TYPE Original Research PUBLISHED 18 July 2023 DOI 10.3389/fvets.2023.1156480 Changes in cortisol and cortisone OPEN ACCESS in hair of pigs reared under heat stress conditions EDITED BY Naceur M'Hamdi, National Agricultural Institute of Tunisia, Tunisia REVIEWED BY Aitor Arrazola, Damián Escribano 1,2, Alexandra Contreras-Jodar 3, Purdue University, United States Marina López-Arjona 4, José Joaquín Cerón 1, Emma Fàbrega 3, Juan Pablo Damián, Universidad de la República, Uruguay Pau Aymerich 5 and Antoni Dalmau 3* *CORRESPONDENCE 1 Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Antoni Dalmau Excellence Campus Mare Nostrum, University of Murcia, Murcia, Spain, 2 Department of Animal antoni.dalmau@irta.es Production, Veterinary School, Regional Campus of International Excellence Campus Mare Nostrum, RECEIVED 01 February 2023 University of Murcia, Murcia, Spain, 3 Animal Welfare Program, IRTA, Monells, Spain, 4 Department of ACCEPTED 23 June 2023 Animal and Food Science, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 Vall Companys Group, PUBLISHED 18 July 2023 Lleida, Spain CITATION Escribano D, Contreras-Jodar A, Heat stress accounts for millions of dollars in losses for swine producers López-Arjona M, Cerón JJ, Fàbrega E, Aymerich P and Dalmau A (2023) Changes in worldwide. The aim of the present study was to determine and evaluate cortisol cortisol and cortisone in hair of pigs reared and cortisone in hair as indicators of thermal stress in growing pigs reared under under heat stress conditions. high environmental temperatures. The study was carried out in two independent Front. Vet. Sci. 10:1156480. doi: 10.3389/fvets.2023.1156480 batches of commercial crosses of Lean Duroc and Pietrain in trials 1 and 2, COPYRIGHT respectively, during the growing period (from 40 to 100 kg; 81 days in trial 1 and © 2023 Escribano, Contreras-Jodar, López- 77 days in trial 2) in the same commercial farm in Spain during the summers Arjona, Cerón, Fàbrega, Aymerich and Dalmau. of 2020 and 2021. In both cases, four rooms were used. In Trial 1, Room 1 had This is an open-access article distributed under the terms of the Creative Commons Attribution cooling and 11 pigs per pen; Room 2 had no cooling and 13 pigs per pen; Room License (CC BY). The use, distribution or 3 had no cooling and 11 pigs per pen, and Room 4 had cooling and 13 pigs per reproduction in other forums is permitted, pen. In Trial 2, Rooms 2 and 3 had cooling and rooms 1 and 4 had no cooling, and provided the original author(s) and the copyright owner(s) are credited and that the all of them had 13 pigs per pen. Mean THI value was higher (p<0.0001) in rooms original publication in this journal is cited, in without cooling systems (75.0 trial 1; 74.9 trial 2) than with them (71.3 trial 1; 71.7 accordance with accepted academic practice. trial 2). A total of four pens per room (16 in total) was selected for analysis of hair No use, distribution or reproduction is permitted which does not comply with these corticoids and all pigs inside were sampled at the end of the study. Fifty percent terms. of the pigs were males (castrated and intact in trial 1 and 2, respectively) and 50% females. In total, 44, 52, 44, and 52 pigs, respectively, were sampled in four rooms from the first trial and 52 for each of four rooms in Trial 2. Cortisol concentrations in hair did not show any significant change in relation to cooling-non-cooling in any trial. However, hair cortisone concentration was 172.3pg./mg and 105.8pg./ mg less (p<0.001) in pigs housed with cooling systems compared to those without them in Trial 1 and 2, respectively. In addition, the cortisone/cortisol ratio, which is an estimator of the activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2, was also greater in rooms without cooling than in rooms with cooling in both trials (p<0.0001 and p=0.0105 for Trials 1 and 2, respectively). In relation to the sex effect, the results showed greater levels in females than in castrated males both in cortisone and the cortisol/cortisone ratio while cortisol hair levels were greater in intact males than in females. Therefore, the use of cortisone and the estimation of 11β-HSD type 2 activity in hair is recommended to evaluate the chronic stress produced by high environmental conditions in pigs instead of using hair cortisol concentrations alone. KEYWORDS animal welfare, corticosteroids, farm conditions, high temperatures, swine Frontiers in Veterinary Science 01 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 1. Introduction The aim of the present study is to determine and evaluate cortisone and cortisol as possible indicators of stress in the hair of Heat stress accounts for millions of dollars in losses for swine growing pigs reared at high environmental temperature conditions producers worldwide (1), and this problem is commonly due to in a commercial Spanish farm during two consecutive high environmental temperatures, which usually persist for several summers. months along the year in warm countries. In addition, climate change is causing global warming, with a higher frequency and duration of heat waves in warm countries and a rise in maximum 2. Methods temperatures and longer summers in colder ones. This global effect of the temperatures combined with the fact that pigs are used to 2.1. Management and husbandry of pigs being reared in intensive conditions with big populations of animals housed in high densities cause the risk of being subjected The study was carried out in the same commercial farm of pigs, to heat stress to increase in pig production nowadays. On the other located in Alcarràs, Lleida, Spain, during two consecutive trials, in hand, in addition to ambient factors, it is important to take into summer 2020 and summer 2021. The experiment was approved by the account that pigs are much more sensitive to heat than are other Institutional Animal Care and Use Committee (IACUC) of IRTA, species due to their physiological limitations. In fact, pigs cannot under Code 8348. The farm has two independent buildings, and each sweat and have little capacity for panting, in comparison to species one has two independent rooms with a capacity of 728 pigs each like dogs, due to smaller lungs, a lesser degree of openness of the (2,912 pigs in total). Each room consisted of a total of 56 pens allocated mouth and not being able to get the tongue out of the mouth, and in four lines of 14 pens each. Between two lines of pens there was a they have a great accumulation of subcutaneous fat (2). Therefore, corridor. Pens on the left side of the corridor allocated only castrated if they do not have a pond or bathing area (3), they have great males (trial 1) or intact males (trial 2), and the right side allocated only limitations for cooling themselves. Furthermore, the use of an air females. Sexes were not mixed in the farm of the present study. Males conditioner is economically unviable for swine farms (2); from trial 1 were castrated in another commercial farm for sows and consequently, in the mid-latitudes, pigs are kept predominantly in piglets following routine processes. This means that pigs were confined livestock buildings with a mechanical ventilation system castrated at 3 days old without anesthesia or analgesia. The two first (4) using evaporation pads. All of these facts make the validation pens of each room were empty and used as hospital pens. The pigs of essential of proper biomarkers of stress to be used in both trials were a commercial cross of Duroc these conditions. Danbred x (Landrace x Largewhite) and reared in the farm. Pigs Blood cortisol is one of the main and most widely used biomarkers entered the farm 3 months old weighing 40 kg and they were reared in pigs (5). Nevertheless, due to the need for handling the animals and until they reached 100 kg as a mean value in the first room at 6 months the related stress response that acts as a confounding factor, in pig old. Once the first room achieved this weight, all rooms were sampled welfare studies (6) it is usually substituted by other non-invasive and the study finished. This occurred from July to September 2020 in samples, such as saliva (7), feces (8), or hair (9). The use of hair Trial 1 and from June to August 2021 in Trial 2. samples for cortisol analysis has advantages, such as: simple handling, particular shipping and storage logistics is not needed, and it offers the possibility of a retrospective analysis of endogenous cortisol exposure 2.2. Experimental design (10, 11), which can be used to assess a chronic state of physiological stress. Cortisol is inactivated in hair by the action of 11β-hydroxysteroid In Trial 1, two management strategies to reduce the consequences dehydrogenase (11β-HSD) type 2 enzyme, converting it to cortisone of heat stress in summer were tested: to provide a cooling system in whereas that 11β-HSD type 1 enzyme is involved in the reduction of Rooms 1 and 4, and to reduce the densities from 13 pigs per pen the 11-keto group to alcohol, converting cortisone to cortisol and both (0.68 m2/pig) to 11 pigs per pen (0.80 m2/pig) in Rooms 1 and 3. isoforms are expressed in skin and hair (12). It has been concluded Consequently, pigs in each room were subjected to a specific that the determination of cortisone in hair in species like sheep could treatment: Room 1 (cooling, low density), Room 2 (no cooling, high be a promising alternative to assess animal welfare in chronic stress density), Room 3 (no cooling, low density) and Room 4 (cooling, high studies (13). However, to the authors’ knowledge, few studies have density). Trial 1 lasted 81 days. In Trial 2, only the use of cooling been conducted cortisone analysis in pig hair as a biomarker of systems was maintained to reduce thermal stress, in this case used in chronic stress in this species. Rooms 2 and 3, while Rooms 1 and 4 had no cooling. In Trial 2, all In order to correctly assess cortisone concentrations, it is pens were maintained at the same density of 13 pigs per pen (0.68 m2/ necessary to use a sensitive assay that is able to differentiate between pig) Trial 2 lasted 77 days. cortisol and cortisone, avoiding cross-reactivity, to prevent erroneous results (14). In a recent study, two sensitive assays were developed and validated for the measurement of cortisol and 2.3. Cooling strategies cortisone, which also enabled the estimation of 11β-HSD type 2 activity, in the hair of sows during different phases of the The cooling system consisted in a mechanical evaporative cooling. reproductive cycle (15). In López-Arjona et al. (15), an increase in This means that the unit used a fan to draw air through a wetted cortisone, possibly due to an increase in activity 11β-HSD type 2, membrane, which provided a large surface area for the evaporation of was found in periods of high temperatures. water. Evaporative cooling exploits the fact that water will absorb a Frontiers in Veterinary Science 02 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 relatively large amount of heat in order to evaporate. The temperature (Precellys Evolution homogeniser, Bertin Technologies; France). of dry air can be dropped significantly through the phase transition of The pulverized hair was incubated with 1 ml of methanol for 18 h liquid water to water vapor (evaporation). For a better assessment of at room temperature with continuous gentle agitation for steroid thermal conditions in pigs, instead of assessing just temperatures or extraction. Samples were then centrifuged 2000 g × 5 min and humidity, the combination of both in an index (THI) was considered. 0.6 mL of each methanol extract was aliquoted into a new The temperature was taken with a probe named “sonda temperature” Eppendorf tube. The samples were evaporated to dryness in a (Exafan, Zaragoza, Spain) and humidity with a probe named “sonda Speed Vac Concentrator (Concentrator 5301, Eppendorf). The dry humedad” (Exafan, Zaragoza, Spain). The THI was calculated extracts were reconstituted with 0.1 ml of phosphate buffer saline according to The National Weather Service Central Region and and stored at −80°C until analysis. Wegner et al. (16, 17) as follows: THI = [(1.8 T) + 32] –[0.55 Cortisol and cortisone were measured in all samples with two (RH/100)] × [((1.8 T) + 32) – 58], where T is the air temperature in °C ® sensitive assays based on AlphaLISA technology (PerkinElmer) that and RH the relative humidity in %, Figures 1, 2. were developed and validated previously for their use in pig hair (15) and the results expressed as pg./mg of hair. In addition, activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 was estimated 2.4. Sampling for hair and corticosteroid by cortisone/cortisol ratio. In the reported validation (15), the assays analysis showed coefficients of variation <12% and the limits of blank were of 1.42 and 0.06 ng/ml and limits of quantification were 6.96 and 2.80 pg./ A total of four pens per room (16 in total per trial) were randomly ml for cortisol and cortisone, respectively. selected and all pigs inside were sampled. Fifty percent of the pigs were males and 50% females. In total, 44, 52, 44, and 52 were sampled in Rooms 1; 2; 3; and 4, respectively in Trial 1, and 52 for each room in 2.5. Statistical analysis Trial 2. An area of around 10 cm × 10 cm located in the pig’s dorsal rump (10 cm from the tail) was selected at the end of the study for Analyses were carried out with the Statistical Analysis System each pig, when pigs weighed 100 kg. Hair was collected by shaving (SAS software, SAS Institute Inc.; Cary, NC, USA). Normality of close to the skin with clippers, trying not to remove the root of the residuals was checked through the Shapiro–Wilk test and QQ plots hair, and avoiding inclusion of the hair follicle in the sample. Once of residuals for each one of the dependent variables studied. The sampled, hair was stored at −21°C inside hermetically sealed bags three variables studied, cortisol, cortisone and the cortisone/ until analysis. cortisol ratio met the normality assumption, so linear models by Cortisol and cortisone extraction from hair was performed as means of Proc Mixed were used. In Trial 1, the variables considered reported before by Daventport et al. (18). The hair samples were were cooling (yes or no), sex (males or female), density (11 pigs per weighed (250 mg), placed in a polypropylene tube and covered pen or 13 pigs per pen) and all the interactions between factors. In with 5 ml of isopropanol. The tube was mixed at room the case of Trial 2, the variables considered were cooling (yes or temperature, centrifuged 1,500 g × 1 min and the isopropanol no), sex (males or females) and the interaction between cooling and discarded. The samples were washed again with isopropanol and sex. In all cases, the pen of origin of the pigs was considered as a left at room temperature until completely dry. Next, the hair random effect. When significant differences were found, the least- samples were cut into small pieces (around 5 mm) with scissors square means of fixed effects (LSMEANS) adjusted to Tukey was and 60 mg from each sample was weighed. The hair was placed in used for multiple comparisons. In all cases, significance was fixed tubes with balls and pulverized to a fine powder in a homogenizer at p < 0.05. FIGURE 1 Mean daily THI values in rooms with and without cooling in Trial 1 (Room 1: cooling and 11 pigs per pen; Room 2: no cooling and 13 pigs per pen; Room 3: no cooling and 11 pigs per pen; and Room 4: cooling and 13 pigs per pen). Mean THI value was higher (p<0.0001) in rooms without cooling systems (75.0) than with them (71.3). Frontiers in Veterinary Science 03 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 FIGURE 2 Mean daily THI values in rooms with (Room 2 and Room 3) and without cooling (Room 1 and Room 4) in Trial 2. Mean THI value was higher (p<0.0001) in rooms without cooling systems (74.9) than with them (71.7). FIGURE 3 Hair cortisone concentration pg./mg of pigs during Trial 1 in relation to treatment (cooling vs. no cooling), sex (male vs. female) and density (11 or 13 pigs per pen). Different letters mean significant differences at p<0.05; NS means not significant differences. 3. Results cooling and density (p = 0.0546; F = 3.74; DF = 1/176) was found (Figure 6). For the cortisone/cortisol ratio, a cooling effect In Trial 1, for cortisone, a cooling effect (p = 0.0005; F = 12.4; (p < 0.0001; F = 17.19; DF = 1/176) and a sex effect (p = 0.0112; DF = 1/176) and a sex effect (p = 0.0094; F = 6.89; DF = 1/176) were F = 6.57; DF = 1/176) were found. In these cases, the cortisone/ found, in addition to a trend for the interaction between cooling and cortisol concentration ratio was greater in pigs reared without a density (p = 0.0905; F = 2.90; DF = 1/176). In fact, the concentration cooling system (10.1 ± 0.60) compared to those reared with of cortisone in hair was greater in the pigs housed without cooling (6.6 ± 0.60) and, also, greater in females (9.4 ± 0.60) than in castrated (hair cortisone concentration was 492.3 ± 34.46 pg./mg) than in pigs males (7.3 ± 0.60; Figure 7). housed with cooling (hair cortisone concentration was In Trial 2, for cortisone a cooling effect was found (p = 0.0003; 320 ± 34.46 pg./mg; Figure 3). In addition, this value was greater as F = 13.85; DF = 1/183), with greater values in pigs within the rooms well in females (hair cortisone concentration was 470 ± 34.42 pg./ without cooling (hair cortisone concentration was 604.6 ± 22.68 pg./ mg) than in castrated males (hair cortisone concentration was mg) than in rooms with cooling (hair cortisone concentration was 342.7 ± 34.42 pg./mg; Figure 3). Finally, the trend shows how Room 498.8 ± 22.28 pg./mg; Figure 8). For cortisol, only a sex effect was 3, the one reaching the highest mean THI (Figure 1) reached, as found (p = 0.0101; F = 6.76; DF = 1/183); with intact males having well, the maximum mean value for cortisone in hair (Figure 4, greater values of cortisol in hair (hair cortisol concentration was p = 0.0905). In Trial 1, for cortisol, no effect of treatment (cooling vs. 439.2 ± 37.94 pg./mg) than with females (hair cortisol concentration no cooling), sex (males vs. female) or density (13 or 11 pigs per pen) was 382.1 ± 37.65 pg./mg; Figure 9). Finally, for the cortisone/cortisol was found, Figure 5, and just a trend for the interaction between ratio, a cooling effect was found (p = 0.0105; F = 6.68; DF = 1/183), Frontiers in Veterinary Science 04 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 FIGURE 4 Hair cortisone concentration pg./mg of pigs of Trial 1 housed in the different rooms (p=0.0904). Different letters mean significant differences at p=0.09. FIGURE 5 Hair cortisol concentration pg./mg of pigs during Trial 1 in relation to treatment (cooling vs. no cooling), sex (male vs. female) and density (11 or 13 pigs per pen). NS means not significant differences. where pigs reared in rooms without a cooling system (1.76 ± 0.109) in growing and finishing pigs, the thermoneutral zone varies had a greater ratio than those reared in rooms with it (1.39 ± 0.107) between dry air temperatures of 15–28°C in a humidity range (Figure 10). between 40 and 80%. In the present study the daily mean temperature reached 29, 30, 31, and 32°C (THI from 76 to 82) in rooms without cooling, while these values were not reached in 4. Discussion rooms with cooling. In both trials of the experiment, cortisone showed greater In both trials the combination of temperature and humidity was concentrations in the rooms without cooling than in rooms with significantly higher in rooms without cooling than in rooms with cooling. In the first trial, the pigs without cooling had a hair cortisone cooling, reaching a mean THI value of 75 in comparison to the concentration of 172.3 pg./mg greater than those with cooling, and in 71.3 in rooms with cooling, which confirmed that pigs reared the second trial this difference was of 105.8 pg./mg. These differences without a cooling system were in risk to be subjected to higher heat are in line with the results shown by López-Arjona et al. (15), where, stress (17, 19). In fact, Wegner et al. (19) defined a threshold of 74 in sows that were at 5 days before farrowing during the warm season, as a starting point based on heat stress for sows housed in closed the reported values of cortisone were 224.4 pg. per mg/hair greater insulated and ventilated barns in Germany. According to EFSA (20), than in sows at 5 days before farrowing during the colder months. Frontiers in Veterinary Science 05 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 FIGURE 6 Hair cortisol concentration pg./mg of pigs of Trial 1 housed in the different rooms (p=0.0546). Different letters mean significant differences at p=0.0546. FIGURE 7 Cortisone/cortisol ratio in Trial 1 in relation to treatment (cooling vs. no cooling), sex (female vs. male) and density (13 vs. 11 pigs per pen). Different letters mean significant differences at p<0.05; NS means not significant differences. Cortisol concentration in hair did not show any significant According to the results of López-Arjona et al. (15), the results of difference in relation to cooling-non-cooling treatments for either of the present study showed that with higher atmospheric temperature both trials. The fact that hair cortisol does not increase under heat there is a greater change in hair cortisone concentrations and in the stress conditions is in accordance with the results of López-Arjona cortisone/cortisol ratio. A possible explanation of these results could et al. (15) in sows subjected to high temperatures. On the other hand, be that at higher temperature the 11β-HSD type 2 presents a higher some authors consider that cortisol in hair is not really a good activity of action. In a kinetic study of 11β-HSD in rat submandibular biomarker of stress. For example, Wiechers et al. (21) showed a lack salivary gland it was found that the NAD-dependent enzyme, the of significant differences in cortisol in hair, when comparing farrowing 11β-HSD type 2, increased its activity as it was subjected to more systems with different situations of chronic stress, and the authors temperature, increasing exponentially up to 40°C temperature, while concluded that measuring hair cortisol might not be the most the activity of the enzyme NADPH-dependent, the 11β-HSD type 1, appropriate method for assessing stress in pigs. In other species, such showed a slight increase (22). Although more studies are needed to as sheep, it was also found that hair cortisone levels were greater than clarify how both isoenzymes act in the case of hair tissue of pig, it was hair cortisol in a model of chronic stress (13). could be postulated that this fact could also explain why no significant Frontiers in Veterinary Science 06 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 FIGURE 8 Hair cortisone concentration pg./mg of pigs during Trial 2 in relation to treatment (cooling vs. no cooling) and sex. Different letters mean significant differences at p<0.05; NS means not significant differences. FIGURE 9 Hair cortisol concentration pg./mg of pigs during Trial 2 in relation to treatment (cooling vs. no cooling) and sex (male vs. female). Different letters mean significant differences at p<0.05; NS means not significant differences. decreases were observed in cortisol, since an increase in the magnitude Overall, the results of the present study would indicate that it is of action of 11β-HSD type 2 does not mean an inactivation of preferable to use cortisone and the cortisone/cortisol ratio (an 11β-HSD type 1, cortisol production continues although in a reduced estimate of 11β-HSD type 2 activity) as indicators of chronic heat amount compared to the production of cortisone induced by 11β-HSD stress in pigs. type 2 (23). In this line, the results of the cortisone/cortisol ratio In relation to the sex effect, the results showed greater levels in indicates a lower activity of this enzyme in the rooms with cooling. In females than in males both in cortisone and the cortisol/cortisone the first trial, the pigs with cooling had a 3.5 pg. cortisone/cortisol ratio, but only in the first trial, where castrated males were used. These ratio per mg/hair less than those without cooling, and in the second results were not repeated in the second trial, where intact males were trial this difference between cooling-non cooling was of 0.4 pg. used and where a sex effect was found in cortisol hair levels, with cortisone/cortisol ratio per mg/hair. These results are also in line with greater levels in male than in female. To the authors’ knowledge, there those shown by López-Arjona et al. (15), where sows assessed during are no studies on the influence of sex in cortisone or cortisol/cortisone the warm season had a greater cortisone/cortisol ratio (7.97 pg), when ratio levels in hair between male and female pigs. However, the compared to sows assessed during the colder months (0.19 pg). influence of sex in cortisol hair levels, although being inconsistent, Frontiers in Veterinary Science 07 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 FIGURE 10 Cortisone/cortisol ratio in Trial 2 in relation to treatment (cooling vs. no cooling) and sex. Different letters mean significant differences at p<0.05; NS means not significant differences. may depend upon numerous factors such as different behavioral significant changes in hair cortisol concentration, plausible due to patterns, body condition and metabolism of gonadal steroids (24). In increased enzymatic activity of 11β-hydroxysteroid dehydrogenase pigs, Trevisan et al. (25) showed that lean sows with poor body type 2 under heat stress. These results suggest that measuring hair condition, had higher hair cortisol concentration compared with cortisone concentration may be a more sensitive biomarker of stress normal-weight sow. In other species such as cats, studies found higher than hair cortisol concentration under stressfully high hair cortisol concentration in aggressive animals, showing a significant environmental temperatures. positive correlation with the agonistic behavioral patterns (26) or lower concentration in castrated female than intact cats (26). In addition, the own sex-specific effects on regulation of HPA axis and Data availability statement the metabolism of gonadal steroids, appear to create different patterns of hair cortisol in male and female of nonhuman primates (27). The raw data supporting the conclusions of this article will Overall, more studies are needed to clarify how all those factors be made available by the authors, without undue reservation. influence on hair cortisol concentration in porcine species, as in this study, intact males had higher levels of cortisol than females. The results of the density effect in Trial 1 should be interpreted Ethics statement with caution, since in our report pigs at a higher density were thermally better (Figure 1) than were pigs at a lower density, and therefore The animal study was reviewed and approved by The Institutional thermal effects could influence the results. Therefore, although the Animal Care and Use Committee (IACUC) of IRTA, under Code animals in lower densities gained 3 kg more at the end of the growing 8348. Written informed consent was obtained from the owners for the period than animals in higher densities (data not shown), these participation of their animals in this study. differences could have been higher if the temperature in the rooms with high densities had not been cooler than in the rooms with lower densities. On the other hand, the combination of both cooling systems Author contributions and lower densities resulted in a gain of 9 kg (data not shown). In addition, it is again a good example to consider in cases where specific DE, AC-J, JC, PA, and AD: conceptualization. DE and AD: stress factors (in this case a higher density) are assessed in conditions writing—original draft preparation. AC-J, ML-A, JC, EF, and PA: of high temperatures, as the use of hair cortisone and cortisol could writing—review and editing. JC and AD: supervision. EF, AD, and PA: result in better biomarkers than the use of cortisol alone, that showed project administration. AD and PA: funding acquisition. All authors just a trend for the combination of cooling systems and lower densities. have read and agreed to the published version of the manuscript. 5. Conclusion Funding Overall, it could be concluded that pigs reared at high This study was included in Vall Companys, S.A.U.’s project environmental temperatures show elevated hair cortisone without WELFARE+ 8IDI-20210216 co-funded by Centre for the Frontiers in Veterinary Science 08 frontiersin.org Escribano et al. 10.3389/fvets.2023.1156480 Development of Industrial Technology (CDTI), a Public Business Conflict of interest Entity, answering to the Ministry of Science and Innovation, and the European Regional Development Fund (ERDF). DE was funded by PA was employed by the company Vall Companys Group. the postdoctoral contract “Generational renewal to promote research” The remaining authors declare that the research was conducted in of the University of Murcia. ML-A (FJC2021-047105-I) by means of a the absence of any commercial or financial relationships that could post-doctoral fellowship, “Juan de la Cierva Formación,” supported by be construed as a potential conflict of interest. the “Ministerio de Ciencia e Innovación.” Publisher’s note Acknowledgments All claims expressed in this article are solely those of the authors The technicians of IRTA and Vallcompanys that participated in and do not necessarily represent those of their affiliated organizations, the sampling process are acknowledged, including Xenia Moles, Aida or those of the publisher, the editors and the reviewers. Xercavins, Aranzazu Varvaró, Anna Moisès, Berta Baulida, Dayane Any product that may be evaluated in this article, or claim that may Teixeria, Ramon Millera, Josep Maria Rebilleda, Mario Durán and be made by its manufacturer, is not guaranteed or endorsed by the Carme Soldevilla. publisher. References 1. St-Pierre NR, Cobanov B, Schnitkey G. 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