy requirement in chickens (Abu-Dieyeh, 2006). However, few studies report on variations to T₃ responses during HS (Scheele et al., 1992; Melesse et al., 2011). From our findings, the elevation in serum T₃ concentration indicates an increased thyroid function and metabolic activity during short-term heat exposure. Increased peripheral conversion of T₄ to T₃ may be necessary to meet the increased demand for heat production which is necessary to achieve hyperthermia during HS.

Stress-induced elevation in corticosterone levels is associated with higher resistance and adjustment to the stress stimuli (Quinteiro-Filho et al., 2010; Xu et al., 2018). However, reports on the hypothalamic-pituitary-adrenal axis activation for the release of corticosterone during HS (Zaboli et al., 2019) are inconsistent. Our study revealed that serum corticosterone levels were unchanged during HS and L-Cit supplementation. This finding is similar to previous reports (Lin et al., 2006; Song et al., 2012; Xie et al., 2015). The inconsistent responses reported on corticosterone release have been attributed to variability in the duration of HS, the intensity of thermal stimuli, pulsatile hormonal activity, species of animal, as well as, tissue-specific responses (Dou et al., 2019; Bai et al., 2020). Furthermore, plasma TP, UA, and TCHO in HS broilers did not differ from control birds, demonstrating minimum alterations in nutrient digestion and absorption. In this study, exposure to acute HS challenge suppressed plasma TG and GLU levels, indicating possible perturbations in lipid and carbohydrate metabolism. Typically, heat challenge elevates plasma GLU levels to permit stress resistance and survival during the “fight or flight” response (Xie et al., 2015). In addition, Lin et al. (2006) had reported that acute HS did not influence glucose levels in broilers, whereas circulating levels of glucose in broilers were diminished during chronic HS. Our findings revealed that L-Cit increased plasma GLU levels but lowered the TG content at TNZ. In line with previous reports, L-Cit had been shown to elicit beneficial effects on plasma TG, and was also implicated in the regulation of glucose and lipid metabolism (Jegatheesan et al., 2015).

Heat stress is closely associated with oxidative stress due to an imbalance between ROS production and the antioxidants defense system (Roushdy et al., 2018). L-Arginine metabolism to L-Cit by NOS allows for the formation of NO, and when in excess can result in oxidative damage, mitochondrial dysfunctions, and lipid peroxidation (Mishra and Jha, 2019). From our study, HS modulated SOD activity, two-fold higher than TNZ, but the effect of L-Cit was evident via increased CAT activity. Controversial reports exist on the influence of HS on antioxidant enzyme activity (Madkour et al., 2020), however in a recent review it has been explained that exposure to high ambient temperature allows for a compensatory increase in the antioxidant enzymes activity such as SOD, GSH-Px, and CAT, in order to afford short term protection against excessive free radicals produced (Goel et al., 2021). Under HS condition, L-Cit increased GSH-Px action to counteract ROS formation, although the plasma MDA and T-AOC contents were not influenced. Previously, L-Cit had been reported to possess potent antioxidant effects via its ability to scavenge hydroxyl radicals and inhibit ROS formation (Coles, 2007). Also, it had been shown that dietary L-Cit improved the antioxidant status via increased SOD, CAT, and T-AOC, but decreased the MDA content in laying hens (Uyanga et al., 2020). Thus, L-Cit stimulation of antioxidant responses may serve to combat HS-induced oxidative damage to certain extents. In addition, stress-induced lymphoid organ involution is a common index for evaluating the immunosuppressive index of stress (Kimura et al., 1996). In this study, acute HS did not affect the spleen index, rather, L-Cit lowered the spleen index, which may be indicative of an altered immune response, such as lymphocyte depletion (Elmore, 2006). Nevertheless, further investigations are needed to understand the influence of L-Cit on lymphoid organ development.

Stress induction leads to the release of HSPs which are critical for thermoprotection, repair of denatured proteins, protein folding, and prevention of subsequent stress effects (Calderwood et al., 2017). The induction of heat shock response during stress condition is primarily anchored on the activation and binding of the HSFs and heat shock elements, to allow for the transcription of HSPs (Creagh et al., 2000; Aramburu et al., 2014). HSF1 and 3 are the two major HSFs responding to heat shock in avian species, thus they are considered as avian-specific HSFs (Xie et al., 2014). Interestingly, our results showed that L-Cit lowered HSF1 expression, which may have resulted in HSP 60 downregulation. More so, L-Cit downregulated HSF3 expression in the hypothalamus under thermoneutral condition. Essentially, during HS, the rapid response of HSFs is necessary for immediate transcription of HSPs, as genes necessary for cell survival are activated, whereas, less essential genes are downregulated (Xie et al., 2014). Since HSPs are constitutively expressed within living organisms, these findings suggest that L-Cit may act to preserve the activity of heat response metabolites under thermoneutrality. Also, it was found that HS induced HSP90 expression, probably to function in protecting the structural and metabolic integrity of tissues against stress-induced injury (Hao and Gu, 2014; Madkour et al., 2021). Furthermore, Zhao et al. (2016) had revealed that there exists a strong positive relationship between NO and HSPs expression (r ≥ 0.9), such that NO had stronger interactions with HSP60 and HSP90, compared to other HSPs. These findings give additional insights into the cross-talk that exists between HSPs and NO-mediated signaling.

Dietary factors can function to modulate the availability or deficiency of NO, rendering important implications for health and disease (Luiking et al., 2010). Previously, L-Cit supplementation was shown to increase the serum-free arginine, citrulline, and ornithine levels, as well as nitric oxide synthesis in laying hens (Uyanga et al., 2020). In this present study, it was revealed that HS significantly modulated circulating NO concentration and arginase activity but inhibited tNOS activity. Alongside this, L-Cit supplementation elicited contrasting effect by decreasing NO concentration and arginase activity but increased tNOS activity. A clear understanding of the opposing action of these two factors with respect to NO generation is yet to be unraveled and necessitates further studies. From existing literature, it is understood that the utilization of L-Cit as an alternative to restore L-arginine homeostasis and NO production may be regulated by feedback mechanisms to limit NO overproduction (Cynober et al., 2010, 2013). Therefore, an increased L-arginine availability arising from L-Cit supplementation may in turn acts as a sensor to prevent potentially neurotoxic levels of NO in the blood circulation (Wu et al., 2009; Cynober et al., 2010). This report is in line with our findings of L-Cit-induced reduction in NO concentration. Additionally, the intracellular NOS activity is reflected by tNOS, which comprises both the constitutive and iNOS isoforms (Uyanga et al., 2020). However, findings from this study revealed that L-Cit-induction of tNOS activity did not correspond with an increase in NO production, whereas, L-Cit was able to lower arginase activity. L-Arginine can be catabolized either by NOS to NO and L-Cit or it is hydrolyzed by arginase to ornithine and urea (Rath et al., 2014). As such, there are multiple cross-inhibitory interactions between these two arginine metabolic pathways which compete for the common substrate, L-arginine (Kocyigit et al., 2004; Rath et al., 2014). It is understood that when excess L-arginine is supplied in the diet, the kidney arginase activity is increased resulting in the rapid degradation of L-arginine to urea and ornithine (via the urea cycle), but where this degradation proceeds too rapidly, L-arginine availability to serve in NO production may be impaired (Ruiz-Feria et al., 2001). L-Cit-induced arginase inhibition had been reported as a protective mechanism against impairment in endothelium-dependent relaxation (El-Bassossy et al., 2013). More so, arginase inhibition restores L-arginine availability for NOS action since both enzymes actively compete for L-arginine (Cynober et al., 2010). Thus, these results demonstrate the regulatory role of L-Cit via arginase inhibition to allow for increased L-arginine availability, tNOS activity and subsequent NO production. Noteworthy is the fact that the L-Cit-induced tNOS activity in this study did not translate for higher NO production. Therefore, we propose that since the control and L-Cit based diets were formulated with equal L-arginine levels, L-arginine availability would have probably contributed to impeding the efficiency of NO regeneration in vivo.

The iNOS isoform is generally stimulated by inflammatory stimuli and contributes to NO production under stressful conditions (Yu et al., 2018). This study revealed that under thermoneutrality, L-Cit supplementation downregulated the central expression of iNOS, and elicited a similar pattern to the COX2-PGE2 expression. Investigation into the iNOS-COX2-PGE2 signaling showed that the hypothalamic expressions of iNOS and COX2 were downregulated by L-Cit under thermoneutral condition, although, L-Cit differentially influenced serum PGE2 at TNZ and HS temperatures. L-Cit initiated an inhibitory role on peripheral PGE2 levels at thermoneutrality but stimulated PGE2 release during HS, which corresponded with the increased hypothalamic PGE2 levels during HS exposure. PGE2 can increase body temperature via coordinated increment in heat production and a concomitant decrease in heat loss (Milton, 1998). Furthermore, the role of PGE2 has been widely investigated in the central control of body temperature regulation (Ayoub et al., 2004), and it has been described as the principal mediating factor for the generation of hyperthermia during fever induction (Osaka, 2010; Conti, 2016). Therefore, PGE2 induction or reduction at different temperatures validates its involvement in body temperature regulation. From our results, it is evident that the decrease in PGE2 levels under thermoneutral condition was mediated by iNOS-dependent downregulation of COX-2 signaling. More so, the L-Cit-suppression of body temperature may be attributed to a modified thermoregulatory set point resulting from the altered activity of thermosensitive neurons in the POA, which is induced by locally released PGE2 (Almeida et al., 2004). However, the unresponsiveness of pro-inflammatory cytokines such as interleukin (IL)-6, and tumor necrosis factor (TNF)-α, would suggest the interplay of other immunomodulatory molecules in activating the iNOS-COX2-PGE2 signaling.

Altogether, findings from this study reveal that acute HS exposure significantly elevated body temperature and altered the redox status, heat shock response, and nitric oxide synthesis in broilers. L-Cit supplementation acted to lower the body temperature and HSFs activation, improved antioxidant enzyme responses, and diminished circulating NO levels. Also, it was evident that inflammatory mediators of the iNOS-COX2-PGE2 signaling have a role to play in the regulation of body temperature at thermoneutrality and during HS conditions. Undoubtedly, these findings provide the knowledge base for further studies to ascertain the role of temperature and nitric oxide in L-Cit’s actions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the College of Animal Science and Veterinary Medicine of Shandong Agricultural University.

VU and HL designed the research. VU, MW, and TT performed the animal experiments and data collection. VU analyzed, interpreted, and presented the data. HJ, JZ, XW, and OO contributed in the research design and results interpretations. All authors read, reviewed and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.