1. Introduction
When climatic conditions are above the animal’s thermal comfort zone (upper critical temperature threshold), animals experience heat stress (HS) as they fail to dissipate heat efficiently to the environment, leading to the initiation of physiological and metabolic adaptation mechanisms. These adaptations invariably compromise their productive potential [
1]. Significant economic losses are associated with HS, as the majority of pork production occurs in regions where pigs are housed either temporarily (during summer) or permanently under HS conditions [
2]
Pigs exposed to HS may experience an increase of up to 2.5 °C in their body temperature (BT) [
3]. A highly conserved animal response to HS present in livestock species, including pigs, is the induction of anorexia (i.e., a voluntary reduction of feed intake) to decrease the internal heat production deriving from nutrient digestion and metabolism, which in turn adversely affects their productivity [
4]. Concurrently, pigs experiencing HS redirect blood flow to the periphery to enhance body heat dissipation [
5]. However, this flow redirection reduces the amount of blood reaching internal organs [
6], leading to reduced oxygen and nutrient delivery to the small intestine and resulting in epithelial damage (shortened intestinal villus height; [
3]). The reduced intestinal villus height in HS pigs has been linked to increased production of reactive oxygen species (ROS) and oxidative stress [
2], leading to mitochondrial damage [
7] and subsequent intestinal cell death further damaging the epithelia and compromising their integrity [
8]. It has been reported that HS-related oxidative stress increment negatively affects the digestive–absorptive function of the small intestine, as evidenced by increased losses of endogenous amino acids (AAs), reduced AA digestibility [
9], depressed expression of genes coding for the synthesis of AA transporters [
10], and lower concentrations of free AAs in serum. Moreover, the permeability of the intestinal epithelia appears to be compromised [
11], which eventually might affect the health of HS pigs. Thus, increasing the activity of the cellular antioxidant system is expected to counteract the negative impact of exposure to HS.
Phytogenic feed additives containing plant secondary metabolites (PSMs) may mitigate the adverse effects of heat stress. The number of studies investigating the effects of PSMs on HS tolerance in growing–finishing pigs is rather limited [
12] compared to other livestock species, including poultry, with variable effects reported [
13,
14]. Of particular interest regarding their capacity to affect thermoregulatory responses in pigs and ruminants are capsaicinoids, especially capsaicin, which are alkaloids that are found in chili peppers (
Capsicum spp.) and are responsible for their burning and irritant effects [
15]. Capsaicin is a prototypical transient receptor potential cation channel subfamily V member 1 (TRVP-1) agonist, which serves as one of the principal thermosensors for the thermoregulatory system, being conserved across mammalian species [
16]. Activation of TRVP-1 by capsaicin lowers body temperature and induces hypothermia via peripheral vasodilation [
17], potentially increasing the animal’s upper critical temperature threshold and therefore its thermal tolerance [
18]. Studies in growing–finishing pigs have shown that capsaicin-containing additives may improve feed efficiency following both short- and long-term HS exposure, although effects on thermoregulatory responses, such as respiration rate (RR), were limited under short-term HS exposure [
19,
20]. Capsaicin-containing additives have also been demonstrated to be effective in improving the productivity of heat-stressed dairy cows [
21,
22,
23] although effects on feed intake (FI) and rectal temperatures have not been consistent [
21,
22]. Further to its actions on thermoregulation, capsaicin may ameliorate oxidative stress and associated inflammatory responses [
24], while it has pronounced effects on nutrient digestion, independent of HS exposure [
25,
26]. These effects are theoretically significant in countering the development of leaky gut [
27] and could potentially alter both AA absorption and utilization, as they are influenced by both the inflamed and HS state in growing pigs [
10,
28], and consequently their performance.
To the best of the authors’ knowledge, this study is the first to concurrently examine the effects of a phytogenic solution derived from Capsicum spp. (PHY) on various aspects of pig health under heat stress (HS). Specifically, it investigates PHY’s impact on pig performance, body temperature, respiratory frequency, the activity of antioxidant enzymes, and the concentration of amino acid metabolites in blood serum. Additionally, it assesses intestinal histomorphology and the expression of genes responsible for synthesizing heat shock protein 90 (HSP90) in the liver and muscle and myosin in muscle. We hypothesized that dietary supplementation with PHY can mitigate some of the adverse effects of HS in pigs. The anticipated benefits include modulating thermoregulatory responses to lower body temperature, reducing oxidative stress, and improving intestinal integrity. Consequently, we expect that the performance of pigs subjected to HS will be enhanced with PHY supplementation.
4. Discussion
Several physiological, metabolic, and behavioral alterations that negatively affect performance occur when pigs are exposed to high ambient temperatures (above 30 °C). The greatest impact occurs during the first 7 days of exposure to HS, also defined as the acute phase [
3,
39]. On the other hand, certain phytogenics, such as capsaicin contained in
Capsicum spp., apparently have the potential to mitigate the negative impacts of HS on pigs. Thus, the present experiment aimed to determine whether a phytogenic solution based on
Capsicum spp. could help pigs to counteract some of the negative effects of HS during the acute phase. In the present study, heat-stressed pigs were exposed to ambient temperature exceeding 30 °C and temperature–humidity index (111) well above the index for TN pigs (77) all the time.
Increased BT and respiration rate are two of the first physiological involuntary and behavioral voluntary alterations, respectively, that occur when pigs are suffering from HS [
3,
40,
41]. Indeed, these variables are commonly used to assess whether pigs are exposed to HS conditions. In the present experiment, the BT (around 39.5 °C) and respiratory frequency (around 40 breaths/min) of TN pigs were within the normal range [
42]. However, the BT of HS pigs exceeded 40.5 °C, increasing up to 41.4 °C for around 16 h every day, and the respiration rate increased up to 94 breaths per minute during the afternoon. The observed values in the present study confirmed that pigs were subjected to HS conditions most of the time.
Interestingly, the BT of HS pigs fed the phytogenic solution (HS-PHY) was 0.5 °C lower than that of pigs fed the control diet (HS-C) from 19:30 h to around 03:00 h the following day. This BT reduction is noteworthy because (a) it occurred when the daily heat load accumulation was the highest, and (b) that was the time when pigs had the greatest difficulty eliminating body heat. Conserving BT within the normal range, which is under the control of the hypothalamus thermoregulatory system, is essential for maintaining homeostasis and normal functioning of the body [
43]. Sensory neurons that innervate the skin and viscera detect changes in both body surface (peripheral) and internal organ temperature due to changes in ambient temperature [
44]. According to Morrison and Nakamura [
45], warm-sensitive neurons with terminals distributed in the skin become activated when ambient temperature increases through the activation of thermoreceptors located in the plasma membrane and transmit this information to the hypothalamic regulatory system. In turn, the hypothalamus triggers a response oriented towards a reduction in body heat production and/or an increase in body heat dissipation in order to maintain normal BT [
44]. Thermoreceptors belong to the transient receptor potential family of ion channels that detect ambient temperature changes, depolarize neurons, and propagate action potentials [
46]. These receptors are capable of converting temperature stimuli into electric potentials [
47] involved in the transmission of information to the hypothalamus. The transient receptor potential type V member 1 (TRPV1) is the most abundant heat-activated non-selective cation channel in sensory neurons [
48]. Importantly, capsaicin is considered to be the prototypical TRPV1 agonist and has pronounced effects on thermoregulation [
49,
50]. The activation of TRPV1 following capsaicin ingestion has been previously shown to lower body temperature and induce hypothermia [
17]. Hence, the reduced BT in the HS pigs fed the diet supplemented with the
Capsicum spp.-based extract may indicate a hypothalamic feedback response triggered by capsaicin, which promoted a reduction in the BT of pigs exposed to HS conditions.
The production of reactive oxygen species (ROS; e.g., superoxide anion, H
2O
2) is an inevitable result of cell metabolism, but the cellular antioxidant system under normal conditions is sufficient to prevent damaging increments in the concentration of ROS [
51]. Components of the system, including the antioxidant enzymes CAT, SOD, and GPX, are also used as oxidative stress markers [
52]. However, oxidative stress may occur if the equilibrium between antioxidants and ROS is lost in favor of the latter [
53], resulting in cell damage [
54]. In line with this, it has been reported that HS exposure increases ROS production in avian muscle cells [
8] and pigs [
41,
55]. In the present experiment, in agreement with those reports, the activity of SOD in the serum of the HS-C pigs decreased by about 24% compared to TN-C pigs. Conversely, certain phytogenic compounds, such as those in
Capsicum spp., possess antioxidant properties [
56]. The analyzed antioxidant activity of the phytogenic solution we used in the present experiment was 56% equivalent to that of vitamin E, whereas the antioxidant activity in the control diet without and with the additive was 9.2 and 18.4%, respectively, equivalent to vitamin E. The analyzed antioxidant activity in the digesta of the HS-PHY pigs was 37% higher than that in the digesta of the HS-C pigs, which confirmed that the differential antioxidant activity between the two groups of pigs was conserved once the diet was consumed. Moreover, the serum activity of both superoxide dismutase and catalase was about 25% higher in the HS-PHY than in the HS-C pigs. In fact, the activity of these enzymes did not differ among HS-PHY and TN-C pigs. A similar response was observed in HS pigs fed a diet supplemented with 20% DL-Met above the requirement [
41]. In addition to their inherent antioxidant activity, capsaicinoids may enhance the oxidative status in mammalian [
57] and avian [
58] tissues by activating intestinal TRPV1 receptors and upregulating the expression of uncoupling protein-2, which serves as an adaptive antioxidant defense mechanism [
59]. Furthermore, capsaicinoids augment antioxidant activities by activating NF-E2-related factor 2 and enhancing mitochondrial antioxidant enzyme production [
58]. These data indicate that the antioxidant activity of phytochemicals contained by
Capsicum spp., which appeared to be conserved in the digesta, contributed to improving the antioxidant status in the serum of the HS-PHY pigs.
Heat shock proteins (HSPs) protect cells against various stressors, including HS, as they participate in the proper assembly of newly synthesized proteins and protein complexes, the disassembly of protein aggregates, the degradation of misfolded proteins, and the functioning of cellular proteins [
60]. HSP90, among all members of the HSP family (HSP100, HSP90, HSP70, HSP60, HSP40, and small ones), plays a crucial role in maintaining normal cellular function under stress conditions [
47]. In the present experiment, the abundance of mRNA coding for the synthesis of HSP90 in the longissimus muscle of HS pigs fed the control diet was 58% higher than that of the TN-C pigs. These results are in agreement with other reports from our lab [
10,
61] and others [
3,
11,
62], showing consistent increments in the expression of HSP because of the exposure of pigs to HS. However, HS pigs fed the diet supplemented with the phytogenic solution had a 56% decrease in the mRNA abundance of HSP90 in muscle, compared to that in HS pigs fed the control diet. Moreover, the mRNA abundance of HSP90 was similar between the TN-C and the HS-PHY pigs. HSP expression is linked to the development of oxidative stress; increased ROS levels lead to higher HSP expression. Thus, the increased antioxidant activity, coupled with the reduced BT, may have contributed to the reduced expression levels of HSP90 in the longissimus muscle of PHY-supplemented pigs [
63].
Alterations in the concentration of several metabolites in serum occurred in HS pigs compared to TN pigs, and those changes may be linked to their HS response. For instance, carnosine, a non-enzymatic free radical scavenger and natural antioxidant [
64] decreased by 45% in HS-C pigs in comparison with TN-C pigs, reflecting the differences observed in the activities of antioxidant enzymes. Also, GABA is the preferred neurotransmitter used by warm-sensitive neurons [
44]; thus, the increased serum concentration of GABA might suggest a higher demand of those neurons for GABA to transmit the information to the hypothalamus. Citrulline and ornithine are precursors for the synthesis of polyamines, whose function consists of stimulating the proliferation of intestinal cells [
65], and increased by about 60% in the serum of HS pigs compared to TN pigs. This alteration was also associated with reduced intestinal villus height in HS pigs, suggesting a higher need for both metabolites. Arg-supplemented diets fed to HS pigs provoked a similar response [
66]. Serum urea usually results from an imbalance in serum amino acids, with higher serum values indicating excess or deficiencies of one or more indispensable amino acids [
67]. Compared to TN-C pigs, serum urea was 2-fold higher in HS-C pigs, suggesting an imbalanced availability of amino acids in HS-C pigs that may be attributed to several factors. Examples include the use of some amino acids such as arginine and methionine as antioxidants [
65] or the use of arginine as a precursor for polyamine synthesis [
65]. It can be attributed also to the reduced expression of the gene coding for the synthesis of myosin in muscle and the apparently increased amino acid oxidation, as indicated by the higher serum concentration of 3-m-His, a biomarker of muscle proteolysis [
68], which coincides with previous reports [
61]. However, serum urea in the HS-PHY pigs was 77% lower than that in HS-C pigs. Similarly, myosin expression was not significantly downregulated in the HS-PHY group. These findings could indicate a more efficient utilization of dietary amino acids by the HS-PHY compared to the HS-C pigs for lean tissue accretion.
The exposure of pigs to HS increases blood flow to the periphery (skin) in an attempt to increase body heat loss, but blood flow to internal organs (small intestine) decreases at the same time [
6]. This translates into lower nutrient and oxygen supply to those organs that, in combination with the depressed antioxidant status, might damage the intestinal epithelium. Increments in BT of up to 1.5 °C in pigs have been associated with small intestine damage, as evidenced by a significant reduction in intestinal villus height [
55,
66,
69]. In the present experiment, villus height in both the jejunum and ileum of the HS-C pigs was about 10% shorter, and intestinal temperature was 1.8 °C higher than that of the TN-C pigs. However, villus height in the jejunum of HS-PHY pigs was 16% higher than in the jejunum of HS-C pigs. Dietary intake of capsaicin has been previously shown to increase the absorptive surface area of the small intestine, especially in the jejunum of rats [
70] and broiler chickens [
71], as well as in the ileum of weaned piglets [
57]. These effects may be attributed to the antioxidant and anti-inflammatory effects of capsaicin in the intestinal tract [
59]. The jejunum is the largest intestinal segment where most of the digestion and absorption of nutrients occurs. Hence, these data indicate adding the phytogenic solution to the diet may help pigs to restore the jejunum villus height observed in the TN-C pigs.
Pigs exposed to HS reduce their voluntary feed intake by 20 to 40% compared to TN pigs [
55,
72] as part of a hypothalamic response, among others, to reduce body heat production and prevent further increments in BT [
44]. In agreement, in the present experiment, the voluntary feed intake of HS pigs fed the control diet was reduced by 40% in comparison with TN-C pigs. However, supplementing the diet with the phytogenic solution helped HS pigs to recover partially their feed intake; the HS-PHY consumed 18% more feed than the HS-C pigs. Although the apparent phytogenic-related decrease in BT of HS-PHY pigs, compared to the HS-C pigs, might explain the higher feed intake of the former pigs, it is still much below the feed intake of TN pigs. In line with the reduction in feed intake, the body weight gain substantially decreased in HS-C pigs, compared to the TN-C pigs; thus, reduced feed intake appears to be the main factor explaining the decreased weight gain. In this regard, it is worth noting that while feed intake was reduced by about 40% in the HS-C pigs compared to the TN-C pigs, body weight gain decreased by about 60%. This differential response between feed intake and weight gain suggests that the reduced weight gain of HS pigs is attributed not solely to the reduced feed intake but also to the other factors. Examples include (a) an apparent deviation of nutrients from growth to a metabolic response that helped pigs fight HS and (b) a growth-related depression of gene expression, as evidenced by the 70% reduction in the expression of the gene coding for myosin in the longissimus muscle. The substantial depression in feed efficiency of HS pigs in comparison with TN pigs seems to support both hypotheses. Similar discrepancies among performance variables were reported previously with pair-fed [
11] and ad libitum fed pigs [
39].
The weight gain of HS-PHY pigs was 24% higher than that of HS-C pigs, indicating the phytogenic solution’s beneficial effect, mostly on feed intake, as it was 18% higher in the HS-PHY pigs. However, the weight gain of the HS-PHY pigs was still about 45% lower than that of the TN-C pigs. Therefore, these data indicate that several factors other than a lower FI may explain the depressed performance of HS pigs fed diets supplemented or not with the phytogenic solution. It is important to note that the control diet was formulated to meet 100% of the requirement of the first five limiting amino acids (lysine, threonine, methionine, tryptophan, and valine) [
32]. However, because HS pigs consumed 30 to 40% less feed than the TN pigs, these amino acids might have become limiting in HS pigs. Actually, we observed an increment in the weight gain of HS pigs fed a diet containing around 25% more of these amino acids or 20% more methionine [
41], in comparison with the non-supplemented pigs. Hence, the combination of strategies aimed at counteracting the negative impact of HS on pigs, such as enriching the diet with limiting amino acids combined with the supplementation of specific phytogenic solutions, may be more effective than a single one.