Relationship between hair cortisol and plasma heat shock proteins in response to heat stress in resilient and sensitive cattle breeds

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Capela, I. Leites, L. Mateus, R. Romão, A. Pereira, R. M.L.N. Pereira, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8492018/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The understanding of the physiological mechanisms of response to heat-stress in different phenotypes is a key feature to future breeding programs that increase cattle breeds resilience to cope with climate change. Cortisol and Heat Shock Proteins release are hallmarks of heat stress response, however the relationship between these three parameters is poorly studied in cattle. This study evaluated the relationship between cortisol and plasma HSP concentrations in response to increased Temperature-Humidity-Index in different phenotypes to identify specific indicators of heat stress. In this study, native breeds activated specific heat loss strategies despite no increment in cortisol levels, calling into question its use as a measure of heat-stress in historically adapted breeds. Plasma HSP60 and HSP90 reveal a specific pattern through an interaction with THI and cortisol, ended to be breed specific indicators for Alentejana and Mertolenga respectively. Plasma HSP70 concentrations, although highly correlated with THI, were independent of cortisol release in all phenotypes, thus, indicating that this protein is a marker of environmental heat exposure rather than a marker of heat-stress. Studies on the adaptive mechanisms governing heat-stress tolerance, are of paramount relevance for the selection of resilient cattle and the profitability of livestock under a scenario of global warming. Biological sciences/Physiology Biological sciences/Zoology Heat Shock Proteins Cortisol Temperature-Humidity Index Heat Stress Bovine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background The increasing demand for animal sourced foods is a worldwide growing challenge, which is aggravated by climate change. Heat-stress (HS) is responsible for productive losses in livestock [ 1 ], which is exacerbated during hot seasons in heat-sensitive selected dairy and beef cattle breeds, where the selection strategy induced a loss of hardiness and adaptive plasticity [ 2 , 3 ]. In this scenario, genetic selection emerges as a cornerstone strategy to mitigate the impact of HS, by selecting thermotolerant phenotypes. However, this requires deepen current knowledge on physiologic HS adaptive mechanisms in both known heat-resilient and heat-sensitive breeds. Ability to adapt to HS depends on species, breeds and life stage, as well as the environment in which they evolved during successive generations [ 4 , 5 ]. The Iberian Peninsula is within world’s most affected places by global warming, due to increased heat waves and intensification of hot seasons [ 6 ]. The native breeds Alentejana (ALT) and Mertolenga (MERT), known for their resilience to heat-stress [ 5 , 7 ], share the same territorial origin and distribution in the Alentejo province of south Portugal, the hottest region. Both ALT and MERT evolved as sub-breeds of the historic Transtagana breed, being Bos primigenius their ancestor [ 8 ], being Bos primigenius their ancestor. However, MERT also displays physical characteristics from Bos desertorum (desert ox) [ 7 ]. The ALT, formerly used for rural work, is a full-brown color heavy breed (600 kg for females, 1000 Kg for males), whereas the MERT is a less heavy (450 Kg for females, 800 Kg for males) breed with a coat color ranging from white to full brown [ 9 , 10 ]. Both breeds are reared in a pasture-based system, under natural edaphoclimatic conditions, using extended natural mating breeding seasons [ 8 – 10 ]. Cortisol release is a physiological response to heat-stress, dependent on breed, individual sensitivity, and stressor intensity [ 11 ]. Cortisol is known to have immunosuppressor modulatory effects, decreasing pro-inflammatory interleukins, such as IL-1β, TNF-α, IL-8, and impairing neutrophil lifespan and function [ 12 , 13 ]. Increased cortisol is linked to poor reproductive performance and to increased risk of clinical disorders in dairy cows [ 14 , 15 ] and goats [ 16 ], namely in the post-partum period. Heat shock response is a universal ancient mechanism, allowing survival when animals face sudden increased temperatures. In cattle, it includes activation of heat shock factor 1 that induces synthesis of Heat Shock Proteins (HSP) [ 17 ], a large family (15 kDa − 110 kDa) of heat-inducible gene products highly preserved in animals, including ruminants [ 18 ]. Their chaperone function consists in protein trafficking and folding, degradation of misfolded proteins, disaggregation of protein complexes and solubilization of aggregated proteins, preserving cellular proteome homeostasis and functionality. Collectively, HSP constitute a ubiquitous complex and versatile network of chaperones [ 19 ], being the most abundant cellular protein family, representing about 5–10% of the cellular protein content [ 20 ]. However, when the cell faces an insult, as in response to HS, this amount increases up to three folds, by up-regulation and expression of inducible forms [ 21 , 22 ]. The relevant role of HSP during heat-stress conducted to the concept that HSP are a primary way to assess the severity of heat-stress [ 23 ]. The HSP60 is mainly found in mitochondria and, in less proportion, in the cytoplasm and cell membrane. It has no inducible isoform but is up-regulated during cell stress, although it is not clear how it appears in bloodstream [ 24 , 25 ]. Less studied in cattle, in humans HSP60 acts as pro-inflammatory at high concentrations, and as anti-inflammatory at low concentrations, mainly interacting with macrophages and dendritic cells [ 26 ], showing a promising value as marker of tissue regeneration, wound healing [ 27 ], and cancer [ 25 ]. Contrary to HSP60, both HSP70 and HSP90 have stress inducible isoforms (HSPA1A and HSP90αA1, respectively) localized in the cytosol [ 21 ]. The HSP70 is the most studied and already linked to HS in cattle [ 28 , 29 ]. It shows a complex immunomodulatory function, being either pro- or anti-inflammatory, depending on concentration, but found in increased concentrations in diseased animals [ 30 ]. In fact, extracellular HSP70 triggers the immune system by TNFα induction and antigen presentation to T cells [ 31 ], and suppresses inflammation via IL-10 increment, being appointed for graft tissue repair, and treatment of human colitis and arthritis [ 32 ]. The HSP90, mostly studied in humans and lab species, also acts as an inflammation modulator, with inhibition linked to inflammation reduction in several diseases [ 33 ]. Most studies in beef and dairy cattle independently analyzed cortisol or HSP during heat-stress episodes. Most studies report increased cortisol concentrations during heat-stress [ 34 – 36 ]. In contrast, in ALT and MERT cattle, Summer HS was not associated to increased hair cortisol concentrations [ 5 ]. HSP studies reported a strong positive correlation between plasma HSP70 concentrations and THI in beef steers [ 37 ], and increased HSP70 (but not HSP60 and HSP90) blood expression during HS in Simmental cattle [ 38 ]. Other studies reported low HSP27, HSP60 and HSP90 concentrations in endometrial tissue of dairy cows, during heat-stress [ 39 ]. Serum and endometrial HSP seasonal variations were also investigated in goats [ 40 , 41 ]. However, in livestock, the relationship between cortisol release and HSP expression in response to HS is poorly understood. In a study in Hanwoo steers [ 42 ], after acute heat-stress, serum cortisol concentrations were not increased, but mRNA HSPP liver expression was increased. The study of this relationship was undertaken in fish, rendering meaningful results [ 43 ]. In thermoneutrality, daily cortisol administration did not change hepatic HSP levels in Sparus sarba [ 44 ], but under HS, high cortisol concentrations impaired the normal increase of HSP70 and HSP90 in Oncorhynchus mykiss [ 43 ], and of HSP30 in Oncorhynchus clarkia [ 45 ]. Also, increased cortisol concentrations suppressed the normal heat induced HSP70 expression in the liver and gill of trout and tilapia [ 46 ]. Considering the results in fish and the few data available in cattle, more research is needed to evaluate the relationship and cross-regulation between cortisol and HSP during HS, as these physiologic mechanisms may reflect biological markers of phenotypes of resistance to HS, and this information can ultimately be incorporated into genetic selection strategies to face sustainable cattle production in a climate change scenario. The objective of the present study was to evaluate the relationship between THI, cortisol and HSP release under thermoneutrality (Winter) or heat-stress (Summer) in heat-resilient and heat-sensitive cattle breeds, and evaluate the associated physiologic mechanisms (body temperatures and metabolic parameters) put forward to respond to heat-stress. These data may have the potential to stand as breed specific phenotypic markers of adaption/resilience to heat stress. The hypothesis to be tested are that: (i) cortisol and HSP interaction modulate the response to THI; and (ii) this modulation is breed specific. 2. Methods 2.1 Experimental procedures 2.1.1 Localization and meteorological data This study was conducted in the Alentejo province of South Portugal, where climate is defined by two well established seasons: Summer, which is dry and hot; and winter, which is rainy and cold [ 47 ]. The location of the herds was characterized by a Csa type Mediterranean climate according to Köppen-Geiger [ 47 ]. IPMA (Portuguese Institute for Sea and Atmosphere, I. P.) gently provided retrospective meteorological data from the nearest weather station (< 8 Km), presented in Table 1 . Cow side meteorological data were registered with a hygro-thermometer (EXTECH-RH101, Rotterdam, Netherlands). Temperature-Humidity Index (THI) was calculated according to National Research Council [ 48 ] formula: THI = (1.8 × Tdb + 32) − (0.55 − 0.0055 × RH) × (1.8 × Tdb − 26) Where, T db =dry-bulb air temperature (°C) an RH = relative humidity. THI60 was calculated as the mean THI value of the previous 60 days. Table 1 Seasonal meteorological data in the location of the herds. Summer Winter Parameter Global (24h) Hottest hours (11am-4pm) Night (9pm-6am) Global (24h) Hottest hours (11am-4pm) Night (9pm-6am) Maximal temperature (°C) 41.6 ± 2.4 41.6 ± 2.4 31.7 ± 3.8 22.9 ± 2.7 22.7 ± 2.5 16.2 ± 1.3 Minimal temperature (°C) 9.9 ± 1.5 19.2 ± 1.7 9.9 ± 1.47 -2.7 ± 1.9 3.1 ± 3.1 -1.9 ± 1.8 Mean temperature (°C) 23.2 ± 1.4 30.2 ± 1.5 18.4 ± 1.4 10.2 ± 1.2 14.5 ± 1.9 7.8 ± 1.6 Relative humidity (%) 63.6 ± 6.1 38.9 ± 5.0 80.9 ± 6.9 81.9 ± 3.6 66.2 ± 8.6 91.0 ± 2.6 Maximal THI 85.5 ± 2.1 85.5 ± 2.1 75.3 ± 6.4 67.6 ± 2.2 67.5 ± 2.1 60.7 ± 1.8 Minimal THI 49.9 ± 2.4 65.3 ± 2.4 49.9 ± 02.4 28.9 ± 3.9 38.3 ± 5.6 29.8 ± 3.6 Mean THI 69.1 ± 1.4 76.3 ± 1.1 63.9 ± 1.9 50.7 ± 1.8 57.7 ± 2.8 46.7 ± 2.7 2.1.2 Animals and handling The study was conducted between the breeding seasons of 2021 to 2022. Three beef herds with purebred ALT and MERT and two dairy herds with pure Holstein-Friesian (HF) breed were enrolled in the study. After sampling all the cows follow their normal productive live. The ALT cows presented the characteristic full brown color and the MERT cows also presented the brown coat. The beef herds had similar management. Briefly, cows grazed on natural pasture under natural edaphoclimatic conditions, with natural shaded areas and ad libitum access to water. Hay supplementation was available ad libitum during seasonal pasture shortage (June to November). Reproductive management consisted of a natural breeding season and a calving season spanning from July to February. The ALT and MERT cows included present the brown coat color. Cows (n = 89) of ALT (n = 34, age: 86.7 ± 26.7 months; parity: 3 ± 0.7) and MERT (n = 55, age: 87 ± 40 months; parity: 3.0 ± 1.6) breeds were enrolled in the study following calving in Summer and Winter. Dairy herds included a medium-yielding farm (A) of 250 Holstein cows with an average of 9150 Kg/cow/305d and a twice daily milking routine, and a high-yielding farm (B) of 500 Holstein-Frisian cows and an average 13500Kg/cow/305d with a thrice daily milking routine. In both herds, housing included free stalls, roof cover to allow continuous shade, sand individual beds, free walking area, and ad libitum access to water. Cows were fed with a TMR formulated to cover maintenance and milk production. The study enrolled 22 cows from herd A (parity: 2.5 ± 1.4) and 44 cows from herd B (parity: 2.2 ± 1.1) In both beef and dairy herds, cows with dystocia or any postpartum clinical disease were not included. Also, cows enrolled in the study did not receive any treatment prior or during the study. 2.1.3 Ocular thermography and body temperatures Ocular thermographs were acquired at 1 meter from the cow, perpendicular to the left eyeball, with the cow placed in the shade to avoid artifacts caused by sun exposure [ 49 ]. Thermographs were taken with a FLIR®EX8 thermographer (Teledyne Flir, Oregon, USA), with an emissivity 0.98, and analyzed with software FLIR Tools TM PC at 76.800 pixel (320x240) resolution and 0.05°C thermal sensitivity (Fig. 1 ). For each thermograph, a circle was drawn around the eyeball, including the skin of the eye cavity and the lacrimal gland in which the maximum and minimum temperatures were measured (OcularMean, OcularMax and OcularMin, respectively). The vaginal and rectal temperatures were acquired with a DIGI-VET SC12 thermometer (Kruuse®, Langeskov, Denmark) with an accuracy of 0.1°C. 2.1.4 Blood metabolic parameters After collection from the coccygeal vein, blood was centrifuged at 2000 x g for 15 min, and aliquoted and stored at -80ºC. Serum BHB was measured using a handheld meter (Freestyle Precision NEO, Abbott®, Fremont, United States) with Optimum β-Ketone strips, as validated by [ 49 ]. Serum T3 and T4 concentrations were measured by chemiluminescence (IMMULITE 1000, Siemens) using LKT31 and LKT41 kits, respectively (Siemens Healthcare Diagnostics Products, Ltd, Gwynedd, UK) [ 50 ]. The intra-assay and inter-assay coefficients of variation were 3.3% and 2.1% for T3 and 5.3% and 3.0% for T4, respectively [ 51 ]. 2.1.5 Plasma Heat Shock Proteins Plasma HSP60, HSP70 and HSP90 were measured with the MyBiosource Bovine HSP60 ELISA kit, Abbexa Cow Heat Shock 70 kDa Protein 1A ELISA kit and MyBiosource Bovine Heat Shock Protein 90 kDa Alpha A1 ELISA kit, respectively (MBS7606407, San Diego, USA; abx150116, Cambridge, United Kingdom; MBS45994, San Diego, USA, respectively). The intra-assay and inter-assay coefficients of variation were 6.2% and 10.4% HSP60, 7.4% and 9.5% for HSP70 and 7.8% and 14.7% for HSP90, respectively [ 51 ]. 2.1.6 Hair cortisol assay Briefly, hair was shaved with an electric clipper, on the left side of the neck and close to the skin, placed into a 2mL Eppendorf, and immediately stored in a cool place protected from light. Dark hair was shaved in an area of approximately 2.5 cm 2 to avoid pigmentation influence in cortisol concentrations [ 52 , 53 ]. Since the hair grows approximately 0.6 mm per day [ 54 ], samples (2–3 cm long) approximately reflected 60 days of cortisol incorporation. For extraction, 200 mg of hair was washed with 3 mL 2-isopropanol (VWR, Radnor, USA), vortexed for 1 min, and the supernatant discharged (repeated three times). Samples were left to dry for 24 h at room temperature, and then manually cut into fragments up to 2 mm. Afterwards, 50 mg of hair was weighed and placed in a glass tube within 1.5 mL methanol (VWR, Radnor, USA) to be extracted for 16 h in a stirring water bath (50°C). Upon extraction, 0.75 mL was evaporated and dried extracts were reconstituted in 0.25 mL PBS at pH8. Cortisol concentrations were measured in duplicate using a commercially available ELISA Kit for Salivary Cortisol (DRG Instruments GmbH, Marburg, Germany). Results were calculated using a 4PL curve fit. The intra-assay CV was 4.5%, and the inter-assay CV was 12.8%. 2.2 Experimental design and statistics 2.2.1 Experimental design The sampling period at the end of Summer (S- August and September) and in the Winter (W- January and February) was established to allow the effect of chronic HS or their total absence, respectively. Cows were examined and sampled in the morning from 10 to 12 am at 38.7 ± 5.3 days postpartum (DPP), in a headlock system. Eye thermography, rectal and vaginal temperatures were immediately accomplished to avoid changes caused by handling. Blood samples were then collected from the coccygeal vein with a 18 G needle into 10 mL dry and EDTA (Vacutest KIMA, Arzegrande, Italy) tubes for measurement of serum beta-hydroxybutyrate (BHB), total T3 and T4 concentrations, and plasma HSP60, HSP70 and HSP90 concentrations. Hair from the neck was collected for cortisol measurement. Body condition score (BCS) was assessed on a scale from 1 to 9 [ 55 ] in ALT and MERT breeds, and in a scale from 1 to 5 [ 56 ] in the HF breed. The three beef herds including both the ALT and MERT breeds and the two HF herds including only HF cows were submitted to similar climatic parameters, providing similar environmental THI for all cows. However, housing conditions differed between beef and dairy cows, which were expected to produce different cow-side THI values (lower for dairy than for beef cows). Management (nutrition, reproduction) was different between beef and dairy herds, also encompassing the suckling versus lactating status. This a priori raised a significant difference between the beef and dairy cows, namely related to the metabolic status. Therefore, the study centered on the effects of THI (Summer vs. Winter) on the physiologic parameters (body temperatures, metabolic indicators, cortisol and HSP concentrations, and their relationships) of each breed. However, a comparison between breeds was also undertaken, as this enabled the evaluation of breed specific responses to THI, and of specific phenotypes of response to heat-stress. Although the comparison between beef breeds is straightforward, the comparison beef-dairy has to be taken with caution due to the intrinsic physiologic and management differences. Nevertheless, in dairy cows, as parameters were measured in both seasons, it is possible to understand the additional effect of the higher THI of Summer over the metabolic effect, on the analyzed parameters, as sampling was carried out at the same postpartum timepoint. For the relationships between THI, cortisol and HSP, the parameters THI60 and HCC were chosen as they provided accumulated data (THI values and cortisol concentrations, respectively) from the previous 60 days, thus referring to the chronic effect of heat-stress in the Summer months, and their absence in the Winter months. 2.2.2 Statistical analysis In this observational study, statistical analysis was performed using the SAS 9.4 version software (SAS Institute Inc 2024). Categorical data were analyzed by Fisher’s exact test. After testing data normal distribution by PROC UNIVARIATE, the variables ocular temperature, rectal and vaginal temperature, serum BHB, T3, T4 and HCC were normally distributed, and the variables plasma HSP60, HSP70 and HSP90 were non-normally distributed. To test the fixed effects of Breed, Season and Breed*Season, for normally distributed data, significant differences were determined using two-way ANOVA or Welch’s ANOVA when necessary, based on Levene’s test. For non-normally distributed data, the Kruskal-Wallis Test was used followed by Friedman test when necessary. Pearson correlations were calculated using PROC CORR to investigate linear relationships. Multiple regression analysis was conducted using PROC GLM and PROC Glimmix to assess the effects of Breed, THI60 and HCC and their interactions on HSP, using the following model: Y = Breed THI60 Cortisol Breed*THI60 Breed*Cortisol THI60*Cortisol Breed*THI-60*Cortisol Since HCC had no effect in HSP70, the final model for plasma HSP70 concentrations was assessed using the following model: Y = Breed THI-60 Breed*THI-60 The best fit model (goodness of fit and complexity) was evaluated using Akaike information criterion (AICC) and coefficient of determination (R 2 ) for normal distributed data, and Pearson Chi-Square/DF for non-normal distributed data. Linear regression graphs were performed in SPSS (IBM SPSS Statistics 27) and 3D multiple regression graphs were performed on Origin2019 (Originlab Corporation, USA) according to best fit models previously found on multiple regression analysis. Values were expressed as Mean ± SEM for normally distributed data and as Median and quartiles (25% and 75%) for non-normally distributed data, and considered statistically different when p ≤ 0.05. 3. Results 3.1 Meteorological data The environmental THI60 retrieved from the meteorological stations was higher (p < 0.0001) in S than in W (S – 70.0 ± 1.4 vs. W – 50.8 ± 2.0). The cow-side THI, retrieved from the environment at sampling was higher (p < 0.0001) in S than in W (S – 73.4 ± 5.4 vs. W – 61.6 ± 5.3). However, as expected from the different housing conditions between breeds, the Summer THI was higher (p < 0.01) in ALT and MERT than in HF (ALT – 75.7 ± 5.9 vs. MERT – 75.5 ± 5.6 vs. HF – 70.7 ± 3.7). The results of the final statistical models used in the analysis of the fixed effects Breed, Season and Breed*Season are shown in supplementary table 1 . 3.2 Body temperatures Vaginal temperature was affected by Breed (p < 0.0001), Season (S – 39.1 ± 0.5°C vs. W – 38.8 ± 0.5°C; p < 0.0001) and Breed*Season (p = 0.01) (Fig. 2 A), as ALT was the only breed without a significant Summer increment (S – 39.2 ± 0.3°C vs. W – 39.2 ± 0.3°C). Rectal temperature followed the same pattern of vaginal temperature. Mean ocular temperature was affected by Breed (p < 0.01), Season (S – 35.6 ± 1.5°C vs. W – 33.2 ± 2.6°C; p < 0.0001), and Breed*Season (p < 0.0001) (Fig. 2 B), as HF was the only breed without a significant increment in Summer (S – 35.3 ± 1.2°C vs. W – 34.9 ± 1.2°C). 3.3 Metabolic parameters The total T4 serum concentrations tended (p = 0.06) to be lower in S than in W (S – 4.0 ± 1.1 µg/dL vs. 4.3 ± 1.4 µg/dL), were affected by Breed (p < 0.0001) and tended (p = 0.09) to show a Breed*Season effect (Fig. 3 A). The ALT was the only breed with a significant (p < 0.05) Summer decrease in serum concentrations (S – 4.6 ± 1.0 µg/dL vs. W – 5.4 ± 1.0 µg/dL). The total T3 serum concentrations were affected by Season (S – 128.9 ± 33.8 ng/dL vs. W – 154.1 ± 41.5 ng/dL; p < 0.0001), and this was significantly affected by Breed (p < 0.05) and Breed*Season (p < 0.01) (Fig. 3 B). The ALT was the only breed with a significant (p < 0.0001) Summer decrease in serum concentrations (S – 119.71 ± 29.40 ng/dL vs. W – 179.3 ± 34.16 ng/dL). The serum BHB concentrations were affected by Season (S – 0.6 ± 0.4 mmol/L vs. W – 0.8 ± 0.8 mmol/L; p < 0.05), by Breed (p < 0.0001) and tended (p = 0.1) to be affected by Breed*Season (Fig. 3 C). The HF was the only breed with a significant (p < 0.001) Summer decrease in serum concentrations (S – 0.8 ± 0.5 mmol/L vs W – 1.3 ± 1.1 mmol/L). 3.4 Hair Cortisol Concentrations Overall HCC was affected by Season (S – 17.4 ± 8.8 pg/mg vs. W – 13.5 ± 8.0 pg/mg, p < 0.01), and by Breed (p = 0.0001), but there was no Season*Breed effect (p = 0.13) (Fig. 4 ). The HF was the only breed with a significant (p < 0.05) Summer increment in HCC (S – 20.2 ± 7.1 pg/mg vs. W – 13.9 ± 5.5 pg/mg). 3.5 Plasma HSP concentrations Plasma HSP concentrations are shown in Table 2 . Plasma HSP60 concentrations were affected by Season [S − 2.5 (1.2–4.1) ng/mL vs. W − 4.4 (1.8–12.7) ng/mL, p < 0.0001)] and by Breed (p < 0.05), as the difference between Summer and Winter was more significant in ALT. The Season*Breed effect was not significant (p = 0.43). Plasma HSP70 concentrations were affected by Season [S – 6.0 (3.0-9.2) ng/mL vs. W − 2.9 (1.9–5.3) ng/mL; p < 0.0001], by Breed (p < 0.05), as the difference between Summer and Winter was more significant in ALT and MERT. The Season*Breed effect was significant (p < 0.0001), as HF was the only breed without a Summer increment [S – 4.5 (2.9–4.5) ng/mL vs. W – 4.7 (2.5–7.6) ng/mL]. Plasma HSP90 concentrations were affected by Season [S – 79.1 (50.7-113.1) ng/mL vs. W – 98.6 (76.9–144.0) ng/mL; p = 0.0001) and by Season*Breed (p < 0.05), as ALT was the only breed with a significant decrease in Summer [S – 76.5 (61.4–91.9) ng/mL vs. W − 145.2 (87.9-163.1) ng/mL]. The independent effect Breed was not significant (p = 0.31). Table 2 Plasma concentrations of HSP60, HSP70 and HSP90 in Alentejana (n = 34), Mertolenga (n = 55) and Holstein-Frisian (n = 66) cows by Season. Values are presented as Median, 0–25% and 75–100% quartiles. For columns within rows with different letters, p < 0.05. Summer Winter Variable ALT MERT HF ALT MERT HF HSP60 0.9 bc (0.2-3.0) 2.5 c (1.4–4.8) 2.6 c (1.4–4.2) 7.4 a (1.8–46.8) 4.5 b (2.1–12.7) 2.6 b (1.2–8.2) HSP70 6.14 ab (4.2–6.9) 8.5 a (3.6–11.4) 4.5 b (2.9–4.5) 1.8 c (1.4–2.6) 3.0 c (2.0-4.5) 4.7 b (2.5–7.6) HSP90 76.5 b (61.4–91.9) 83.6 b (40.6-127.2) 80.1 b (50.7-113.1) 145.2 a (87.9-163.1) 98.8 b (68.23–138.4) 97.1 b (78.17–122.2) 3.6 Relationship effects of Breed, THI60 and cortisol on HSP plasma concentrations The results of the final models for interactions (PROC GLM and GlimmixnegBin) are presented in Supplementary Table 1. Plasma HSP60 concentrations were affected by Breed (p < 0.05), Cortisol (p = 0.01), THI60 (p < 0.001) and by Breed*THI60*Cortisol (p < 0.05). This latter interaction only showed a significant increase in HSP60 concentrations in ALT cows, 2% for each THI60*Cortisol unit increment (p = 0.01) (Fig. 5 A), but had no effect in MERT and HF cows (p = 0.95 and p = 0.5, respectively). However, Cortisol and THI60, independently, decreased HSP60 plasma concentrations in ALT cows (p = 0.01 and p = 0.001, respectively). In MERT and HF, plasma HSP60 was affected independently by THI60 and Cortisol. Plasma HSP60 concentrations were decreased (p < 0.0001) by THI60 in MERT and HF cows (5% and 1% per THI60 increment, respectively) (Fig. 5 B). Plasma HSP60 concentrations were also decreased (p < 0.001) by Cortisol in MERT cows (9% per cortisol increment), but were increased (p < 0.001) (2% per cortisol increment) in HF cows (Fig. 5 C). However, this Cortisol effect was not significantly different between MERT and HF breeds (p = 0.63). In contrast, plasma HSP70 concentrations were affected by Breed (p < 0.0001), THI60 (p < 0.0001) and THI60*Breed (p = 0.0001), but not by Cortisol (p = 0.63), THI60*Cortisol (p = 0.17) or Breed*THI60*Cortisol (p = 0.6). Therefore, these non-significant effects were removed from the model. The THI60*Breed effect is illustrated in Fig. 6 . The THI60 increased (p < 0.0001) HSP70 plasma concentrations in ALT and MERT cows (6% and 5% per THI60 increment, respectively), however had no effect in HF cows (p = 0.63) (Fig. 6 ). Plasma HSP90 concentrations were affected by Breed (p < 0.05), Breed*THI60 (p < 0.01), Breed*Cortisol (p < 0.05), and Breed*THI60*Cortisol (p = 0.01). This latter interaction (Breed*THI60*Cortisol) significantly (p < 0.05) decreased HSP90 plasma concentrations in MERT cows (by 1% per THI60*Cortisol increment) (Fig. 7 A), but had no effect in ALT and HF cows (p = 0.62 and p = 0.1, respectively). In these breeds, THI60 and Cortisol showed independent effects in HSP plasma concentrations. In ALT, THI60 decreased (p < 0.0001) HSP90 plasma concentrations by 4% per THI60 unit increment, whereas in HF cows only a decrease tendency (p = 0.07) was observed, resulting in a significant effect (p < 0.05) between the two (Fig. 7 B). Cortisol significantly decreased (p < 0.01) HSP90 plasma concentrations in ALT and HF cows, equally by 2% per HCC unit increment (Fig. 7 C). Independently, THI60 and Cortisol increased (p < 0.05) HSP90 plasma concentrations in MERT cows. 4. Discussion The present study evaluated the relationship between THI, cortisol and HSP release under thermoneutrality (Winter) or heat-stress (Summer) in heat-resilient and heat-sensitive cattle breeds, and the associated physiologic mechanisms (body temperatures and metabolic parameters) of response to heat-stress. Results showed that native breeds (ALT and MERT) grazed on pasture under natural edaphoclimatic conditions, although submitted to similar environmental THI60, were affected by higher cow-side THI at sampling during Summer, compared to the HF breed. This was expected, as although the three breeds were located in the same region, the HF cows were housed and received permanent shade and cooling measures, whereas the native breeds were under natural conditions. Therefore, ALT and MERT cows were expected to suffer higher levels of heat-stress than HF cows. Results evidence that the breeds ALT, MERT and HF developed specific strategies to cope with heat-stress, including the physiologic response of body temperatures, metabolic parameters, cortisol release and HSP production, as well as different relationships between THI stimuli and cortisol and HSP response. Regarding body temperatures, HF cows showed no increase in body external (ocular) temperature in Summer compared to Winter, which could be related to the housing conditions, including the presence of cooling systems and the protection from solar radiation, which is an important component of heat-stress [ 57 , 58 ]. However, HF cows had increased body internal (vaginal and rectal) temperature in Summer compared to Winter, denoting the accumulated effect of THI over the metabolic endogenous heat production due to milk yield. In fact, this increase in internal body temperatures, as well as the variation observed in other parameters, observed between Summer and Winter, may be assumed as heat-stress (THI) induced, as measurements obtained in both seasons were at the same postpartum time-point (around 40 DPP), thus reflecting a similar metabolic scenario, and in similar management conditions. Therefore, the comparison of the rate of change of the analyzed parameters between Summer (high THI, under heat-stress) versus Winter (low THI, under thermoneutrality), under similar postpartum metabolic scenario and management conditions, may validate the apparent limitation of comparing breeds with intrinsically different endogenous metabolic rate (ALT and MERT versus HF). Contrary to HF cows, ALT and MERT cows showed an increase in ocular temperature in Summer compared to Winter, potentially reflecting the direct effects of the THI in pasture conditions. This effect was most noticeable in MERT (4.5°C higher in Summer compared to Winter). This increment in ocular temperature enables heat loss by vasodilatation of superficial vessels [ 59 ]. In MERT, this efficient heat loss resulted in only a non-significant 0.3°C increase in vaginal (and rectal) temperature in Summer compared to Winter. Although less efficient in the ocular heat loss, ALT cows showed no significant internal temperature increment in Summer, compared to Winter. This was related to the higher capacity in decreasing internal heat production through the metabolic rate (see below). Therefore, although evolving in the same edaphoclimatic scenario and with a presumed common ancestor, ALT and MERT, as heat-resilient breeds, developed different physiologic strategies to mitigate heat-stress. This breed’s strategical differences emerge as relevant phenotypes for the genetic selection towards resilience to climatic change. The metabolic response to high THI during heat-stress was also different in the three breeds. Serum concentrations of BHB were low and had no variation between seasons in ALT and MERT cows. This was expected as, by 40 DPP, suckling does not configure a significant metabolic insult to the cows. By contrary, in HF cows, serum concentrations of BHB were much higher than in ALT and MERT cows, dealing with the metabolic challenge of lactation. The higher concentrations in Winter compared to Summer may reflect a higher milk production in Winter versus Summer, as heat-stress negative effects in milk yield of dairy cows are well documented [ 60 ]. In fact, the high metabolic rate of HF cows is necessary for the production of milk, and the decrease of the metabolic rate to face heat-stress effects also lead to decrease in milk production. Serum concentrations of total T4 and T3 were not different between seasons in MERT and HF cows. By contrary, ALT cows showed a significant decrease in serum concentrations of both metabolic indicators in Summer, compared to Winter, most noticeably in the case of total T3 serum concentrations. This effect is shown through the Breed*Season effect in serum T3 concentrations. In Summer, HF and MERT cows non-significantly decreased T3 concentrations by 8% and 14%, respectively, whereas ALT cows showed a significant decrease of 33%. A similar trend was found for serum T4 concentrations. Thyroid hormones are the main players in metabolic adjustment [ 61 ], and it is well documented that an increment in environmental temperature leads to a decrease in T3 and T4 blood concentrations [ 62 , 63 ], which attains a notable efficiency in Bos indicus heat adaptability [ 64 ]. These results evidence that ALT and MERT breeds are more efficient in the above metabolic mechanism than the HF breed and confirms their heat-resilient status and adaptive mechanism under heat-stress, as previously reported [ 5 , 7 , 65 ]. Plasma cortisol concentrations were not different between seasons in ALT and MERT cows, confirming previous studies from the team that cortisol concentrations are not reliable makers of heat-stress in these breeds [ 5 ]. In contrast, HF cows showed a significant increase in blood cortisol release in Summer compared to Winter. This reflects a more intense response to heat-stress in HF cows, reinforcing that this breed is more sensitive to heat-stress due to their higher metabolic rate [ 66 – 68 ]. Plasma concentrations of HSP also evidenced specific trends between seasons and breeds. In the three breeds, concentrations of HSP60 were significantly lower in Summer than in Winter, but the season effect was more noticeable in ALT and MERT than in HF. Plasma concentrations of HSP60 increase following an injury and loss of cellular integrity [ 43 ], as this protein function as a potent chemoattractant for neutrophils, being released by injured cells [ 25 ]. The way HSP60 reaches the bloodstream is not yet fully understood [ 24 , 25 ]. One hypothesis put forward, is that peripheral vasodilation in response to heat-stress leads to poor internal blood perfusion and to damage in the intestinal barrier causing leakage of some HSP to the bloodstream [ 37 ]. Results from the present study do not support this theory, at least in MERT. These cows experienced a significant increase in body external temperature in Summer, and therefore an expected inherent superficial blood vasodilation and redistribution of blood volume [ 59 ] but had lower plasma HSP60 concentrations in Summer than in Winter. Results are in accordance with studies that reported decreased endometrial HSP60 expression in healthy cows during Summer [ 39 ], and that found a decreased HSP60 expression in the afternoon, compared to early morning in Simmental cattle [ 38 ]. Contrary to HSP60, plasma concentrations of HSP70 were higher in Summer than in Winter in ALT and MERT, but not in HF cows. An elevation of HSP70 expression protects the cell from thermal injuries [ 69 ]. Early studies on the response to heat-stress [ 70 , 71 ], showed that long term heat-acclimated rats showed 200% more HSP70 expression than rats not heat-acclimated, but this was only associated with environmental temperatures and not with the rat’s body temperature. This was also observed in Angus steers, where plasma HSP70 concentrations were related with environmental temperatures but not with body temperature [ 72 ]. Other studies in calves [ 37 , 72 ] found increased HSP70 expression in blood mononuclear cells and in hair follicles associated with increased environmental THI. The above results indicate that HSP70 expression increases cell thermal tolerance, not related with body temperature increment. This concept is well illustrated in ALT, which display high HSP70 plasma concentrations in Summer, without a significant increment in internal body temperature. Therefore, HSP70 emerge as a reliable cattle heat-stress indicator [ 37 , 38 , 72 ], but a breed specific value must be evaluated. The effects of heat-stress on cattle HSP90 plasma concentrations are conflicting, either revealing a positive blood expression [ 38 ] or a negative endometrial expression [ 39 ]. In the present study, the season effect was only significant in ALT, indicating a decrease in plasma concentrations in Summer. The analysis of the interrelationships between Breed, THI60 and HCC on HSP, delivered relevant results, and again evidenced breed specific effects. The THI60 and HCC variables are well related, as the type and amount of hair used for cortisol extraction roughly entails the accumulated content of the previous 60 days, and the mean THI60 calculated at the end of Summer and Winter, reflects the accumulated effect of heat-stress or thermoneutrality. Plasma HSP60 concentrations were affected by the Breed*THI-60*Cortisol effect, revealing that ALT cows showed a 2% increase in concentrations per each THI60*Cortisol unit increment. Therefore, an increase in concentrations under heat-stress were dependent on the simultaneous increase in cortisol concentrations. Indeed, without this cortisol effect, THI60 decreased the HSP60 plasma concentrations, which was noticeable in the ALT Summer concentrations, as HCC were similar in both seasons. By contrast, in MERT and HF cows, the effects of THI60 and Cortisol on HSP60 were independent: Increased THI60 decreased HSP60 concentrations in both breeds, whereas Cortisol decreased HSP60 concentrations in MERT, and increased concentrations in HF. In HF cows under heat-stress, this increased HSP60 concentrations induced by cortisol, may result from a higher incidence of postpartum disease [ 73 ], which could result in the release of HSP60 from damaged endometrial cells, or from new synthesis due to immune modulation [ 25 , 43 ] or wound tissue repair [ 27 ]. In contrast to HSP60, plasma concentrations of HSP70 were affected by THI60, but not by HCC. Also, THI60 increased HSP70 concentrations in ALT and MERT, but not in HF cows. These results corroborate the increased HSP70 expression in response to increased ambient temperatures, but evidences that, as in rats, this is independent of HCC, and therefore independent of the stress experienced by the animal. This supports the hypothesis that environmental temperature stimuli received by subcutaneous thermoreceptors trigger the release of HSP70, which in turn lead the adjustment in animal thermoregulation [ 74 ]. Interestingly, plasma concentrations of HSP90 behaved similarly to HSP60, exhibiting a Breed*THI60*Cortisol effect. In this way, only in MERT, a decrease of HSP90 plasma concentrations induced by increased THI60 was only observed in presence of a concomitant increase in HCC. In ALT and HF cows, the effects of THI60 and HCC were independent, but both effects decreased HSP90 concentrations. 5. Conclusion The present study highlights the existence of a cattle breed specific response to heat-stress, including the type and magnitude of the physiologic parameters put forward, as well as the relationship between them. Heat-stress induced different heat loss strategies, considering body temperatures and metabolic parameters, and even in close related native breeds under the same climatic stimuli, differences were noticeable. In heat-resilient cattle, MERT cows attained heat loss through a body external (ocular) temperature increase and associated peripheric vasodilation, and in a less degree in lowering the internal metabolic rate. ALT cows displayed a high efficiency in decreasing the internal metabolic rate, through modulation of total T4 and T3 secretion. In contrast, HF cows under similar climatic stimuli had lower heat loss efficiency through the above mechanisms, turning it a more heat-sensitive breed. The relationship between THI and HCC on HSP secretion showed marked differences between breeds. The high Summer THI induced a decrease in HSP60 plasma concentrations, however in ALT cows, a simultaneous high cortisol release showed the opposite effect. By contrary, in MERT and HF cows, the effects of THI and cortisol secretion were independent, and both decreased plasma HSP60 concentrations. Therefore, plasma HSP60 concentrations emerge as a potential marker of heat-stress in the ALT breed. The high Summer THI induced a decrease in HSP90 plasma concentrations, however in MERT cows this was only accomplished when associated to a simultaneous increase in cortisol secretion. By contrary, in ALT and HF cows the effects of THI and cortisol secretion were independent, and both decreased plasma HSP90 concentrations. Therefore, plasma HSP90 concentrations emerge as a potential marker of heat-stress in the MERT breed. Notably, HSP70 concentrations were correlated with THI, but this was independent of cortisol release, which indicates that plasma HSP70 concentrations are a marker of environmental heat exposure rather than a marker of heat-stress. Altogether, these results evidence that response strategies to heat-stress evolved differently in cattle breeds, even in close related breeds under the same environment, and phenotypes of heat-resilience must be evaluated for each species, as one apparently universal marker of heat-resilience may not be appropriate for a given breed. Further studies are needed in order to understand the adaptive mechanisms governing heat-stress tolerance, which can be included in selection programs, to ensure the resilience of cattle and the profitability of livestock under a scenario of global warming. Abbreviations ALT Alentejana breed BCS Body Condition Score BHB Beta-hydroxybutyrate DPP Days Postpartum HCC Hair Cortisol Concentrations HF Holstein-Friesian breed HS Heat Stress HSP60 Heat Shock Protein 60kDa HSP70 Heat Shock Protein 70kDa HSP90 Heat Shock Protein 90kDa IPMA Portuguese Institute for Sea and Atmosphere, I. P. MERT Mertolenga breed OcularMax Maximal Ocular Temperature OcularMean Mean Ocular Temperature OcularMin Minimal Ocular Temperature S Summer THI60 Temperature-Humidity-Index of the previous 60 days T3 Triiodothyronine T4 Thyroxine W Winter Declarations Acknowledgements We thank the staff of Associação de Criadores de Bovinos Mertolengos (ACBM) and Associação de Criadores de Bovinos de Raça Alentejana (ACBRA) for their help and assistance. We thank to Barão&Barão, and Vacaria da Bica companies. We thank the team at CIISA’s Reproduction and Development Laboratory, as well as the technical assistance of Professor Cristina Mateus from CIISA’s Quality of Animal Products Laboratory. Funding sources This study was funded by projects FCT PTDC/CVT-CVT/6932/2020, FCT UIDB/00276/2020 (CIISA), UIDB/05183/2020 (MED), LA/P/0059/2020 (AL4Animals) and PDR2020-101- 03112; PhD grant SFRH/BD/148804/2019 (Capela L) . CRediT authorship contribution statement Luís G Capela – Conceptualization, Methodology, Writing, Review and Editing; Inês C Leites – Methodology, sampling; Luísa M Mateus – Methodology, hormone assays, Review; Ricardo P Romão – Methodology, sampling, Review and Editing; Alfredo Pereira – Funding acquisition; Rosa MLN Pereira – Conceptualization, Review and Editing, Funding acquisition; Luís Lopes-da-Costa - Conceptualization, Methodology, Writing, Review and Editing, Funding acquisition. Ethics approval for animal use The experiments were conducted in compliance with the Portuguese legislation for the use of animals for experimental purposes (Decreto-Lei nº129/92 and Portaria nº1005/92, DR nº245, série I-B, 4930-42) and with the European Union legislation (Directive 2010/63/UE). The research protocol was approved by the Institutional Animal Care and Use Committee (Reference CEIE nº008/2022). Animal technical procedures were performed by licensed veterinarians, with informed consent from the management of the respective associations. All procedures were performed in accordance with the ARRIVE guidelines and the institutional guidelines for animal research. The reporting of all experimental methods follows in accordance with the ARRIVE guidelines. Data and model availability statement The datasets generated during the current study are not publicly available because they are still being analyzed for further work on the first author's PhD, being confidential until the end of PhD. Data is available from the corresponding author on reasonable request. Declaration of Generative AI use The authors declare that they did not use any generative AI or AI-assisted technology tool throughout the writing process. Declaration of interest statement The authors declare no conflicts of competing interest. Consent to Publish declaration Not applicable. 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09:39:47","extension":"xml","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181850,"visible":true,"origin":"","legend":"","description":"","filename":"201538d041094ac289e07dc8ce4967fd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/0829de7feefa9ecdb22bdbeb.xml"},{"id":100967569,"identity":"43cb470c-61fa-4de8-a39c-f2c7107dbeea","added_by":"auto","created_at":"2026-01-23 09:36:48","extension":"html","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202848,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/6a3b388418ea5e10bf01c92f.html"},{"id":101202784,"identity":"4e082b6b-61c1-4917-b529-713ae7e86205","added_by":"auto","created_at":"2026-01-27 09:37:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":382059,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative thermographic image under thermoneutrality in native Alentejana (A) and Holstein-Friesian (B) cows.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/7fddca8dce42317fb20a043a.png"},{"id":100967532,"identity":"2a3f55e5-4043-43cb-9add-49c477221125","added_by":"auto","created_at":"2026-01-23 09:36:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188104,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the vaginal and ocular temperatures Breed*Season effect. ALT: Alentejana, HF: Holstein Frisian, MERT: Mertolenga. \u003cstrong\u003eA\u003c/strong\u003e – bc and dc, p \u0026lt; 0.05; bd, p \u0026lt; 0.0001. \u003cstrong\u003eB\u003c/strong\u003e – ab, p \u0026lt; 0.01; ac and bc, p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/4e834ea768d210f7abe77f91.png"},{"id":101204065,"identity":"137cd1af-076b-442d-bf92-eb6e31fc3876","added_by":"auto","created_at":"2026-01-27 09:41:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":190623,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the Breed*Season effect on serum metabolic indicators, total thyroxine (T4), total triiodothyronine (T3) and β-hydroxybutyrate (BHB). ALT: Alentejana, HF: Holstein Frisian, MERT: Mertolenga. \u003cstrong\u003eA\u003c/strong\u003e – ab, p \u0026lt; 0.05; ac, p \u0026lt; 0.0001; bc, p \u0026lt; 0.01. \u003cstrong\u003eB\u003c/strong\u003e – ab, p \u0026lt; 0.001; ac, p \u0026lt; 0.0001; cb, p \u0026lt; 0.01; \u003cstrong\u003eC\u003c/strong\u003e ab p \u0026lt; 0.01; ac, p \u0026lt; 0.0001; bc, p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/b328fb51fe93112f2dffb39b.png"},{"id":101202790,"identity":"58d8d4e6-cce1-4936-9663-ce2d2c0f18f1","added_by":"auto","created_at":"2026-01-27 09:37:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135941,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the Breed*Season effect on hair cortisol concentrations. ALT: Alentejana, HF: Holstein Frisian, MERT: Mertolenga). ab and bd, p \u0026lt; 0.01; ad, p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/47918d9d0ca9a1788b9430ec.png"},{"id":101203005,"identity":"b0bc22b4-3ac0-412e-bcb1-e55789d1a7a7","added_by":"auto","created_at":"2026-01-27 09:38:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":304278,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical illustration of models for (A) interaction THI60*Cortisol in Alentejana (ALT) plasma HSP60 concentrations; (B) independent effect of THI60 in Mertolenga (MERT) and Holstein-Friesian (HF) plasma HSP60 concentrations; (C) independent effect of Cortisol in MERT and HF plasma HSP60 concentrations. Vales are in natural Log.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/fa3b9dd693b0e684dc03b547.png"},{"id":101751334,"identity":"44ccc999-f472-42d6-b53a-f1b87564d295","added_by":"auto","created_at":"2026-02-03 10:19:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141694,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression for the effect of Breed*THI60 in plasma HSP70 concentrations in Alentejana (ALT), Mertolenga (MERT) and Holstein-Friesian (HF) breeds. Values are in natural Log.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/42ca80e65ec71e94847967b7.png"},{"id":100967538,"identity":"fa14debe-7c64-4599-97d5-fda74fff91de","added_by":"auto","created_at":"2026-01-23 09:36:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":321514,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical illustration of models for the interaction THI60*Cortisol on plasma HSP90 concentrations. (A) Effect Breed*THI60*Cortisol in Mertolenga (MERT); (B) THI60 effect in Alentejana (ALT) and Holstein-Friesian (HF); (C) Cortisol effect in ALT and HF. Values are in natural Log.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/46f7abd8ee9692f654b9f05c.png"},{"id":102398944,"identity":"004b01de-8b41-4253-8d08-37fdce0bd718","added_by":"auto","created_at":"2026-02-11 10:31:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2889015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/df9f2359-10d8-4d9c-bb73-250bea0a2fbd.pdf"},{"id":101202575,"identity":"152dee7c-1c47-4abc-9adf-3f7f714c7841","added_by":"auto","created_at":"2026-01-27 09:36:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23462,"visible":true,"origin":"","legend":"","description":"","filename":"suplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8492018/v1/4f43226ede56e21a36ff7486.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Relationship between hair cortisol and plasma heat shock proteins in response to heat stress in resilient and sensitive cattle breeds","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe increasing demand for animal sourced foods is a worldwide growing challenge, which is aggravated by climate change. Heat-stress (HS) is responsible for productive losses in livestock [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which is exacerbated during hot seasons in heat-sensitive selected dairy and beef cattle breeds, where the selection strategy induced a loss of hardiness and adaptive plasticity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this scenario, genetic selection emerges as a cornerstone strategy to mitigate the impact of HS, by selecting thermotolerant phenotypes. However, this requires deepen current knowledge on physiologic HS adaptive mechanisms in both known heat-resilient and heat-sensitive breeds. Ability to adapt to HS depends on species, breeds and life stage, as well as the environment in which they evolved during successive generations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Iberian Peninsula is within world\u0026rsquo;s most affected places by global warming, due to increased heat waves and intensification of hot seasons [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The native breeds Alentejana (ALT) and Mertolenga (MERT), known for their resilience to heat-stress [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], share the same territorial origin and distribution in the Alentejo province of south Portugal, the hottest region. Both ALT and MERT evolved as sub-breeds of the historic Transtagana breed, being \u003cem\u003eBos primigenius\u003c/em\u003e their ancestor [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], being \u003cem\u003eBos primigenius\u003c/em\u003e their ancestor. However, MERT also displays physical characteristics from \u003cem\u003eBos desertorum\u003c/em\u003e (desert ox) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The ALT, formerly used for rural work, is a full-brown color heavy breed (600 kg for females, 1000 Kg for males), whereas the MERT is a less heavy (450 Kg for females, 800 Kg for males) breed with a coat color ranging from white to full brown [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Both breeds are reared in a pasture-based system, under natural edaphoclimatic conditions, using extended natural mating breeding seasons [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCortisol release is a physiological response to heat-stress, dependent on breed, individual sensitivity, and stressor intensity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cortisol is known to have immunosuppressor modulatory effects, decreasing pro-inflammatory interleukins, such as IL-1β, TNF-α, IL-8, and impairing neutrophil lifespan and function [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Increased cortisol is linked to poor reproductive performance and to increased risk of clinical disorders in dairy cows [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and goats [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], namely in the post-partum period.\u003c/p\u003e \u003cp\u003eHeat shock response is a universal ancient mechanism, allowing survival when animals face sudden increased temperatures. In cattle, it includes activation of heat shock factor 1 that induces synthesis of Heat Shock Proteins (HSP) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], a large family (15 kDa \u0026minus;\u0026thinsp;110 kDa) of heat-inducible gene products highly preserved in animals, including ruminants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their chaperone function consists in protein trafficking and folding, degradation of misfolded proteins, disaggregation of protein complexes and solubilization of aggregated proteins, preserving cellular proteome homeostasis and functionality. Collectively, HSP constitute a ubiquitous complex and versatile network of chaperones [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], being the most abundant cellular protein family, representing about 5\u0026ndash;10% of the cellular protein content [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, when the cell faces an insult, as in response to HS, this amount increases up to three folds, by up-regulation and expression of inducible forms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The relevant role of HSP during heat-stress conducted to the concept that HSP are a primary way to assess the severity of heat-stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe HSP60 is mainly found in mitochondria and, in less proportion, in the cytoplasm and cell membrane. It has no inducible isoform but is up-regulated during cell stress, although it is not clear how it appears in bloodstream [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Less studied in cattle, in humans HSP60 acts as pro-inflammatory at high concentrations, and as anti-inflammatory at low concentrations, mainly interacting with macrophages and dendritic cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], showing a promising value as marker of tissue regeneration, wound healing [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and cancer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContrary to HSP60, both HSP70 and HSP90 have stress inducible isoforms (HSPA1A and HSP90αA1, respectively) localized in the cytosol [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The HSP70 is the most studied and already linked to HS in cattle [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It shows a complex immunomodulatory function, being either pro- or anti-inflammatory, depending on concentration, but found in increased concentrations in diseased animals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In fact, extracellular HSP70 triggers the immune system by TNFα induction and antigen presentation to T cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and suppresses inflammation via IL-10 increment, being appointed for graft tissue repair, and treatment of human colitis and arthritis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The HSP90, mostly studied in humans and lab species, also acts as an inflammation modulator, with inhibition linked to inflammation reduction in several diseases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost studies in beef and dairy cattle independently analyzed cortisol or HSP during heat-stress episodes. Most studies report increased cortisol concentrations during heat-stress [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, in ALT and MERT cattle, Summer HS was not associated to increased hair cortisol concentrations [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. HSP studies reported a strong positive correlation between plasma HSP70 concentrations and THI in beef steers [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and increased HSP70 (but not HSP60 and HSP90) blood expression during HS in Simmental cattle [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Other studies reported low HSP27, HSP60 and HSP90 concentrations in endometrial tissue of dairy cows, during heat-stress [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Serum and endometrial HSP seasonal variations were also investigated in goats [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, in livestock, the relationship between cortisol release and HSP expression in response to HS is poorly understood. In a study in Hanwoo steers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], after acute heat-stress, serum cortisol concentrations were not increased, but mRNA HSPP liver expression was increased. The study of this relationship was undertaken in fish, rendering meaningful results [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In thermoneutrality, daily cortisol administration did not change hepatic HSP levels in \u003cem\u003eSparus sarba\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], but under HS, high cortisol concentrations impaired the normal increase of HSP70 and HSP90 in \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and of HSP30 in \u003cem\u003eOncorhynchus clarkia\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Also, increased cortisol concentrations suppressed the normal heat induced HSP70 expression in the liver and gill of trout and tilapia [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Considering the results in fish and the few data available in cattle, more research is needed to evaluate the relationship and cross-regulation between cortisol and HSP during HS, as these physiologic mechanisms may reflect biological markers of phenotypes of resistance to HS, and this information can ultimately be incorporated into genetic selection strategies to face sustainable cattle production in a climate change scenario.\u003c/p\u003e \u003cp\u003eThe objective of the present study was to evaluate the relationship between THI, cortisol and HSP release under thermoneutrality (Winter) or heat-stress (Summer) in heat-resilient and heat-sensitive cattle breeds, and evaluate the associated physiologic mechanisms (body temperatures and metabolic parameters) put forward to respond to heat-stress. These data may have the potential to stand as breed specific phenotypic markers of adaption/resilience to heat stress. The hypothesis to be tested are that: (i) cortisol and HSP interaction modulate the response to THI; and (ii) this modulation is breed specific.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental procedures\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Localization and meteorological data\u003c/h2\u003e \u003cp\u003eThis study was conducted in the Alentejo province of South Portugal, where climate is defined by two well established seasons: Summer, which is dry and hot; and winter, which is rainy and cold [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The location of the herds was characterized by a Csa type Mediterranean climate according to K\u0026ouml;ppen-Geiger [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. IPMA (Portuguese Institute for Sea and Atmosphere, I. P.) gently provided retrospective meteorological data from the nearest weather station (\u0026lt;\u0026thinsp;8 Km), presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Cow side meteorological data were registered with a hygro-thermometer (EXTECH-RH101, Rotterdam, Netherlands). Temperature-Humidity Index (THI) was calculated according to National Research Council [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] formula:\u003c/p\u003e \u003cp\u003eTHI = (1.8 \u0026times; Tdb\u0026thinsp;+\u0026thinsp;32) \u0026minus; (0.55\u0026thinsp;\u0026minus;\u0026thinsp;0.0055 \u0026times; RH) \u0026times; (1.8 \u0026times; Tdb\u0026thinsp;\u0026minus;\u0026thinsp;26)\u003c/p\u003e \u003cp\u003eWhere, T\u003csub\u003edb\u003c/sub\u003e=dry-bulb air temperature (\u0026deg;C) an RH\u0026thinsp;=\u0026thinsp;relative humidity.\u003c/p\u003e \u003cp\u003eTHI60 was calculated as the mean THI value of the previous 60 days.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSeasonal meteorological data in the location of the herds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlobal\u003c/p\u003e \u003cp\u003e(24h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHottest hours\u003c/p\u003e \u003cp\u003e(11am-4pm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNight\u003c/p\u003e \u003cp\u003e(9pm-6am)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlobal\u003c/p\u003e \u003cp\u003e(24h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHottest hours\u003c/p\u003e \u003cp\u003e(11am-4pm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNight\u003c/p\u003e \u003cp\u003e(9pm-6am)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximal temperature (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimal temperature (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean temperature (\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative humidity (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximal THI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimal THI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;02.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean THI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Animals and handling\u003c/h2\u003e \u003cp\u003eThe study was conducted between the breeding seasons of 2021 to 2022. Three beef herds with purebred ALT and MERT and two dairy herds with pure Holstein-Friesian (HF) breed were enrolled in the study. After sampling all the cows follow their normal productive live. The ALT cows presented the characteristic full brown color and the MERT cows also presented the brown coat. The beef herds had similar management. Briefly, cows grazed on natural pasture under natural edaphoclimatic conditions, with natural shaded areas and \u003cem\u003ead libitum\u003c/em\u003e access to water. Hay supplementation was available \u003cem\u003ead libitum\u003c/em\u003e during seasonal pasture shortage (June to November). Reproductive management consisted of a natural breeding season and a calving season spanning from July to February. The ALT and MERT cows included present the brown coat color. Cows (n\u0026thinsp;=\u0026thinsp;89) of ALT (n\u0026thinsp;=\u0026thinsp;34, age: 86.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.7 months; parity: 3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7) and MERT (n\u0026thinsp;=\u0026thinsp;55, age: 87\u0026thinsp;\u0026plusmn;\u0026thinsp;40 months; parity: 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6) breeds were enrolled in the study following calving in Summer and Winter.\u003c/p\u003e \u003cp\u003eDairy herds included a medium-yielding farm (A) of 250 Holstein cows with an average of 9150 Kg/cow/305d and a twice daily milking routine, and a high-yielding farm (B) of 500 Holstein-Frisian cows and an average 13500Kg/cow/305d with a thrice daily milking routine. In both herds, housing included free stalls, roof cover to allow continuous shade, sand individual beds, free walking area, and \u003cem\u003ead libitum\u003c/em\u003e access to water. Cows were fed with a TMR formulated to cover maintenance and milk production. The study enrolled 22 cows from herd A (parity: 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4) and 44 cows from herd B (parity: 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1)\u003c/p\u003e \u003cp\u003eIn both beef and dairy herds, cows with dystocia or any postpartum clinical disease were not included. Also, cows enrolled in the study did not receive any treatment prior or during the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Ocular thermography and body temperatures\u003c/h2\u003e \u003cp\u003eOcular thermographs were acquired at 1 meter from the cow, perpendicular to the left eyeball, with the cow placed in the shade to avoid artifacts caused by sun exposure [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thermographs were taken with a FLIR\u0026reg;EX8 thermographer (Teledyne Flir, Oregon, USA), with an emissivity 0.98, and analyzed with software FLIR Tools\u003csup\u003eTM\u003c/sup\u003ePC at 76.800 pixel (320x240) resolution and 0.05\u0026deg;C thermal sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For each thermograph, a circle was drawn around the eyeball, including the skin of the eye cavity and the lacrimal gland in which the maximum and minimum temperatures were measured (OcularMean, OcularMax and OcularMin, respectively). The vaginal and rectal temperatures were acquired with a DIGI-VET SC12 thermometer (Kruuse\u0026reg;, Langeskov, Denmark) with an accuracy of 0.1\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4 Blood metabolic parameters\u003c/h2\u003e \u003cp\u003eAfter collection from the coccygeal vein, blood was centrifuged at 2000 x g for 15 min, and aliquoted and stored at -80\u0026ordm;C. Serum BHB was measured using a handheld meter (Freestyle Precision NEO, Abbott\u0026reg;, Fremont, United States) with Optimum β-Ketone strips, as validated by [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Serum T3 and T4 concentrations were measured by chemiluminescence (IMMULITE 1000, Siemens) using LKT31 and LKT41 kits, respectively (Siemens Healthcare Diagnostics Products, Ltd, Gwynedd, UK) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The intra-assay and inter-assay coefficients of variation were 3.3% and 2.1% for T3 and 5.3% and 3.0% for T4, respectively [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.1.5 Plasma Heat Shock Proteins\u003c/h2\u003e \u003cp\u003ePlasma HSP60, HSP70 and HSP90 were measured with the MyBiosource Bovine HSP60 ELISA kit, Abbexa Cow Heat Shock 70 kDa Protein 1A ELISA kit and MyBiosource Bovine Heat Shock Protein 90 kDa Alpha A1 ELISA kit, respectively (MBS7606407, San Diego, USA; abx150116, Cambridge, United Kingdom; MBS45994, San Diego, USA, respectively). The intra-assay and inter-assay coefficients of variation were 6.2% and 10.4% HSP60, 7.4% and 9.5% for HSP70 and 7.8% and 14.7% for HSP90, respectively [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.1.6 Hair cortisol assay\u003c/h2\u003e \u003cp\u003eBriefly, hair was shaved with an electric clipper, on the left side of the neck and close to the skin, placed into a 2mL Eppendorf, and immediately stored in a cool place protected from light. Dark hair was shaved in an area of approximately 2.5 cm\u003csup\u003e2\u003c/sup\u003e to avoid pigmentation influence in cortisol concentrations [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Since the hair grows approximately 0.6 mm per day [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], samples (2\u0026ndash;3 cm long) approximately reflected 60 days of cortisol incorporation.\u003c/p\u003e \u003cp\u003eFor extraction, 200 mg of hair was washed with 3 mL 2-isopropanol (VWR, Radnor, USA), vortexed for 1 min, and the supernatant discharged (repeated three times). Samples were left to dry for 24 h at room temperature, and then manually cut into fragments up to 2 mm. Afterwards, 50 mg of hair was weighed and placed in a glass tube within 1.5 mL methanol (VWR, Radnor, USA) to be extracted for 16 h in a stirring water bath (50\u0026deg;C). Upon extraction, 0.75 mL was evaporated and dried extracts were reconstituted in 0.25 mL PBS at pH8. Cortisol concentrations were measured in duplicate using a commercially available ELISA Kit for Salivary Cortisol (DRG Instruments GmbH, Marburg, Germany). Results were calculated using a 4PL curve fit. The intra-assay CV was 4.5%, and the inter-assay CV was 12.8%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design and statistics\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Experimental design\u003c/h2\u003e \u003cp\u003eThe sampling period at the end of Summer (S- August and September) and in the Winter (W- January and February) was established to allow the effect of chronic HS or their total absence, respectively. Cows were examined and sampled in the morning from 10 to 12 am at 38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 days postpartum (DPP), in a headlock system. Eye thermography, rectal and vaginal temperatures were immediately accomplished to avoid changes caused by handling. Blood samples were then collected from the coccygeal vein with a 18 G needle into 10 mL dry and EDTA (Vacutest KIMA, Arzegrande, Italy) tubes for measurement of serum beta-hydroxybutyrate (BHB), total T3 and T4 concentrations, and plasma HSP60, HSP70 and HSP90 concentrations. Hair from the neck was collected for cortisol measurement. Body condition score (BCS) was assessed on a scale from 1 to 9 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] in ALT and MERT breeds, and in a scale from 1 to 5 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] in the HF breed.\u003c/p\u003e \u003cp\u003eThe three beef herds including both the ALT and MERT breeds and the two HF herds including only HF cows were submitted to similar climatic parameters, providing similar environmental THI for all cows. However, housing conditions differed between beef and dairy cows, which were expected to produce different cow-side THI values (lower for dairy than for beef cows). Management (nutrition, reproduction) was different between beef and dairy herds, also encompassing the suckling versus lactating status. This a priori raised a significant difference between the beef and dairy cows, namely related to the metabolic status. Therefore, the study centered on the effects of THI (Summer vs. Winter) on the physiologic parameters (body temperatures, metabolic indicators, cortisol and HSP concentrations, and their relationships) of each breed. However, a comparison between breeds was also undertaken, as this enabled the evaluation of breed specific responses to THI, and of specific phenotypes of response to heat-stress. Although the comparison between beef breeds is straightforward, the comparison beef-dairy has to be taken with caution due to the intrinsic physiologic and management differences. Nevertheless, in dairy cows, as parameters were measured in both seasons, it is possible to understand the additional effect of the higher THI of Summer over the metabolic effect, on the analyzed parameters, as sampling was carried out at the same postpartum timepoint. For the relationships between THI, cortisol and HSP, the parameters THI60 and HCC were chosen as they provided accumulated data (THI values and cortisol concentrations, respectively) from the previous 60 days, thus referring to the chronic effect of heat-stress in the Summer months, and their absence in the Winter months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Statistical analysis\u003c/h2\u003e \u003cp\u003eIn this observational study, statistical analysis was performed using the SAS 9.4 version software (SAS Institute Inc 2024). Categorical data were analyzed by Fisher\u0026rsquo;s exact test. After testing data normal distribution by PROC UNIVARIATE, the variables ocular temperature, rectal and vaginal temperature, serum BHB, T3, T4 and HCC were normally distributed, and the variables plasma HSP60, HSP70 and HSP90 were non-normally distributed. To test the fixed effects of Breed, Season and Breed*Season, for normally distributed data, significant differences were determined using two-way ANOVA or Welch\u0026rsquo;s ANOVA when necessary, based on Levene\u0026rsquo;s test. For non-normally distributed data, the Kruskal-Wallis Test was used followed by Friedman test when necessary. Pearson correlations were calculated using PROC CORR to investigate linear relationships. Multiple regression analysis was conducted using PROC GLM and PROC Glimmix to assess the effects of Breed, THI60 and HCC and their interactions on HSP, using the following model:\u003c/p\u003e \u003cp\u003eY\u0026thinsp;=\u0026thinsp;Breed THI60 Cortisol Breed*THI60 Breed*Cortisol THI60*Cortisol\u003c/p\u003e \u003cp\u003eBreed*THI-60*Cortisol\u003c/p\u003e \u003cp\u003eSince HCC had no effect in HSP70, the final model for plasma HSP70 concentrations was assessed using the following model:\u003c/p\u003e \u003cp\u003eY\u0026thinsp;=\u0026thinsp;Breed THI-60 Breed*THI-60\u003c/p\u003e \u003cp\u003eThe best fit model (goodness of fit and complexity) was evaluated using Akaike information criterion (AICC) and coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) for normal distributed data, and Pearson Chi-Square/DF for non-normal distributed data. Linear regression graphs were performed in SPSS (IBM SPSS Statistics 27) and 3D multiple regression graphs were performed on Origin2019 (Originlab Corporation, USA) according to best fit models previously found on multiple regression analysis. Values were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for normally distributed data and as Median and quartiles (25% and 75%) for non-normally distributed data, and considered statistically different when p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Meteorological data\u003c/h2\u003e \u003cp\u003eThe environmental THI60 retrieved from the meteorological stations was higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in S than in W (S \u0026ndash; 70.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 vs. W \u0026ndash; 50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0). The cow-side THI, retrieved from the environment at sampling was higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in S than in W (S \u0026ndash; 73.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 vs. W \u0026ndash; 61.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3). However, as expected from the different housing conditions between breeds, the Summer THI was higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in ALT and MERT than in HF (ALT \u0026ndash; 75.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 vs. MERT \u0026ndash; 75.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6 vs. HF \u0026ndash; 70.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7).\u003c/p\u003e \u003cp\u003eThe results of the final statistical models used in the analysis of the fixed effects Breed, Season and Breed*Season are shown in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Body temperatures\u003c/h2\u003e \u003cp\u003eVaginal temperature was affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), Season (S \u0026ndash; 39.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C vs. W \u0026ndash; 38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and Breed*Season (p\u0026thinsp;=\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), as ALT was the only breed without a significant Summer increment (S \u0026ndash; 39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C vs. W \u0026ndash; 39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C). Rectal temperature followed the same pattern of vaginal temperature.\u003c/p\u003e \u003cp\u003eMean ocular temperature was affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Season (S \u0026ndash; 35.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u0026deg;C vs. W \u0026ndash; 33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u0026deg;C; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and Breed*Season (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), as HF was the only breed without a significant increment in Summer (S \u0026ndash; 35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026deg;C vs. W \u0026ndash; 34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u0026deg;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Metabolic parameters\u003c/h2\u003e \u003cp\u003eThe total T4 serum concentrations tended (p\u0026thinsp;=\u0026thinsp;0.06) to be lower in S than in W (S \u0026ndash; 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u0026micro;g/dL vs. 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 \u0026micro;g/dL), were affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and tended (p\u0026thinsp;=\u0026thinsp;0.09) to show a Breed*Season effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The ALT was the only breed with a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Summer decrease in serum concentrations (S \u0026ndash; 4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026micro;g/dL vs. W \u0026ndash; 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 \u0026micro;g/dL).\u003c/p\u003e \u003cp\u003eThe total T3 serum concentrations were affected by Season (S \u0026ndash; 128.9\u0026thinsp;\u0026plusmn;\u0026thinsp;33.8 ng/dL vs. W \u0026ndash; 154.1\u0026thinsp;\u0026plusmn;\u0026thinsp;41.5 ng/dL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and this was significantly affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and Breed*Season (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The ALT was the only breed with a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) Summer decrease in serum concentrations (S \u0026ndash; 119.71\u0026thinsp;\u0026plusmn;\u0026thinsp;29.40 ng/dL vs. W \u0026ndash; 179.3\u0026thinsp;\u0026plusmn;\u0026thinsp;34.16 ng/dL).\u003c/p\u003e \u003cp\u003eThe serum BHB concentrations were affected by Season (S \u0026ndash; 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L vs. W \u0026ndash; 0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mmol/L; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and tended (p\u0026thinsp;=\u0026thinsp;0.1) to be affected by Breed*Season (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The HF was the only breed with a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) Summer decrease in serum concentrations (S \u0026ndash; 0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mmol/L vs W \u0026ndash; 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 mmol/L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Hair Cortisol Concentrations\u003c/h2\u003e \u003cp\u003eOverall HCC was affected by Season (S \u0026ndash; 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 pg/mg vs. W \u0026ndash; 13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0 pg/mg, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and by Breed (p\u0026thinsp;=\u0026thinsp;0.0001), but there was no Season*Breed effect (p\u0026thinsp;=\u0026thinsp;0.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The HF was the only breed with a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Summer increment in HCC (S \u0026ndash; 20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 pg/mg vs. W \u0026ndash; 13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 pg/mg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Plasma HSP concentrations\u003c/h2\u003e \u003cp\u003ePlasma HSP concentrations are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Plasma HSP60 concentrations were affected by Season [S \u0026minus;\u0026thinsp;2.5 (1.2\u0026ndash;4.1) ng/mL vs. W \u0026minus;\u0026thinsp;4.4 (1.8\u0026ndash;12.7) ng/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)] and by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as the difference between Summer and Winter was more significant in ALT. The Season*Breed effect was not significant (p\u0026thinsp;=\u0026thinsp;0.43).\u003c/p\u003e \u003cp\u003ePlasma HSP70 concentrations were affected by Season [S \u0026ndash; 6.0 (3.0-9.2) ng/mL vs. W \u0026minus;\u0026thinsp;2.9 (1.9\u0026ndash;5.3) ng/mL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as the difference between Summer and Winter was more significant in ALT and MERT. The Season*Breed effect was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as HF was the only breed without a Summer increment [S \u0026ndash; 4.5 (2.9\u0026ndash;4.5) ng/mL vs. W \u0026ndash; 4.7 (2.5\u0026ndash;7.6) ng/mL].\u003c/p\u003e \u003cp\u003ePlasma HSP90 concentrations were affected by Season [S \u0026ndash; 79.1 (50.7-113.1) ng/mL vs. W \u0026ndash; 98.6 (76.9\u0026ndash;144.0) ng/mL; p\u0026thinsp;=\u0026thinsp;0.0001) and by Season*Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as ALT was the only breed with a significant decrease in Summer [S \u0026ndash; 76.5 (61.4\u0026ndash;91.9) ng/mL vs. W \u0026minus;\u0026thinsp;145.2 (87.9-163.1) ng/mL]. The independent effect Breed was not significant (p\u0026thinsp;=\u0026thinsp;0.31).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlasma concentrations of HSP60, HSP70 and HSP90 in Alentejana (n\u0026thinsp;=\u0026thinsp;34), Mertolenga (n\u0026thinsp;=\u0026thinsp;55) and Holstein-Frisian (n\u0026thinsp;=\u0026thinsp;66) cows by Season. Values are presented as Median, 0\u0026ndash;25% and 75\u0026ndash;100% quartiles. For columns within rows with different letters, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" morerows=\"1\" nameend=\"c5\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" morerows=\"1\" nameend=\"c9\" namest=\"c7\" rowspan=\"2\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariable\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eALT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMERT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eHF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eALT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eMERT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eHF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHSP60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.9\u003c/b\u003e \u003csup\u003e\u003cb\u003ebc\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.2-3.0)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2.5\u003c/b\u003e \u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1.4\u0026ndash;4.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e2.6\u003c/b\u003e \u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1.4\u0026ndash;4.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e7.4\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1.8\u0026ndash;46.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e4.5\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2.1\u0026ndash;12.7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e2.6\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1.2\u0026ndash;8.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHSP70\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6.14\u003c/b\u003e\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(4.2\u0026ndash;6.9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e8.5\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(3.6\u0026ndash;11.4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e4.5\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2.9\u0026ndash;4.5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e1.8\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1.4\u0026ndash;2.6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3.0\u003c/b\u003e\u003csup\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2.0-4.5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e4.7\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(2.5\u0026ndash;7.6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHSP90\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e76.5\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(61.4\u0026ndash;91.9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003e83.6\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(40.6-127.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e80.1\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(50.7-113.1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e145.2\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(87.9-163.1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e98.8\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(68.23\u0026ndash;138.4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e97.1\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(78.17\u0026ndash;122.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Relationship effects of Breed, THI60 and cortisol on HSP plasma concentrations\u003c/h2\u003e \u003cp\u003eThe results of the final models for interactions (PROC GLM and GlimmixnegBin) are presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003ePlasma HSP60 concentrations were affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Cortisol (p\u0026thinsp;=\u0026thinsp;0.01), THI60 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and by Breed*THI60*Cortisol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This latter interaction only showed a significant increase in HSP60 concentrations in ALT cows, 2% for each THI60*Cortisol unit increment (p\u0026thinsp;=\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), but had no effect in MERT and HF cows (p\u0026thinsp;=\u0026thinsp;0.95 and p\u0026thinsp;=\u0026thinsp;0.5, respectively). However, Cortisol and THI60, independently, decreased HSP60 plasma concentrations in ALT cows (p\u0026thinsp;=\u0026thinsp;0.01 and p\u0026thinsp;=\u0026thinsp;0.001, respectively). In MERT and HF, plasma HSP60 was affected independently by THI60 and Cortisol. Plasma HSP60 concentrations were decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) by THI60 in MERT and HF cows (5% and 1% per THI60 increment, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Plasma HSP60 concentrations were also decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) by Cortisol in MERT cows (9% per cortisol increment), but were increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (2% per cortisol increment) in HF cows (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, this Cortisol effect was not significantly different between MERT and HF breeds (p\u0026thinsp;=\u0026thinsp;0.63).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, plasma HSP70 concentrations were affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), THI60 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and THI60*Breed (p\u0026thinsp;=\u0026thinsp;0.0001), but not by Cortisol (p\u0026thinsp;=\u0026thinsp;0.63), THI60*Cortisol (p\u0026thinsp;=\u0026thinsp;0.17) or Breed*THI60*Cortisol (p\u0026thinsp;=\u0026thinsp;0.6). Therefore, these non-significant effects were removed from the model. The THI60*Breed effect is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The THI60 increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) HSP70 plasma concentrations in ALT and MERT cows (6% and 5% per THI60 increment, respectively), however had no effect in HF cows (p\u0026thinsp;=\u0026thinsp;0.63) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlasma HSP90 concentrations were affected by Breed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Breed*THI60 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Breed*Cortisol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Breed*THI60*Cortisol (p\u0026thinsp;=\u0026thinsp;0.01). This latter interaction (Breed*THI60*Cortisol) significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decreased HSP90 plasma concentrations in MERT cows (by 1% per THI60*Cortisol increment) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), but had no effect in ALT and HF cows (p\u0026thinsp;=\u0026thinsp;0.62 and p\u0026thinsp;=\u0026thinsp;0.1, respectively). In these breeds, THI60 and Cortisol showed independent effects in HSP plasma concentrations. In ALT, THI60 decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) HSP90 plasma concentrations by 4% per THI60 unit increment, whereas in HF cows only a decrease tendency (p\u0026thinsp;=\u0026thinsp;0.07) was observed, resulting in a significant effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the two (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Cortisol significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) HSP90 plasma concentrations in ALT and HF cows, equally by 2% per HCC unit increment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Independently, THI60 and Cortisol increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) HSP90 plasma concentrations in MERT cows.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present study evaluated the relationship between THI, cortisol and HSP release under thermoneutrality (Winter) or heat-stress (Summer) in heat-resilient and heat-sensitive cattle breeds, and the associated physiologic mechanisms (body temperatures and metabolic parameters) of response to heat-stress. Results showed that native breeds (ALT and MERT) grazed on pasture under natural edaphoclimatic conditions, although submitted to similar environmental THI60, were affected by higher cow-side THI at sampling during Summer, compared to the HF breed. This was expected, as although the three breeds were located in the same region, the HF cows were housed and received permanent shade and cooling measures, whereas the native breeds were under natural conditions. Therefore, ALT and MERT cows were expected to suffer higher levels of heat-stress than HF cows.\u003c/p\u003e \u003cp\u003eResults evidence that the breeds ALT, MERT and HF developed specific strategies to cope with heat-stress, including the physiologic response of body temperatures, metabolic parameters, cortisol release and HSP production, as well as different relationships between THI stimuli and cortisol and HSP response. Regarding body temperatures, HF cows showed no increase in body external (ocular) temperature in Summer compared to Winter, which could be related to the housing conditions, including the presence of cooling systems and the protection from solar radiation, which is an important component of heat-stress [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. However, HF cows had increased body internal (vaginal and rectal) temperature in Summer compared to Winter, denoting the accumulated effect of THI over the metabolic endogenous heat production due to milk yield. In fact, this increase in internal body temperatures, as well as the variation observed in other parameters, observed between Summer and Winter, may be assumed as heat-stress (THI) induced, as measurements obtained in both seasons were at the same postpartum time-point (around 40 DPP), thus reflecting a similar metabolic scenario, and in similar management conditions. Therefore, the comparison of the rate of change of the analyzed parameters between Summer (high THI, under heat-stress) versus Winter (low THI, under thermoneutrality), under similar postpartum metabolic scenario and management conditions, may validate the apparent limitation of comparing breeds with intrinsically different endogenous metabolic rate (ALT and MERT versus HF).\u003c/p\u003e \u003cp\u003eContrary to HF cows, ALT and MERT cows showed an increase in ocular temperature in Summer compared to Winter, potentially reflecting the direct effects of the THI in pasture conditions. This effect was most noticeable in MERT (4.5\u0026deg;C higher in Summer compared to Winter). This increment in ocular temperature enables heat loss by vasodilatation of superficial vessels [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In MERT, this efficient heat loss resulted in only a non-significant 0.3\u0026deg;C increase in vaginal (and rectal) temperature in Summer compared to Winter. Although less efficient in the ocular heat loss, ALT cows showed no significant internal temperature increment in Summer, compared to Winter. This was related to the higher capacity in decreasing internal heat production through the metabolic rate (see below). Therefore, although evolving in the same edaphoclimatic scenario and with a presumed common ancestor, ALT and MERT, as heat-resilient breeds, developed different physiologic strategies to mitigate heat-stress. This breed\u0026rsquo;s strategical differences emerge as relevant phenotypes for the genetic selection towards resilience to climatic change.\u003c/p\u003e \u003cp\u003eThe metabolic response to high THI during heat-stress was also different in the three breeds. Serum concentrations of BHB were low and had no variation between seasons in ALT and MERT cows. This was expected as, by 40 DPP, suckling does not configure a significant metabolic insult to the cows. By contrary, in HF cows, serum concentrations of BHB were much higher than in ALT and MERT cows, dealing with the metabolic challenge of lactation. The higher concentrations in Winter compared to Summer may reflect a higher milk production in Winter versus Summer, as heat-stress negative effects in milk yield of dairy cows are well documented [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In fact, the high metabolic rate of HF cows is necessary for the production of milk, and the decrease of the metabolic rate to face heat-stress effects also lead to decrease in milk production. Serum concentrations of total T4 and T3 were not different between seasons in MERT and HF cows. By contrary, ALT cows showed a significant decrease in serum concentrations of both metabolic indicators in Summer, compared to Winter, most noticeably in the case of total T3 serum concentrations. This effect is shown through the Breed*Season effect in serum T3 concentrations. In Summer, HF and MERT cows non-significantly decreased T3 concentrations by 8% and 14%, respectively, whereas ALT cows showed a significant decrease of 33%. A similar trend was found for serum T4 concentrations. Thyroid hormones are the main players in metabolic adjustment [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and it is well documented that an increment in environmental temperature leads to a decrease in T3 and T4 blood concentrations [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], which attains a notable efficiency in \u003cem\u003eBos indicus\u003c/em\u003e heat adaptability [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. These results evidence that ALT and MERT breeds are more efficient in the above metabolic mechanism than the HF breed and confirms their heat-resilient status and adaptive mechanism under heat-stress, as previously reported [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlasma cortisol concentrations were not different between seasons in ALT and MERT cows, confirming previous studies from the team that cortisol concentrations are not reliable makers of heat-stress in these breeds [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, HF cows showed a significant increase in blood cortisol release in Summer compared to Winter. This reflects a more intense response to heat-stress in HF cows, reinforcing that this breed is more sensitive to heat-stress due to their higher metabolic rate [\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlasma concentrations of HSP also evidenced specific trends between seasons and breeds. In the three breeds, concentrations of HSP60 were significantly lower in Summer than in Winter, but the season effect was more noticeable in ALT and MERT than in HF. Plasma concentrations of HSP60 increase following an injury and loss of cellular integrity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], as this protein function as a potent chemoattractant for neutrophils, being released by injured cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The way HSP60 reaches the bloodstream is not yet fully understood [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. One hypothesis put forward, is that peripheral vasodilation in response to heat-stress leads to poor internal blood perfusion and to damage in the intestinal barrier causing leakage of some HSP to the bloodstream [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Results from the present study do not support this theory, at least in MERT. These cows experienced a significant increase in body external temperature in Summer, and therefore an expected inherent superficial blood vasodilation and redistribution of blood volume [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] but had lower plasma HSP60 concentrations in Summer than in Winter. Results are in accordance with studies that reported decreased endometrial HSP60 expression in healthy cows during Summer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and that found a decreased HSP60 expression in the afternoon, compared to early morning in Simmental cattle [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContrary to HSP60, plasma concentrations of HSP70 were higher in Summer than in Winter in ALT and MERT, but not in HF cows. An elevation of HSP70 expression protects the cell from thermal injuries [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Early studies on the response to heat-stress [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], showed that long term heat-acclimated rats showed 200% more HSP70 expression than rats not heat-acclimated, but this was only associated with environmental temperatures and not with the rat\u0026rsquo;s body temperature. This was also observed in Angus steers, where plasma HSP70 concentrations were related with environmental temperatures but not with body temperature [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Other studies in calves [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] found increased HSP70 expression in blood mononuclear cells and in hair follicles associated with increased environmental THI. The above results indicate that HSP70 expression increases cell thermal tolerance, not related with body temperature increment. This concept is well illustrated in ALT, which display high HSP70 plasma concentrations in Summer, without a significant increment in internal body temperature. Therefore, HSP70 emerge as a reliable cattle heat-stress indicator [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], but a breed specific value must be evaluated.\u003c/p\u003e \u003cp\u003eThe effects of heat-stress on cattle HSP90 plasma concentrations are conflicting, either revealing a positive blood expression [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] or a negative endometrial expression [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, the season effect was only significant in ALT, indicating a decrease in plasma concentrations in Summer.\u003c/p\u003e \u003cp\u003eThe analysis of the interrelationships between Breed, THI60 and HCC on HSP, delivered relevant results, and again evidenced breed specific effects. The THI60 and HCC variables are well related, as the type and amount of hair used for cortisol extraction roughly entails the accumulated content of the previous 60 days, and the mean THI60 calculated at the end of Summer and Winter, reflects the accumulated effect of heat-stress or thermoneutrality. Plasma HSP60 concentrations were affected by the Breed*THI-60*Cortisol effect, revealing that ALT cows showed a 2% increase in concentrations per each THI60*Cortisol unit increment. Therefore, an increase in concentrations under heat-stress were dependent on the simultaneous increase in cortisol concentrations. Indeed, without this cortisol effect, THI60 decreased the HSP60 plasma concentrations, which was noticeable in the ALT Summer concentrations, as HCC were similar in both seasons. By contrast, in MERT and HF cows, the effects of THI60 and Cortisol on HSP60 were independent: Increased THI60 decreased HSP60 concentrations in both breeds, whereas Cortisol decreased HSP60 concentrations in MERT, and increased concentrations in HF. In HF cows under heat-stress, this increased HSP60 concentrations induced by cortisol, may result from a higher incidence of postpartum disease [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], which could result in the release of HSP60 from damaged endometrial cells, or from new synthesis due to immune modulation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] or wound tissue repair [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to HSP60, plasma concentrations of HSP70 were affected by THI60, but not by HCC. Also, THI60 increased HSP70 concentrations in ALT and MERT, but not in HF cows. These results corroborate the increased HSP70 expression in response to increased ambient temperatures, but evidences that, as in rats, this is independent of HCC, and therefore independent of the stress experienced by the animal. This supports the hypothesis that environmental temperature stimuli received by subcutaneous thermoreceptors trigger the release of HSP70, which in turn lead the adjustment in animal thermoregulation [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, plasma concentrations of HSP90 behaved similarly to HSP60, exhibiting a Breed*THI60*Cortisol effect. In this way, only in MERT, a decrease of HSP90 plasma concentrations induced by increased THI60 was only observed in presence of a concomitant increase in HCC. In ALT and HF cows, the effects of THI60 and HCC were independent, but both effects decreased HSP90 concentrations.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study highlights the existence of a cattle breed specific response to heat-stress, including the type and magnitude of the physiologic parameters put forward, as well as the relationship between them. Heat-stress induced different heat loss strategies, considering body temperatures and metabolic parameters, and even in close related native breeds under the same climatic stimuli, differences were noticeable. In heat-resilient cattle, MERT cows attained heat loss through a body external (ocular) temperature increase and associated peripheric vasodilation, and in a less degree in lowering the internal metabolic rate. ALT cows displayed a high efficiency in decreasing the internal metabolic rate, through modulation of total T4 and T3 secretion. In contrast, HF cows under similar climatic stimuli had lower heat loss efficiency through the above mechanisms, turning it a more heat-sensitive breed.\u003c/p\u003e \u003cp\u003eThe relationship between THI and HCC on HSP secretion showed marked differences between breeds. The high Summer THI induced a decrease in HSP60 plasma concentrations, however in ALT cows, a simultaneous high cortisol release showed the opposite effect. By contrary, in MERT and HF cows, the effects of THI and cortisol secretion were independent, and both decreased plasma HSP60 concentrations. Therefore, plasma HSP60 concentrations emerge as a potential marker of heat-stress in the ALT breed. The high Summer THI induced a decrease in HSP90 plasma concentrations, however in MERT cows this was only accomplished when associated to a simultaneous increase in cortisol secretion. By contrary, in ALT and HF cows the effects of THI and cortisol secretion were independent, and both decreased plasma HSP90 concentrations. Therefore, plasma HSP90 concentrations emerge as a potential marker of heat-stress in the MERT breed. Notably, HSP70 concentrations were correlated with THI, but this was independent of cortisol release, which indicates that plasma HSP70 concentrations are a marker of environmental heat exposure rather than a marker of heat-stress.\u003c/p\u003e \u003cp\u003eAltogether, these results evidence that response strategies to heat-stress evolved differently in cattle breeds, even in close related breeds under the same environment, and phenotypes of heat-resilience must be evaluated for each species, as one apparently universal marker of heat-resilience may not be appropriate for a given breed. Further studies are needed in order to understand the adaptive mechanisms governing heat-stress tolerance, which can be included in selection programs, to ensure the resilience of cattle and the profitability of livestock under a scenario of global warming.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlentejana breed\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBody Condition Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBHB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBeta-hydroxybutyrate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDays Postpartum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHair Cortisol Concentrations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHolstein-Friesian breed\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat Stress\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSP60\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat Shock Protein 60kDa\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSP70\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat Shock Protein 70kDa\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSP90\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat Shock Protein 90kDa\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePortuguese Institute for Sea and Atmosphere, I. P.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMERT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMertolenga breed\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOcularMax\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaximal Ocular Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOcularMean\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean Ocular Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOcularMin\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimal Ocular Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTHI60\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTemperature-Humidity-Index of the previous 60 days\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriiodothyronine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThyroxine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of Associação de Criadores de Bovinos Mertolengos (ACBM) and Associação de Criadores de Bovinos de Raça Alentejana (ACBRA) for their help and assistance. We thank to Barão\u0026amp;Barão, and Vacaria da Bica companies. \u0026nbsp;We thank the team at CIISA’s Reproduction and Development Laboratory, as well as the technical assistance of Professor Cristina Mateus from CIISA’s Quality of Animal Products Laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by projects FCT PTDC/CVT-CVT/6932/2020, FCT UIDB/00276/2020 (CIISA), UIDB/05183/2020 (MED), LA/P/0059/2020 (AL4Animals) and PDR2020-101- 03112; PhD grant SFRH/BD/148804/2019 (Capela L)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuís G Capela – Conceptualization, Methodology, Writing, Review and Editing; Inês C Leites – Methodology, sampling; Luísa M Mateus – Methodology, hormone assays, Review; Ricardo P Romão – Methodology, sampling, Review and Editing; Alfredo Pereira – Funding acquisition; Rosa MLN Pereira – Conceptualization, Review and Editing, Funding acquisition; Luís Lopes-da-Costa - Conceptualization, Methodology, Writing, Review and Editing, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval for animal use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were conducted in compliance with the Portuguese legislation for the use of animals for experimental purposes (Decreto-Lei nº129/92 and Portaria nº1005/92, DR nº245, série I-B, 4930-42) and with the European Union legislation (Directive 2010/63/UE). The research protocol was approved by the Institutional Animal Care and Use Committee (Reference CEIE nº008/2022). Animal technical procedures were performed by licensed veterinarians, with informed consent from the management of the respective associations. All procedures were performed in accordance with the ARRIVE guidelines and the institutional guidelines for animal research. The reporting of all experimental methods follows in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and model availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are not publicly available because they are still being analyzed for further work on the first author's PhD, being confidential until the end of PhD. Data is available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they did not use any generative AI or AI-assisted technology tool throughout the writing process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThornton, P., Nelson, G., Mayberry, D. \u0026amp; Herrero, M. Impacts of heat stress on global cattle production during the 21st century: A modelling study. \u003cem\u003eLancet Planet. 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Physiol.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e (1), 285\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-physiol-020518-114546\u003c/span\u003e\u003cspan address=\"10.1146/annurev-physiol-020518-114546\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Heat Shock Proteins, Cortisol, Temperature-Humidity Index, Heat Stress, Bovine","lastPublishedDoi":"10.21203/rs.3.rs-8492018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8492018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe understanding of the physiological mechanisms of response to heat-stress in different phenotypes is a key feature to future breeding programs that increase cattle breeds resilience to cope with climate change. Cortisol and Heat Shock Proteins release are hallmarks of heat stress response, however the relationship between these three parameters is poorly studied in cattle. This study evaluated the relationship between cortisol and plasma HSP concentrations in response to increased Temperature-Humidity-Index in different phenotypes to identify specific indicators of heat stress. In this study, native breeds activated specific heat loss strategies despite no increment in cortisol levels, calling into question its use as a measure of heat-stress in historically adapted breeds. Plasma HSP60 and HSP90 reveal a specific pattern through an interaction with THI and cortisol, ended to be breed specific indicators for Alentejana and Mertolenga respectively. Plasma HSP70 concentrations, although highly correlated with THI, were independent of cortisol release in all phenotypes, thus, indicating that this protein is a marker of environmental heat exposure rather than a marker of heat-stress. Studies on the adaptive mechanisms governing heat-stress tolerance, are of paramount relevance for the selection of resilient cattle and the profitability of livestock under a scenario of global warming.\u003c/p\u003e","manuscriptTitle":"Relationship between hair cortisol and plasma heat shock proteins in response to heat stress in resilient and sensitive cattle breeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 09:36:42","doi":"10.21203/rs.3.rs-8492018/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-16T10:07:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T00:13:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T11:50:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60540182029491225567113774158369967890","date":"2026-01-31T11:36:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200053171648098401789533830320348591218","date":"2026-01-22T10:20:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T17:30:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T17:29:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-20T14:15:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T10:45:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-19T10:38:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2827caec-90f0-49a8-b1b2-e33f9a7b0ed6","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61571666,"name":"Biological sciences/Physiology"},{"id":61571667,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-05-06T07:24:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 09:36:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8492018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8492018","identity":"rs-8492018","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}