Identical thermal stress coupled with different temperature and humidity combinations affects nutrient digestibility and gut metabolites of laying hens

ABSTRACT The present study investigated whether the same temperature-humidity index (THI) values under different conditions of air temperature and relative humidity (RH) would affect the thermoregulatory, nutritional, and behavioral responses of laying hens. One hundred twenty Hy-Line Brown laying hens (60-weeks-old) were divided equally in two environmental chambers: 26 °C with 70% RH (hRH75) and 30 °C with 30% RH (hT75) for 28 days. The two ambient environments (hRH75 and hT75) had an identical THI value of 75, calculated using an empirical formula for laying hens. Neither hRH75 nor hT75 affected rectal and body-surface temperatures and heart and respiratory rates. The concentration of volatile fatty acids in fecal excreta were altered by the thermal treatments. hT75 vs. hRH75 decreased the proportion of acetate and increased the proportion of propionate in fecal samples. hT75 vs. hRH75 lowered the digestibility of dry matter, crude protein, and neutral detergent fiber at 14 days. Thermal treatments did not affect heat stress-associated behavioral responses including feeding, drinking, panting, and wing elevation at any stage. Laying hens exposed to the same THI at different temperatures and RH exhibit equal physiological responses including rectal and body-surface temperatures, heart and respiratory rates, and behavioral responses. Nonetheless, high-temperature treatment (hT75; 30 °C and 30% RH) vs. low temperature treatment (hRH75; 26 °C and 70% RH) affects nutrient digestibility and gut metabolites, suggesting that there are negligible but discernable responses to temperature in the gut physiology.


Introduction
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: THI = 0.6T db + 0.4T wb (1) 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.

Ethical matters
Research on animals was conducted according to ethical guidelines provided by the Institutional Committee on Animal Use (KU19008).
One hundred twenty 60-week-old laying hens (Hy-Line Brown) were housed in two identical environmental chambers.Each chamber had one tier of 20 cages, placed 1 m above the ground.The cage dimensions were 41 × 37 × 40 cm (length × width × height), with each cage containing water nipples and a trough feeder and equipped with a heater (MCP-300; MAXCON Co., Bucheon, Korea), an air-conditioner (AR07J5174HA, SAMSUNG, Suwon, Korea), a humidifier (MH-601A; mtechwin Co., Gimhae, Korea), a dehumidifier (NED-062P; NAWOOEL Co., Gimpo, Korea), and a main controller.There were three hens per cage.Two cages were considered a replicate and each hen was provided with 506 cm 2 of floor space.Hens were initially adapted to the chambers for two weeks prior to the experiment, at an ambient temperature of 24±1.5 °C with 50±3.0%(THI of 70) RH and a 16L:8D photoperiod.At the end of the adaptation period, the ambient temperature in the first chamber was adjusted to 26±1.2 °C with 70±2.2%RH (hRH75), whereas the second chamber was adjusted to 30±1.0 °C and 30±2.5% RH (hT75) for 28 days.The air-conditioner and the heater were used to maintain the indicated temperature while the humidifier and the dehumidifier were employed to maintain the indicated relative humidity in the chambers.Temperature and humidity data were recorded at 10 min intervals during the experimental period.Both thermal treatments had a THI of 75.The THI was calculated using the formula (equation 1) proposed by Zulovich and DeShazer (1990).
Temperature and humidity loggers (MHT-381SD; Lutron Electronic Enterprise Co., Taipei, Taiwan) were placed in each chamber.Corn-soybean meal-based commercial laying hen diets were used (Table 1).Laying hens had ad libitum access to clean water and feed throughout the experimental period.On days 7, 14, 21, and 28, following thermal treatment, one hen per cage was randomly selected, and rectal temperature was measured by inserting a rectal thermometer to a depth of 3 cm into the rectum.Body-surface temperature was recorded at three different sites (i.e., head, chest, and leg) using a thermal imaging camera (Cat ® S60: equipped with a FLIR™ Lepton, FLIR Systems Inc., Wilsonville, OR, USA) as previously described by Cangar et al. (2008) and Jeong et al. (2020).
In brief, the birds were picked up and placed on the floor, and the images were taken at a distance of about 30 cm as fast as possible to reduce stress effects.The birds were handled by wearing latex gloves to avoid influences of heat and moisture of the hands on the temperature of the feathers.The heart rate (beats per minute) was determined in one chicken per replicate using a stethoscope placed on the right Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al. side of the breast, and the respiratory rate (breaths per minute) was measured by counting the number of breaths of each chosen chicken; a stopwatch was used to time 1 min for both measurements.

Fecal nutrient digestibility
During the last three days of weeks 2 and 4, the weighed quantities of feed provided and fresh fecal excretion samples per replicate were quantitatively collected to determine the apparent total tract digestibility of dry matter (DM), crude protein (CP), crude fat, neutral detergent fiber (NDF), and crude ash.Excreta from each replicate were collected four times daily, stored at −20 ℃, and pooled per replicate.The excreta samples were dried in a drying oven at 55 ℃ for 72 h and ground for chemical analysis.Feed and excreta samples were analyzed in duplicate for DM (Method 930.15;AOAC, 2005), crude fat (Method 920.39;AOAC, 2005), CP (Method 990.03;AOAC, 2005), NDF (Goering and Van Soest, 1970), and crude ash (Method 942.05;AOAC, 2005).

Fecal volatile fatty acid analysis
Fresh excreta samples freed from uric acid and cecal droppings were collected fortnightly and processed to measure the volatile fatty acids (VFA) contents on the same day of sampling.Approximately 1 g of excreta was added to 9 mL of cold distilled water and homogenized using an Ultra Turrax homogenizer (Digital Ultra-Turrax T25, IKA, Staufen, Germany), and 0.05 mL of saturated HgCl 2 , 1 mL of 25% H 3 PO 4 , and 0.2 mL of 2% pivalic acid were added to the mixture and centrifuged at 1,000 × g at 4 ℃ for 20 min.The supernatant (1.5 mL) was then collected and stored at −20 ℃ for later analysis.The concentrations of VFA in the samples were measured using gas chromatography (6890 Series GC System, HP, Palo Alto, CA, USA) as previously described by Kim et al. (2018).

Assessment of behavioral response
Behavioral observations were performed on days 7, 14, 21, and 28.High-definition video cameras (HDR-XR160, Sony Corp., Tokyo, Japan) were set up to record the behavioral responses of the hens (6 replicates per treatment).Behavioral responses were observed using 10 min video sampling from 20:00 to 22:00 h (before the lights were turned off).Behavioral responses were recorded according to a predefined ethogram (Table 2) and were presented following a previously published method (Mack et al., 2013;Kim et al., 2021a).

Sitting
Most of the ventral region of the hen's body is in contact with cage floor.No space is visible between the cage floor and the hen.

Action
Feeding Head is located inside feeder.

Drinking
Beak is in contact with a nipple drinker.

Preening
Gently pecking or scratching its own feathers.

Aggression pecking
Beak contacts another hen.

Wing position
Elevated A space can be seen between hen's wings and body.

Not elevated
There is no observable space between the hen's wings and body.

Panting
Beak is open and respiration rate is abnormally rapid.

Not panting
Beak is closed, and respiration rate is normal.
Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al. 5

Statistical analysis
Two adjacent cages were used as experimental units.The results are presented as least square means and standard deviation.All data were analyzed using the paired t-test procedure in SAS (Statistical Analysis System, version 9.4).The following mathematical model was adopted: in which Y ij = dependent variable, μ = mean of the variable, α i = effect of treatments, and ε ij = random error.The observed behavioral data collected weekly over 28 days were combined for statistical analysis.For all analyses, P<0.05 and 0.05<P<0.10 were considered as statistical significance and tendency.

Results
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.
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).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%.Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al. 7 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.

Discussion
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(Kim et al., , 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 Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al. 8 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  Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al. 9 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 Identical thermal stress coupled with different temperature and humidity combinations affects nutrient... Kim et al.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.

Conclusions
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.

Figure 1 -
Figure 1 -Effect of heat stress on behavioral responses.

Table 2 -
Behavioral ethogram of laying hens PostureStandingBoth feet are in contact with the cage floor; no other body part is in contact with cage floor.

Table 5 -
Effect of different ambient temperatures with an equal temperature-humidity index (THI = 75) on concentration of fecal volatile fatty acids 1 (VFA; % of total VFA 2 )