Stress is a biological adaptive response that restores homeostasis and alters the normal physiological state of animals. Heat stress usually occurs in animals when there is an imbalance resulting in the generation of more heat than its dissipation (Renaudeau et al., 2012). The heat stress caused by environmental factors such as temperature, relative humidity (RH), and thermal irradiation (Akbarian et al., 2016) is considered a major challenge affecting the global poultry industry. It deteriorates egg production (Ebeid et al., 2012), antioxidant parameters (Ibtisham et al., 2019; Khan et al., 2023), and nutrient digestibility (Kim et al., 2020) and alters behavioral patterns (Mack et al., 2013). Laying hens are particularly vulnerable to heat stress because of their extended long production cycle (up to 74 weeks of age), lack of sweat glands, feathered bodies, and inherently high metabolic rates (Narinç et al., 2016).

The temperature-humidity index (THI) is an indicator of thermal stress and has been used to predict the impact of the thermal environment on the thermoregulatory status of animals (Zulovich and DeShazer, 1990; Tao and Xin, 2003). The THI chart was developed using an empirical equation factoring in temperature and RH that determine the quantitative and qualitative aspects of heat stress in animals (Zulovich and DeShazer, 1990). The THI for laying chickens is defined by the equation:

in which T_db = dry-bulb temperature (℉) and T_wb = wet-bulb temperature (℉) (Zulovich and DeShazer, 1990). The THI chart has been classified into four stress zones—comfort zone (THI < 70), alert zone (THI 70–75), danger zone (THI 76–81), and emergency zone (THI > 81)—depending on the severity of heat stress imposed. This chart implies that laying hens would receive identical heat stress intensity from the environment if they were exposed to high temperatures with low humidity, or low temperatures with high humidity. There is a lack of literature on the effects of equal THI values coupled with different combinations of temperature and RH on the nutrition, physiology, and behavior of laying hens. It is hypothesized that equal THI values result in the same thermoregulatory responses, but with different effects on the other evaluated responses. Previously, we reported that high temperature deteriorates the stress indicators including performance, physiology, and behavior of laying hens (Kim et al., 2020, Kim et al., 2021a) and that thermal environments (equal THI of 75) with different combinations of temperature and humidity do not affect laying production and stress indicators including corticosterone in yolk, albumen, and plasma samples in laying hens (Kim et al., 2021b). The present study was conducted to monitor the thermoregulatory responses (i.e., nutrition, physiology, and behavior indices) of laying hens exposed to the same THI values.

Laying hens exposed to either treatments, hRH75 or hT75 (THI = 75), exhibited no differences in rectal temperature and heart and respiratory rates (Table 3). Hens in both treatments hRH75 and hT75 exhibited no changes in body-surface temperatures at any stage (Table 4), although hRH75-exposed hens had slightly higher (P = 0.060) chest temperatures than hT75-exposed hens on day 7.

Table 3

	Thermal treatment2					P-value
	hRH75			hT75
	Mean	SD		Mean	SD
Rectal temperature
Day 7	41.11	0.448		41.26	0.389	0.435
Day 14	40.92	0.204		41.05	0.227	0.195
Day 21	41.16	0.151		41.10	0.082	0.283
Day 28	41.18	0.132		41.17	0.142	0.872
Heart rate
Day 7	377.4	25.05		363.6	26.68	0.249
Day 14	302.1	18.71		312.3	25.04	0.316
Day 21	321.0	36.82		309.6	27.16	0.441
Day 28	291.0	14.76		298.2	24.17	0.432
Respiratory rate
Day 7	28.80	3.795		27.60	6.450	0.618
Day 14	25.20	3.795		29.10	10.397	0.280
Day 21	27.00	5.099		28.80	4.733	0.424
Day 28	24.60	3.406		25.20	3.795	0.714

SD - standard deviation.

Table 4

	Thermal treatment2					P-value
	hRH75			hT75
	Mean	SD		Mean	SD
Day 7
Head	30.50	2.071		30.60	1.883	0.915
Chest	26.90	2.421		25.00	1.710	0.060
Leg	26.70	2.539		26.80	2.364	0.926
Day 14
Head	31.80	0.595		31.90	0.934	0.806
Chest	25.92	1.542		26.97	1.141	0.101
Leg	28.81	1.665		29.41	2.140	0.493
Day 21
Head	32.03	1.266		32.27	1.506	0.704
Chest	28.34	1.234		27.74	2.704	0.531
Leg	27.59	2.323		26.69	2.979	0.461
Day 28
Head	31.70	1.284		32.20	1.503	0.434
Chest	25.68	1.130		25.74	1.546	0.922
Leg	27.57	2.512		27.76	1.981	0.853

SD - standard deviation.

Acetate was proportionally the most dominant VFA on days 14 and 28 (Table 5). Interestingly, total VFA ranged from 36.3 to 45.9 mmol/g feces on day 14, and 78.9 to 91.0 mmol/g feces on day 28 (data not shown). The proportion of acetate in fecal samples was higher in hRH75-exposed hens than in hT75-exposed hens (P = 0.061). In contrast, treatments hRH75 vs. hT75 lowered the proportion of propionate in fresh fecal samples (P = 0.046).

Table 5

	Thermal treatment3					P-value
	hRH75			hT75
	Mean	SD		Mean	SD
Day 14
Acetate	83.30	9.068		75.07	4.35	0.061
Propionate	10.09	4.339		14.55	2.47	0.046
Butyrate	9.071	3.598		10.37	2.73	0.490
Day 28
Acetate	88.14	3.785		89.16	3.703	0.577
Propionate	5.548	2.543		5.300	2.130	0.829
Butyrate	6.311	2.075		5.544	1.796	0.421

SD - standard deviation.

Treatments hT75 vs. hRH75 lowered the digestibility of DM (P = 0.023), CP (P = 0.010), and NDF (P = 0.052) (Table 6). However, the digestibility of crude fat and crude ash were not affected by the thermal treatments by day 14. At 28 days, none of the thermal treatments affected nutrient digestibility, although treatments hT75 vs. hRH75 tended to decrease the digestibility of CP (P = 0.114) and crude ash (P = 0.072) by approximately 10.5%.

Table 6

	Thermal treatment2					P-value
	hRH75			hT75
	Mean	SD		Mean	SD
Day 14
Dry matter (%)	79.54	3.87		74.96	4.33	0.023
Crude protein (%)	77.71	6.40		63.79	13.76	0.010
Crude fat (%)	92.99	1.57		92.03	4.00	0.489
Neutral detergent fiber (%)	56.39	8.27		47.64	10.42	0.052
Ash (%)	50.06	7.79		43.97	12.90	0.217
Day 28
Dry matter (%)	76.38	2.25		73.09	8.31	0.242
Crude protein (%)	72.39	7.81		65.14	11.39	0.114
Crude fat (%)	93.62	1.26		93.36	2.83	0.791
Neutral detergent fiber (%)	56.01	2.08		48.26	17.17	0.173
Ash (%)	75.95	4.30		67.91	11.88	0.072

SD - standard deviation.

As an indicator of heat stress for laying hens (Kim et al., 2021a), behavioral patterns were measured using video recordings on days 7, 14, 21, and 28 (Figure 1A-1H). None of the thermal treatments affected the behavioral patterns (data not shown). Thus, the behavioral patterns obtained at the four time points were pooled per replicate to simplify presentation. When all the observations monitored during the experimental period were pooled, three behavioral patterns (i.e., drinking, feeding, and preening) tended to be altered by thermal treatments—hT75 vs. hRH75 increased drinking and preening and decreased feeding.

Figure 1

RH - relative humidity.

hRH75: temperature = 26 ℃ and RH = 70%; hT75: temperature = 30 ℃ and RH = 30%.

Values are least squares means.

Dots represent the 24 pooled observations per treatment.

In a previous study, we observed that exposure to high temperatures lowered egg production, impaired gut functions, increased stress indicators including an increase of corticosterone in plasma and eggs, heterophil to lymphocyte ratio, altered heat-stress associated behaviors, and elevated physiological responses such as body temperature and respiratory rate (Kim et al., 2020, 2021a). In a subsequent study (Kim et al., 2021b), we reported that laying hens raised in the equal thermal environment (THI = 75) with different combinations of temperature and RH (30 °C and 30% RH; 26 °C and 70% RH) exhibited no differences in egg production and stress physiology (i.e., blood heterophil to lymphocyte ratio and corticosterone levels in plasma, and egg yolk and albumen). Here, we explored the effects of equal thermal environments (THI = 75) combined with different temperatures and RH on cardiac and digestive physiology and behavioral patterns of laying hens. As previously observed, the equal thermal environment (THI = 75) coupled with different combinations of temperature and RH (i.e., hRH75 or hT75) did not affect the rectal and body temperature, cardiac physiology including heart and respiratory rate, and behavioral patterns. Thus, our results confirmed that the THI chart can predict the thermal stress of laying hens (Zulovich and DeShazer, 1990; Purswell et al., 2012). However, we found that equal thermal treatments affected the indicators of gut physiology (i.e., gut metabolites and nutrient digestibility). In both thermal treatments (hT75 vs. hRT75), acetate levels decreased, but propionate levels increased in fresh fecal samples at day 14. Additionally, in both chambers, the digestibility of nutrients in the hens decreased by day 14 of heat treatment. Taken together, our study provides evidence that equal thermal treatments (THI = 75) with different combinations of temperature and RH had identical effects on the behavior and cardiac physiology, but exhibited an unequal effect on the gut physiology of laying hens.

In the chambers, we regulated temperature (26 ℃ vs. 30 ℃) and RH (70% vs. 30%) to create thermal alert zone (THI = 75) using equation 1 generated for laying hens (Zulovich and DeShazer, 1990). It could be argued that the lack of cardiac and behavioral responses in this study might be attributed to the difference in environmental temperature being only 4 ℃ (THI = 75; from 26 vs. 30 ℃). Yahav (2000) reported that temperature, not RH, is the dominant driving factor of heat stress affecting the laying performance and physiology of laying hens. However, Cai et al. (2020) and Kim et al. (2020) found that air temperature differing by 5 or 6 ℃ caused a reduction in laying production and nutrient digestibility, increase in body-surface temperature, and alterations in behavioral patterns of laying hens. Thus, if temperature is considered to be the single determinant of heat stress for laying hens, the parameters for cardiac and digestive functions, and behavioral patterns could have been detected. Therefore, the lack of physiological and behavioral responses is not because of the temperature between the hT75 and hRT75 treatments, but because both thermal treatments were maintained at the same THI value. In future experiments, a clear explanation could be given if two environmental chambers with conditions of 24 ℃ with 95% RH and 32 ℃ with 15% RH, respectively (THI = 75), could be constructed, but this is difficult to achieve in practical experimental settings.

It is well documented that high environmental temperatures induce a cascade of physiological and metabolic changes (i.e., elevated rectal and body-surface temperatures) in laying hens (Mashaly et al., 2004; Rozenboim et al., 2007; Xie et al., 2015; Chang et al., 2018; Barrett et el., 2019; Xing et al., 2019; Kim et al., 2021a). Additionally, the heart rate of chickens elevates to increase blood flow to the capillary vessels to dissipate the body-generated sensible heat (Yahav, 2009). Finally, increasing their respiratory rate is the main mechanism used by poultry to dissipate latent heat (Sahin et al., 2009). All cardiac functions described as the indicators of heat stress were not altered between the two thermal treatments in this study; this supports our view that the THI values can be accepted as reliable indicators of heat stress in laying hens.

Extended heat exposure is a major environmental stressor that has a negative effect on nutrient digestibility (He et al., 2018). We observed that the digestibility of DM, CP, and NDF were significantly impaired in laying hens exposed to treatments hT75 vs. hRH75, and this effect was noted at 14 days, but not at 28 days. It is not yet clear whether the observed reduction in nutrient digestibility by hT75 vs. hRH75 can be attributed to hT75-induced inhibition in either the digestion or absorption processes, or both. Khan et al. (2012) reported that heat stress could inhibit the activity of digestive enzymes. In addition, it has been reported that heat stress inhibits the absorption of nutrients in the small intestine (Bonnet et al., 1997; Zhang et al., 2017). Thus, it is likely that digestion and nutrient absorption processes in the gastrointestinal tract may be more affected by treatment hT75 than by hRH75, although both had an equal THI value.

It can be postulated that impaired CP and NDF digestibility by hT75 vs. hRH75 noted in this study could have increased the substrate for gut microbiota and led to the altered VFA concentrations. As previously observed by Kim et al. (2020), heat stress lowered the digestibility of NDF but increased the VFA concentration in fecal droppings of laying hens. Similarly, Wang et al. (2019) observed elevated concentration of acetate, propionate, and butyrate in heat stressed laying hens exposed to an environmental temperature of 38 ℃ compared with those exposed to 28 ℃. In this study, we observed that the equal thermal treatments affected the proportion of acetate and propionate, which indicated a close link between the thermal treatments and gut physiology. In contrast, hT75 vs. hRH75 did not increase the percentage of VFA equally. Therefore, the hT75-mediated increase in substrates for bacterial fermentation cannot be fully responsible for the observed VFA percentage. However, a clear explanation for this was not obtained and needs to be addressed. Further studies are necessary to investigate the concentration of digesta in the ileum or ceca and gut microbiome profiles, which can help clarify the observed effects on gut physiology. Our study tentatively suggests that the equal thermal environments (THI = 75) combined with different temperature and RH had an impact on gut microbiome and metabolites in laying hens and manifested predominantly as changes in fecal VFA concentration.

Many researchers have suggested that walking and standing behavioral responses and feed intake are reduced during heat stress conditions, while behavioral responses such as sitting, drinking, wing elevation, and panting are increased to dissipate excess heat (Gowe and Fairfull, 2008; Lara and Rostagno, 2013; Mack et al., 2013; Sohail et al., 2013; Rostagno, 2020). In this study, no differences in behavioral responses were observed between the thermal treatments, confirming that equal THI had no effect on heat stress-associated behaviors. Interestingly, when each recorded behavioral pattern was pooled, hT75 vs. hRH75 increased drinking but decreased feeding behavior, indicating that temperature might affect the behavior of chickens in a discernible manner under chronic heat stress experimental settings.

The results of this study support the conclusion that the equal thermal environments (THI = 75), created with different temperature-RH combinations, do not affect behaviors and cardiac physiology, but altered the gut physiology of laying hens.