Seasonal Heat Stress Effects on the Physiological, Hematological, Hormonal, and Biochemical Responses of Tunisian Sheep Breeds

Seasonal Heat Stress Effects on the Physiological, Hematological, Hormonal, and Biochemical Responses of Tunisian Sheep Breeds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seasonal Heat Stress Effects on the Physiological, Hematological, Hormonal, and Biochemical Responses of Tunisian Sheep Breeds Walid Mâaoui, Jihen Toumi, Mounir Nafti, Mohamed Amine Ferchichi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8610819/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Heat stress is a major environmental challenge for small ruminant production in Mediterranean and arid areas. This study investigated the effects of seasonal heat stress on physiological, hematological, hormonal, and biochemical parameters in Tunisian sheep raised in northern Tunisia. Thirty multiparous ewes (10 per breed) from White Fat-tailed Tunisian Barbarin (BTR), Fine-tailed Tunisian Western (QFO), and Fine-tailed Thibar Black (NTB) breeds were evaluated during a thermo-neutral period and a heat stress period characterized by elevated temperature–humidity index (THI) values. Rectal temperature, respiration rate, heart rate, and skin temperature were recorded, with skin temperature measured at selected anatomical sites. Blood samples collected during both periods were analyzed for hematological indices, plasma concentrations of thyroxine, triiodothyronine, cortisol, and thyroid-stimulating hormone, as well as biochemical parameters including glucose, cholesterol, total proteins, and alkaline phosphatase. Data were analyzed using analysis of variance. Heat stress significantly increased rectal temperature, respiration rate, and heart rate in all breeds, indicating activation of thermoregulatory mechanisms. The NTB breed exhibited the greatest increase in rectal temperature, suggesting higher sensitivity to heat stress. Hematological and biochemical responses to increasing THI included elevated red blood cell count, hemoglobin, cholesterol, and total protein concentrations, along with decreases in mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose, and alkaline phosphatase. Heat stress significantly reduced thyroxine and cortisol concentrations, whereas triiodothyronine remained unaffected. Thyroid-stimulating hormone was significantly altered only in NTB ewes. These findings demonstrate breed-specific adaptive responses to heat stress and provide relevant information for sheep management and selection under Mediterranean climatic conditions. Heat stress Sheep breeds Physiological responses Hematological and biochemical parameters Hormonal changes Tunisia Introduction For many years, climate has been described as a set of physical states of the climatic system, which consists of interrelated elements such as the atmosphere, biosphere, hydrosphere, lithosphere, and cryosphere. Hence, it is defined by the perception of the collection of time-averaged quantities underlying the pattern and performance of numerous components and the correlations between them (Peixoto and Oort, 1992 ). Peel et al. ( 2007 ) reported that the world is divided into 6 climatic zones: the tropical zone, the dry zone, the temperate zone, the continental zone, the polar zone, and the alpine zone. Tunisia is located on the Mediterranean coast of northwest Africa, where it borders Libya to the southeast and Algeria to the west. Tunisia is situated between 30° and 38° north latitudes and between 7° and 12° east longitudes. It features a Mediterranean climate in the north with mild, rainy winters and hot, dry summers; a semiarid climate in the central part of the country; and an arid climate in the south. Both arid and semiarid regions worldwide are facing critical water supply problems (Adham et al., 2019 ) because of erratic rainfall (Weischet and Endlicher, 2000 ; Lionello et al., 2006 ). One of the most serious risks to our planet's population and economy is climate change (Skuce et al., 2013 ). According to the International Panel on Climate Change (IPCC, 2013 ), global average temperatures are projected to increase by 1.8 to 4.0°C over the next 90 years. This shift directly and indirectly affects livestock systems as well as human and livestock health. The Middle East and North Africa (MENA) regions are anticipated to be severely impacted by climate change, altering already hot and dry environmental conditions (Sanchez et al., 2004 ; Giorgi and Lionello, 2008 ; Lelieveld et al., 2012 ; Ozturk et al., 2015 ). Therefore, according to Lelieveld et al. ( 2016 ), the occurrence of hot days and nights will increase significantly. Over the Middle East and North Africa, the maximal temperature on a hot day is approximately 43°C and is projected to reach 46°C by 2050 and 50°C by the end of the century. At high temperatures and humidities, the ability of animals to dissipate heat is reduced, leading to an increase in body temperature. In such cases, water evaporation, which is generated by the respiratory system, and perspiration are the most efficient methods for dissipating heat. Nevertheless, when the environmental heat load exceeds a critical temperature, animals are unable to cool and maintain their physiological functions (Silanikove, 2000 ; Salama et al., 2014 ). Recently, the temperature–humidity index has been used by several authors to evaluate the effects of thermal stress on the physiological parameters of animals, such as temperature, pulse rate, reaction speed and time (Naqvi et al., 2004 ; Sejian et al., 2012 ; Silva et al., 2016 ; Mehaba et al., 2021 ), as well as blood parameters (Sivakumar et al., 2010 ; Sejian et al., 2012 ; Rana et al., 2014 ). Various studies have investigated the relationships between heat stress and the physiological parameters of sheep worldwide (Silanikove, 2000 ; Sivakumar et al., 2010 ; Rana et al., 2014 ; Mehaba et al., 2021 ). However, there are no published reports on the effects of heat stress on the physiological and hematological parameters of Tunisian sheep breeds. Therefore, the aim of this study was to investigate the influence of natural thermal stress on the physiological and hematological parameters of three Tunisian sheep breeds raised in northern Tunisia. Materials and methods Ethical approval This study was approved by the Official Committee of Protection and Use of Animals of the Tunisian National Institute of Agronomy (protocol n°05/15). Site description and methodological measurements This experiment took place on the farm of the Agricultural High School of Mateur in northern Tunisia, which is located at latitude 37°04' N and longitude 9°62' E. The experiment was carried out over 13 weeks from 25 March 2021 until 20 June 2021. Both the air temperature (Ta) and relative humidity (RH) throughout the experiment were recorded via a thermohygrometer (Testo 608-H1, Entech Industrial Solution Co., Ltd., Thailand) to calculate the THI according to the following formula described by Thom (1959): THI = 0.8 × Ta + ((RH/100) × (Ta - 14.3)) + 46.4 where Ta is the ambient temperature during the measurements in °C, and RH is the relative humidity during the measurements in (%). In accordance with Moran (2005), the neutral thermal zone (THZ) is defined by a THI lower than 72. The range of values of 72–77 corresponds to mild heat stress (MIZ), 78–89 corresponds to moderate thermal stress (MOZ), and values over 90 indicate severe thermal stress. Experimental procedure, animals and management The survey was conducted on 30 nonlactating, nonpregnant multiparous ewes. The breeds were divided into 3 groups of 10 each as follows: (1) the first group consisted of White Fat-tailed Tunisian Barbarin (BTR) sheep, with average body weights (BWs) of 37.1 ± 2.69 kg and 5.3 years, respectively; (2) the second group consisted of Fine-tailed Tunisian Western (QFO) sheep of white color, with an average BW = 35.5 ± 5.27 kg and an average age = 5.3 years; and (3) the third group consisted of Fine-tailed Thibar Black (NTB) sheep. The animals were housed in well-ventilated sheds, opened on one side, with an asbestos roof 3.0 m in height and maintained under hygienic conditions, and each group of ewes was housed in a pen (L = 4 m × l = 2 m, 0.8 m²/animal). The feeding and watering hours were 08:00 and 18:00; 2.5 kg/day/animal oat hay was distributed, and 250 g/day/animal commercial concentrated feed was provided. To ensure that the animals were healthy during the trial, prophylactic precautions were taken against sheep illnesses, such as sheep pox, peste des petits ruminants, enterotoxemia, and endo- and ectoparasitic infestations, as stipulated by the institute's health calendar. The animals were kept in a shed for the entire duration of the experiment and were not exposed to the sun. Physiological parameters Physiological parameters such as RT, HR, RR, and ST (forehead (FO), rump (RS), left shoulder (LS), top of shoulder (TS), rump (RU), chest floor (CF), and udder (UD)) of certain parts of the body of an animal were measured from 11:00 am to 2:00 pm, which was the time when we recorded the highest temperature of the day. Consequently, this is typically the time when the highest thermal stress is experienced by the animals. RT was measured via a digital transrectal thermometer; within 15 s of its insertion, the thermometer beeped and displayed the detected temperature to one decimal place. HR was measured via a veterinary stethoscope by placing the chestpiece on the left side of the chest floor, counting the number of beats for 15 seconds, multiplying by 4, and expressing in beats/min. Skin temperature was measured in different regions of the body with an infrared thermometer (Berrcom JXB-178, Berrcom Factory, China). Blood collection and parameters studied At the same time, blood samples were collected from the jugular vein in 5 ml vacutainer tubes in two periods (no-stress period and thermal stress period), with 3 tubes per animal per period (two vacutainer tubes with lithium heparin and one tube with EDTA). However, as a result of time constraints, the blood was immediately transported to the physiology laboratory and stored at a low temperature in an ice holder. Blood parameters such as red blood cell count (RBC), white blood cell count (WBC), hemoglobin (Hb), hematocrit (Ht), platelet (Pl), packed cell volume (PCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were subsequently determined. An automated hematology analyzer (Rayto RT-7600) was used to analyze the blood metabolites. Lithium heparin-containing tubes were centrifuged for 5–10 minutes at 3000 rpm to recover the plasma. Blood plasma was analyzed with Siemens IMMULITE 1000 Immunoassay systems for thyrotropin (TSH) and free thyroxine (T4) concentrations. Additionally, an enzyme-linked fluorescence assay (ELFA) was used to determine the concentrations of cortisol (COR) and free triiodothyronine (T3). In addition, glucose (GLU), cholesterol (CHO), total protein (TOP), and alkaline phosphatase (ALP) concentrations were determined via an RT-2100C microplate reader. Statistical analysis After the analysis, all the data were subjected to analysis of variance with the SAS GLM procedure (SAS, 2010), and multiple comparisons among means were conducted via LSMEANS, with hypothesis testing at a significance level of 5%. Specifically, the model was defined as follows: Results Climate conditions The experimental period was characterized by three distinct thermal phases. The first phase corresponded to a thermoneutral and stress-free period, with an average ambient temperature (Ta) of 18.27 ± 3.67 °C and a temperature–humidity index (THI) of 62.84 ± 4.54. During this period, environmental conditions remained within the comfort zone for sheep. The second phase was defined as a mild heat stress period. Ambient temperature increased to approximately 30.00 ± 3.50 °C, while THI reached an average value of 74.51 ± 2.23. These conditions marked the onset of thermal challenge. The third phase corresponded to moderate heat stress. Mean Ta reached 36.83 ± 2.08 °C, and THI increased to 81.24 ± 1.09. During this period, maximum recorded values reached 38.5 °C for ambient temperature and 82 units for THI. Overall climatic conditions recorded between March and June are summarized in Table 1. The average minimum temperature was 16.86 ± 3.93 °C, while the average maximum temperature was 21.44 ± 5.05 °C. Mean relative humidity reached 75.42 ± 10.83%, with an average THI of 65.08 ± 6.43 across the study period Physiological parameters Physiological responses to increasing heat load are presented in Table 2. Heat stress resulted in clear changes in heart rate (HR), respiratory rate (RR), rectal temperature (RT), and skin temperature across all breeds. As THI increased from thermoneutral to moderate heat stress conditions, RT increased significantly within each breed. The magnitude of this increase varied among breeds. NTB ewes showed the highest rectal temperatures under moderate heat stress conditions. Respiratory rate followed a similar pattern. RR increased progressively with rising THI levels in all breeds. The increase was more pronounced under moderate heat stress, with higher values observed for NTB and QFO ewes compared to BTR. Heart rate also increased under heat stress conditions. Differences among breeds were observed, particularly under moderate heat stress, although breed effects were not significant for all physiological traits. Skin temperature measurements, recorded at multiple body sites (forehead, thorax side, rump side, loin side, udder, carpal fold, and under the tail), increased consistently with increasing THI (Table 2). For all anatomical locations, skin temperature was significantly higher under moderate heat stress than under thermoneutral or mild heat stress conditions. No significant breed effect was detected for skin temperature at the different sites. Hematological parameters Hematological responses to heat stress are shown in Table 3. Red blood cell count (RBC), hemoglobin concentration (Hb), and hematocrit (Ht) increased significantly under heat stress in QFO and NTB ewes. Packed cell volume (PCV) increased significantly only in QFO ewes. White blood cell counts (WBC) did not show significant changes across THI levels or breeds. Platelet counts also remained stable under heat stress conditions. Mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) decreased under heat stress in all breeds. These decreases were significant when comparing thermoneutral and moderate heat stress conditions within breeds. Breed comparisons under moderate heat stress conditions showed significant differences for some hematological parameters, as indicated by the contrast tests presented in Table 3. Hormonal parameters Hormonal responses to heat stress are summarized in Table 4. Thyroxine (T4) concentrations decreased significantly in all three breeds under heat stress conditions. In contrast, triiodothyronine (T3) concentrations remained relatively stable across THI levels and breeds. Cortisol concentrations decreased significantly under heat stress in all breeds. Thyroid-stimulating hormone (TSH) showed a significant decrease only in NTB ewes, while no significant changes were observed in BTR and QFO ewes. No significant breed differences were detected for most hormonal parameters, except where indicated by within-breed comparisons between thermoneutral and moderate heat stress conditions. Biochemical parameters Biochemical parameters measured in response to heat stress are presented in Table 5. Plasma glucose concentration decreased significantly under heat stress in QFO ewes, whereas no significant changes were observed in BTR and NTB ewes. Cholesterol concentration increased significantly in BTR and NTB ewes under heat stress conditions. Total protein concentration showed a slight decrease in BTR ewes, while it increased in QFO and NTB ewes. Alkaline phosphatase (ALP) activity decreased significantly under heat stress in all breeds. This decrease was consistent across thermoneutral and moderate heat stress conditions. Breed comparisons under moderate heat stress conditions revealed significant differences for selected biochemical traits, as indicated by the contrast analyses in Table 5. Discussion The present study demonstrates that seasonal heat stress induces marked physiological, hematological, hormonal, and biochemical changes in Tunisian sheep breeds, consistent with the thermoregulatory and metabolic adaptations observed in other small ruminants exposed to high ambient temperatures (Sejian et al., 2012; Silva et al., 2016). Physiological responses The observed increases in HR, RR, and RT under mild and moderate heat stress confirm the activation of thermoregulatory mechanisms, including evaporative cooling and enhanced peripheral blood flow (Naqvi et al., 2004; Mehaba et al., 2021). The NTB breed consistently showed higher RT than BTR and QFO ewes, suggesting reduced heat tolerance and a higher susceptibility to hyperthermia. These differences likely reflect breed-specific variations in metabolic rate, coat characteristics, and skin pigmentation, which influence heat dissipation efficiency (Hansen, 2004; Collier et al., 2012). The significant rise in RR under heat stress also indicates increased respiratory heat loss as a compensatory mechanism, which may be accompanied by subtle changes in blood gas composition and acid-base balance (West, 2003). These physiological adjustments highlight the importance of monitoring both RT and RR in field conditions to detect early signs of heat strain. Hematological adaptations Heat stress significantly increased RBC, Hb, and Ht in QFO and NTB ewes, and PCV in QFO ewes only. Such hematological adjustments suggest hemoconcentration due to reduced plasma volume, a common adaptive response to maintain oxygen delivery under thermal stress (Al-Haidary, 2004; Sejian et al., 2013). The decrease in MCH and MCHC observed in all breeds may indicate alterations in hemoglobin synthesis or red cell morphology during prolonged heat exposure. The lack of significant change in platelet counts suggests that acute thermal stress does not substantially affect thrombopoiesis in these breeds. However, long-term exposure or concurrent nutritional stress could amplify hematological disturbances. Hormonal regulation The decrease in T4 across all breeds reflects a downregulation of basal metabolic rate, consistent with thermoregulatory strategies aimed at reducing endogenous heat production (Nazifi et al., 2003; Dhanda & Kundu, 2001). T3 levels remained relatively stable, which may indicate the maintenance of essential metabolic functions despite reduced thyroid activity. Cortisol, a key stress hormone, decreased significantly in all breeds, contrasting with some reports of elevated cortisol under acute heat stress (Abilay et al., 1975; Christison & Johnson, 1972). This may reflect chronic adaptation, where prolonged exposure leads to attenuation of the hypothalamic–pituitary–adrenal axis response. TSH changes were breed-specific, significant only in NTB ewes, suggesting differential endocrine sensitivity and highlighting the importance of considering breed when evaluating thermal stress resilience. Biochemical adjustments Metabolic shifts under heat stress were evident from glucose, cholesterol, total protein, and ALP changes. The decline in glucose in QFO ewes suggests increased peripheral utilization or decreased gluconeogenesis, aligning with reports that heat stress can impair energy metabolism (Macías-Cruz et al., 2016). Elevated cholesterol in BTR and NTB may reflect lipid mobilization or altered hepatic lipid metabolism, a common feature under heat stress. Total protein alterations likely reflect both changes in plasma volume and protein turnover. The reduction in ALP activity across all breeds may indicate modulation of enzymatic function to conserve energy and maintain homeostasis under stress. Implications for breeding and management The physiological, hematological, and biochemical responses observed in North African sheep under heat stress carry direct implications for breeding and management. Animals with lower rectal temperatures, stable respiratory rates, and favorable blood profiles cope better with thermal stress. These traits can guide selective breeding. By choosing animals that maintain homeostasis under heat, producers can gradually improve flock resilience (Sejian et al., 2012; Macías-Cruz et al., 2016). Local breeds display adaptations shaped by arid environments. Preserving these adaptive traits is crucial. Introducing them into breeding programs can enhance heat tolerance while maintaining productivity. Genetic selection should focus not only on production traits but also on indicators of thermotolerance. Such integration supports sustainable management in regions with seasonal extremes (Silva et al., 2016). Management practices complement breeding strategies. Providing shade, ventilation, or cooling reduces the thermal load on animals. Adequate water supply is essential. Without sufficient hydration, thermoregulation is impaired. Diet also plays a key role. Supplementing feed with antioxidants, electrolytes, or high-quality protein helps maintain metabolic balance and mitigate oxidative stress induced by heat (Nazifi et al., 2003; Sejian et al., 2012). Reproductive performance is sensitive to temperature. Heat stress can alter estrus expression, conception, and fetal development. Adjusting breeding schedules to cooler periods or applying targeted management during hot months can improve outcomes (Macías-Cruz et al., 2016). Monitoring is another critical tool. Recording rectal temperature, respiratory rate, and key metabolites allows early detection of stress. Timely interventions in feeding, watering, or housing conditions can preserve animal welfare and productivity (Silva et al., 2016; Mehaba et al., 2021). In practice, a multifaceted approach is most effective. Genetic selection, environmental adjustments, nutritional support, and reproductive planning must work together. Aligning management with the natural adaptive capacity of each breed ensures animals remain healthy and productive, even under intense thermal stress. Conclusion This study comprehensively evaluated the impact of seasonal heat stress on three Tunisian sheep breeds. The results indicate that a higher temperature‒humidity index (THI) disrupts homeostasis, leading to significant physiological, hematological, biochemical, and hormonal changes. Key thermoregulatory measures, including the respiration rate, heart rate, and rectal temperature, increased notably under heat stress, highlighting activated heat dissipation mechanisms. Breed-specific responses were evident, with the NTB breed showing the highest rectal temperature, suggesting lower thermal tolerance than the BTR and QFO breeds. Physiological stress responses are linked to marked alterations in blood parameters, such as increased red blood cell count, hemoglobin, cholesterol, and total protein, in contrast with decreases in mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), glucose, and alkaline phosphatase (ALP) levels. These findings indicate metabolic reprioritization and potential hemoconcentration due to heat stress. Hormonal adaptations to chronic heat exposure were also apparent. All the breeds presented consistent decreases in both thyroxine (T4) and cortisol levels, indicating metabolic downregulation and a modified stress response, diverging from acute stress indicators. T3 and thyroid-stimulating hormone (TSH) levels remained stable, although significant TSH variation was noted in the NTB breed, indicating complex endocrine adjustments. In summary, this study identified distinct adaptive mechanisms and vulnerabilities of Tunisian sheep breeds under heat stress. The findings on breed-specific differences in rectal temperature and hormonal responses provide critical biomarkers for assessing heat tolerance. This research emphasizes the need for breed-specific management strategies and the selection of resilient genotypes, contributing to sustainable sheep production in Tunisia and analogous Mediterranean and arid regions facing climate change challenges . Declarations Acknowledgments This work was made possible thanks to generous financial support from ADIPARA Research Laboratory. The authors gratefully acknowledge the technical staff of the Higher School of Agriculture of Mateur for their assistance with blood sample collection. We also sincerely thank all members of the ADIPARA Research Laboratory for their valuable technical support and assistance throughout the study. Funding This work was conducted within the research activities of the ADIPARA Research Laboratory “Integrated Improvement and Development of Animal Productivity and Feed Resources”-LR13AGR02), IRESA–University of Carthage, Higher School of Agriculture of Mateur (Tunisia), as part of the research project “Management of Animal Genetic Resources (GRGA)”, funded by the Ministry of Higher Education and Scientific Research of Tunisia. The funding body had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript. Competing interests The authors declare that they have no competing interests. Authors' contributions Walid Maâoui, Bayrem Jemmali, and Abderrahmen Ben Gara conceived and designed the study. Material preparation, data collection, and primary data analysis were carried out by Walid Maâoui and Jihen Toumi. Mohamed Amine Ferchichi conducted the statistical analyses and contributed to the interpretation of the results. The first draft of the manuscript was prepared by Walid Maâoui and Zahran Khaldi. Bayrem Jemmali, Mounir Nafti, and Zahran Khaldi provided technical input and critical suggestions during the revision of the manuscript. Hamadi Rouissi and Abderrahmen Ben Gara ensured scientific supervision and overall guidance of the study. All authors critically reviewed the manuscript and approved the final version. Data Availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. 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Silva TPD, Da Costa Torreão JN, Torreão Marques CA, De Araújo MJ, Rocha Bezerra L, Dhanasekaran K, Sejian V (2016) Effect of multiple stress factors on adaptive capability of native ewes. J Therm Biol 59:39–46. https://doi.org/10.1016/j.jtherbio.2016.05.001. Singh KM, Singh S, Ganguly I, Ganguly A, Nachiappan RK, Chopra A, Narula HK (2016) Evaluation of Indian sheep breeds of arid zone under heat stress condition. Small Rumin Res 141:113–117. https://doi.org/10.1016/j.smallrumres.2016.07.008. Sivakumar VN, Singh G, Varshney VP (2010) Antioxidants supplementation on acid–base balance during heat stress in goats. Asian-Australas J Anim Sci 23(11):1462–1468. https://doi.org/10.5713/ajas.2010.90471. Skuce PJ, Morgan ER, van Dijk J, Mitchell M (2013) Animal health aspects of adaptation to climate change. Animal 7:333–345. https://doi.org/10.1017/S175173111300075X. Temizel EM, Yesilbag K, Batten C, Senturk S, Maan NS, Clement-Mertens PP, Batmaz H (2009) Epizootic hemorrhagic disease in cattle, western Turkey. Emerg Infect Dis 15:317–319. https://doi.org/10.3201/eid1502.080572. Thom EC (1959) The discomfort index. Weatherwise 12:57–59. Weischet W, Endlicher W (2000) Regionale Klimatologie – Teil 2: Die Alte Welt . Teubner, Stuttgart. Wise ME, Armstrong DV, Huber JT, Hunter R, Wiersma F (1988) Hormonal alterations in lactating dairy cows in response to thermal stress. J Dairy Sci 71(9):2480–2485. https://doi.org/10.3168/jds.s0022-0302(88)79834-3. Tables Tables 1 to 5 are available in the Supplementary Files section. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8610819","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592720641,"identity":"4d4970c1-a5af-4be7-8091-dd7d0b826dac","order_by":0,"name":"Walid Mâaoui","email":"","orcid":"","institution":"Institut National Agronomique de Tunis","correspondingAuthor":false,"prefix":"","firstName":"Walid","middleName":"","lastName":"Mâaoui","suffix":""},{"id":592720642,"identity":"3ee52a16-9da4-42a8-b5f5-0b2c3699daf3","order_by":1,"name":"Jihen Toumi","email":"","orcid":"","institution":"Central Analytical Laboratory of AnimalFeed (LCAAB)","correspondingAuthor":false,"prefix":"","firstName":"Jihen","middleName":"","lastName":"Toumi","suffix":""},{"id":592720643,"identity":"9593f69d-0c23-4281-b6a8-d127d6f9dfa7","order_by":2,"name":"Mounir Nafti","email":"","orcid":"","institution":"Regional Center of Research in Oases Agriculture: Centre Regional de Recherche en Agriculture Oasienne","correspondingAuthor":false,"prefix":"","firstName":"Mounir","middleName":"","lastName":"Nafti","suffix":""},{"id":592720649,"identity":"60d6b80c-d626-4595-a42a-e15d6c1ccbe0","order_by":3,"name":"Mohamed Amine Ferchichi","email":"","orcid":"","institution":"École Supérieure d'Agriculture Mateur: Ecole Superieure d'Agriculture Mateur","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Amine","lastName":"Ferchichi","suffix":""},{"id":592720651,"identity":"70dced88-568d-4c15-bf69-192c3c72cd72","order_by":4,"name":"Zahran 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Mateur","correspondingAuthor":false,"prefix":"","firstName":"Bayrem","middleName":"","lastName":"Jemmali","suffix":""},{"id":592720653,"identity":"d2f51e61-7874-4b9c-b414-022d44aa75eb","order_by":6,"name":"Hamadi Rouissi","email":"","orcid":"","institution":"École Supérieure d'Agriculture Mateur: Ecole Superieure d'Agriculture Mateur","correspondingAuthor":false,"prefix":"","firstName":"Hamadi","middleName":"","lastName":"Rouissi","suffix":""},{"id":592720654,"identity":"9bbb887c-9a21-4e0f-8d89-931ba6e49ae0","order_by":7,"name":"Abderrahmen Ben Gara","email":"","orcid":"","institution":"École Supérieure d'Agriculture Mateur: Ecole Superieure d'Agriculture Mateur","correspondingAuthor":false,"prefix":"","firstName":"Abderrahmen","middleName":"Ben","lastName":"Gara","suffix":""}],"badges":[],"createdAt":"2026-01-15 13:13:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8610819/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8610819/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103505598,"identity":"3bbe88ba-f4d1-47ae-b91e-926b65c4befe","added_by":"auto","created_at":"2026-02-26 13:32:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":713150,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8610819/v1/b7bb0739-464d-4e1f-9e61-0c394f481e24.pdf"},{"id":103256765,"identity":"97687f83-3426-4cea-ac78-9787c04be181","added_by":"auto","created_at":"2026-02-23 17:07:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28394,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8610819/v1/4ef8aef9f08c903026a3c018.docx"}],"financialInterests":"","formattedTitle":"Seasonal Heat Stress Effects on the Physiological, Hematological, Hormonal, and Biochemical Responses of Tunisian Sheep Breeds","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor many years, climate has been described as a set of physical states of the climatic system, which consists of interrelated elements such as the atmosphere, biosphere, hydrosphere, lithosphere, and cryosphere. Hence, it is defined by the perception of the collection of time-averaged quantities underlying the pattern and performance of numerous components and the correlations between them (Peixoto and Oort, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Peel et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) reported that the world is divided into 6 climatic zones: the tropical zone, the dry zone, the temperate zone, the continental zone, the polar zone, and the alpine zone. Tunisia is located on the Mediterranean coast of northwest Africa, where it borders Libya to the southeast and Algeria to the west. Tunisia is situated between 30\u0026deg; and 38\u0026deg; north latitudes and between 7\u0026deg; and 12\u0026deg; east longitudes. It features a Mediterranean climate in the north with mild, rainy winters and hot, dry summers; a semiarid climate in the central part of the country; and an arid climate in the south. Both arid and semiarid regions worldwide are facing critical water supply problems (Adham et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) because of erratic rainfall (Weischet and Endlicher, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Lionello et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). One of the most serious risks to our planet's population and economy is climate change (Skuce et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to the International Panel on Climate Change (IPCC, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), global average temperatures are projected to increase by 1.8 to 4.0\u0026deg;C over the next 90 years. This shift directly and indirectly affects livestock systems as well as human and livestock health. The Middle East and North Africa (MENA) regions are anticipated to be severely impacted by climate change, altering already hot and dry environmental conditions (Sanchez et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Giorgi and Lionello, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lelieveld et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ozturk et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, according to Lelieveld et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the occurrence of hot days and nights will increase significantly. Over the Middle East and North Africa, the maximal temperature on a hot day is approximately 43\u0026deg;C and is projected to reach 46\u0026deg;C by 2050 and 50\u0026deg;C by the end of the century.\u003c/p\u003e \u003cp\u003eAt high temperatures and humidities, the ability of animals to dissipate heat is reduced, leading to an increase in body temperature. In such cases, water evaporation, which is generated by the respiratory system, and perspiration are the most efficient methods for dissipating heat. Nevertheless, when the environmental heat load exceeds a critical temperature, animals are unable to cool and maintain their physiological functions (Silanikove, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Salama et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Recently, the temperature\u0026ndash;humidity index has been used by several authors to evaluate the effects of thermal stress on the physiological parameters of animals, such as temperature, pulse rate, reaction speed and time (Naqvi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sejian et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mehaba et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), as well as blood parameters (Sivakumar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sejian et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rana et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious studies have investigated the relationships between heat stress and the physiological parameters of sheep worldwide (Silanikove, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sivakumar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rana et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mehaba et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there are no published reports on the effects of heat stress on the physiological and hematological parameters of Tunisian sheep breeds. Therefore, the aim of this study was to investigate the influence of natural thermal stress on the physiological and hematological parameters of three Tunisian sheep breeds raised in northern Tunisia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Official Committee of Protection and Use of Animals of the Tunisian National Institute of Agronomy (protocol n\u0026deg;05/15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSite description and methodological measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment took place on the farm of the Agricultural High School of Mateur in northern Tunisia, which is located at latitude 37\u0026deg;04\u0026apos; N and longitude 9\u0026deg;62\u0026apos; E. The experiment was carried out over 13 weeks from 25 March 2021 until 20 June 2021. Both the air temperature (Ta) and relative humidity (RH) throughout the experiment were recorded via a thermohygrometer (Testo 608-H1, Entech Industrial Solution Co., Ltd., Thailand) to calculate the THI according to the following formula described by Thom (1959):\u003c/p\u003e\n\u003cp\u003eTHI = 0.8 \u0026times; Ta + ((RH/100) \u0026times; (Ta - 14.3)) + 46.4\u003c/p\u003e\n\u003cp\u003ewhere Ta is the ambient temperature during the measurements in \u0026deg;C, and RH is the relative humidity during the measurements in (%).\u003c/p\u003e\n\u003cp\u003eIn accordance with Moran (2005), the neutral thermal zone (THZ) is defined by a THI lower than 72. The range of values of 72\u0026ndash;77 corresponds to mild heat stress (MIZ), 78\u0026ndash;89 corresponds to moderate thermal stress (MOZ), and values over 90 indicate severe thermal stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental procedure, animals and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survey was conducted on 30 nonlactating, nonpregnant multiparous ewes. The breeds were divided into 3 groups of 10 each as follows: (1) the first group consisted of White Fat-tailed Tunisian Barbarin (BTR) sheep, with average body weights (BWs) of 37.1 \u0026plusmn; 2.69 kg and 5.3 years, respectively; (2) the second group consisted of Fine-tailed Tunisian Western (QFO) sheep of white color, with an average BW = 35.5 \u0026plusmn; 5.27 kg and an average age = 5.3 years; and (3) the third group consisted of Fine-tailed Thibar Black (NTB) sheep. The animals were housed in well-ventilated sheds, opened on one side, with an asbestos roof 3.0 m in height and maintained under hygienic conditions, and each group of ewes was housed in a pen (L = 4 m \u0026times; l = 2 m, 0.8 m\u0026sup2;/animal). The feeding and watering hours were 08:00 and 18:00; 2.5 kg/day/animal oat hay was distributed, and 250 g/day/animal commercial concentrated feed was provided. To ensure that the animals were healthy during the trial, prophylactic precautions were taken against sheep illnesses, such as sheep pox, peste des petits ruminants, enterotoxemia, and endo- and ectoparasitic infestations, as stipulated by the institute\u0026apos;s health calendar. The animals were kept in a shed for the entire duration of the experiment and were not exposed to the sun.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysiological parameters such as RT, HR, RR, and ST (forehead (FO), rump (RS), left shoulder (LS), top of shoulder (TS), rump (RU), chest floor (CF), and udder (UD)) of certain parts of the body of an animal were measured from 11:00 am to 2:00 pm, which was the time when we recorded the highest temperature of the day. Consequently, this is typically the time when the highest thermal stress is experienced by the animals. RT was measured via a digital transrectal thermometer; within 15 s of its insertion, the thermometer beeped and displayed the detected temperature to one decimal place. HR was measured via a veterinary stethoscope by placing the chestpiece on the left side of the chest floor, counting the number of beats for 15 seconds, multiplying by 4, and expressing in beats/min. Skin temperature was measured in different regions of the body with an infrared thermometer (Berrcom JXB-178, Berrcom Factory, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood collection and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eparameters\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;studied\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the same time, blood samples were collected from the jugular vein in 5 ml vacutainer tubes in two periods (no-stress period and thermal stress period), with 3 tubes per animal per period (two vacutainer tubes with lithium heparin and one tube with EDTA). However, as a result of time constraints, the blood was immediately transported to the physiology laboratory and stored at a low temperature in an ice holder. Blood parameters such as red blood cell count (RBC), white blood cell count (WBC), hemoglobin (Hb), hematocrit (Ht), platelet (Pl), packed cell volume (PCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were subsequently determined. An automated hematology analyzer (Rayto RT-7600) was used to analyze the blood metabolites. Lithium heparin-containing tubes were centrifuged for 5\u0026ndash;10 minutes at 3000 rpm to recover the plasma. Blood plasma was analyzed with Siemens IMMULITE 1000 Immunoassay systems for thyrotropin (TSH) and free thyroxine (T4) concentrations. Additionally, an enzyme-linked fluorescence assay (ELFA) was used to determine the concentrations of cortisol (COR) and free triiodothyronine (T3). In addition, glucose (GLU), cholesterol (CHO), total protein (TOP), and alkaline phosphatase (ALP) concentrations were determined via an RT-2100C microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the analysis, all the data were subjected to analysis of variance with the SAS GLM procedure (SAS, 2010), and multiple comparisons among means were conducted via LSMEANS, with hypothesis testing at a significance level of 5%. Specifically, the model was defined as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1771866056.png\" width=\"755\" height=\"542\"\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClimate conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental period was characterized by three distinct thermal phases. The first phase corresponded to a thermoneutral and stress-free period, with an average ambient temperature (Ta) of 18.27 \u0026plusmn; 3.67 \u0026deg;C and a temperature\u0026ndash;humidity index (THI) of 62.84 \u0026plusmn; 4.54. During this period, environmental conditions remained within the comfort zone for sheep.\u003c/p\u003e\n\u003cp\u003eThe second phase was defined as a mild heat stress period. Ambient temperature increased to approximately 30.00 \u0026plusmn; 3.50 \u0026deg;C, while THI reached an average value of 74.51 \u0026plusmn; 2.23. These conditions marked the onset of thermal challenge.\u003c/p\u003e\n\u003cp\u003eThe third phase corresponded to moderate heat stress. Mean Ta reached 36.83 \u0026plusmn; 2.08 \u0026deg;C, and THI increased to 81.24 \u0026plusmn; 1.09. During this period, maximum recorded values reached 38.5 \u0026deg;C for ambient temperature and 82 units for THI.\u003c/p\u003e\n\u003cp\u003eOverall climatic conditions recorded between March and June are summarized in Table 1. The average minimum temperature was 16.86 \u0026plusmn; 3.93 \u0026deg;C, while the average maximum temperature was 21.44 \u0026plusmn; 5.05 \u0026deg;C. Mean relative humidity reached 75.42 \u0026plusmn; 10.83%, with an average THI of 65.08 \u0026plusmn; 6.43 across the study period\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysiological responses to increasing heat load are presented in Table 2. Heat stress resulted in clear changes in heart rate (HR), respiratory rate (RR), rectal temperature (RT), and skin temperature across all breeds.\u003c/p\u003e\n\u003cp\u003eAs THI increased from thermoneutral to moderate heat stress conditions, RT increased significantly within each breed. The magnitude of this increase varied among breeds. NTB ewes showed the highest rectal temperatures under moderate heat stress conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRespiratory rate followed a similar pattern. RR increased progressively with rising THI levels in all breeds. The increase was more pronounced under moderate heat stress, with higher values observed for NTB and QFO ewes compared to BTR.\u003c/p\u003e\n\u003cp\u003eHeart rate also increased under heat stress conditions. Differences among breeds were observed, particularly under moderate heat stress, although breed effects were not significant for all physiological traits.\u003c/p\u003e\n\u003cp\u003eSkin temperature measurements, recorded at multiple body sites (forehead, thorax side, rump side, loin side, udder, carpal fold, and under the tail), increased consistently with increasing THI (Table 2). For all anatomical locations, skin temperature was significantly higher under moderate heat stress than under thermoneutral or mild heat stress conditions. No significant breed effect was detected for skin temperature at the different sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHematological responses to heat stress are shown in Table 3. Red blood cell count (RBC), hemoglobin concentration (Hb), and hematocrit (Ht) increased significantly under heat stress in QFO and NTB ewes. Packed cell volume (PCV) increased significantly only in QFO ewes. White blood cell counts (WBC) did not show significant changes across THI levels or breeds. Platelet counts also remained stable under heat stress conditions. Mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) decreased under heat stress in all breeds. These decreases were significant when comparing thermoneutral and moderate heat stress conditions within breeds.\u003c/p\u003e\n\u003cp\u003eBreed comparisons under moderate heat stress conditions showed significant differences for some hematological parameters, as indicated by the contrast tests presented in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHormonal parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHormonal responses to heat stress are summarized in Table 4. Thyroxine (T4) concentrations decreased significantly in all three breeds under heat stress conditions. In contrast, triiodothyronine (T3) concentrations remained relatively stable across THI levels and breeds. Cortisol concentrations decreased significantly under heat stress in all breeds. Thyroid-stimulating hormone (TSH) showed a significant decrease only in NTB ewes, while no significant changes were observed in BTR and QFO ewes. No significant breed differences were detected for most hormonal parameters, except where indicated by within-breed comparisons between thermoneutral and moderate heat stress conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiochemical parameters measured in response to heat stress are presented in Table 5. Plasma glucose concentration decreased significantly under heat stress in QFO ewes, whereas no significant changes were observed in BTR and NTB ewes. Cholesterol concentration increased significantly in BTR and NTB ewes under heat stress conditions. Total protein concentration showed a slight decrease in BTR ewes, while it increased in QFO and NTB ewes.\u003c/p\u003e\n\u003cp\u003eAlkaline phosphatase (ALP) activity decreased significantly under heat stress in all breeds. This decrease was consistent across thermoneutral and moderate heat stress conditions. Breed comparisons under moderate heat stress conditions revealed significant differences for selected biochemical traits, as indicated by the contrast analyses in Table 5.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates that seasonal heat stress induces marked physiological, hematological, hormonal, and biochemical changes in Tunisian sheep breeds, consistent with the thermoregulatory and metabolic adaptations observed in other small ruminants exposed to high ambient temperatures (Sejian et al., 2012; Silva et al., 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe observed increases in HR, RR, and RT under mild and moderate heat stress confirm the activation of thermoregulatory mechanisms, including evaporative cooling and enhanced peripheral blood flow (Naqvi et al., 2004; Mehaba et al., 2021). The NTB breed consistently showed higher RT than BTR and QFO ewes, suggesting reduced heat tolerance and a higher susceptibility to hyperthermia. These differences likely reflect breed-specific variations in metabolic rate, coat characteristics, and skin pigmentation, which influence heat dissipation efficiency (Hansen, 2004; Collier et al., 2012).\u003c/p\u003e\n\u003cp\u003eThe significant rise in RR under heat stress also indicates increased respiratory heat loss as a compensatory mechanism, which may be accompanied by subtle changes in blood gas composition and acid-base balance (West, 2003). These physiological adjustments highlight the importance of monitoring both RT and RR in field conditions to detect early signs of heat strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematological adaptations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeat stress significantly increased RBC, Hb, and Ht in QFO and NTB ewes, and PCV in QFO ewes only. Such hematological adjustments suggest hemoconcentration due to reduced plasma volume, a common adaptive response to maintain oxygen delivery under thermal stress (Al-Haidary, 2004; Sejian et al., 2013). The decrease in MCH and MCHC observed in all breeds may indicate alterations in hemoglobin synthesis or red cell morphology during prolonged heat exposure.\u003c/p\u003e\n\u003cp\u003eThe lack of significant change in platelet counts suggests that acute thermal stress does not substantially affect thrombopoiesis in these breeds. However, long-term exposure or concurrent nutritional stress could amplify hematological disturbances.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHormonal regulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe decrease in T4 across all breeds reflects a downregulation of basal metabolic rate, consistent with thermoregulatory strategies aimed at reducing endogenous heat production (Nazifi et al., 2003; Dhanda \u0026amp; Kundu, 2001). T3 levels remained relatively stable, which may indicate the maintenance of essential metabolic functions despite reduced thyroid activity.\u003c/p\u003e\n\u003cp\u003eCortisol, a key stress hormone, decreased significantly in all breeds, contrasting with some reports of elevated cortisol under acute heat stress (Abilay et al., 1975; Christison \u0026amp; Johnson, 1972). This may reflect chronic adaptation, where prolonged exposure leads to attenuation of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal axis response. TSH changes were breed-specific, significant only in NTB ewes, suggesting differential endocrine sensitivity and highlighting the importance of considering breed when evaluating thermal stress resilience.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical adjustments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetabolic shifts under heat stress were evident from glucose, cholesterol, total protein, and ALP changes. The decline in glucose in QFO ewes suggests increased peripheral utilization or decreased gluconeogenesis, aligning with reports that heat stress can impair energy metabolism (Mac\u0026iacute;as-Cruz et al., 2016). Elevated cholesterol in BTR and NTB may reflect lipid mobilization or altered hepatic lipid metabolism, a common feature under heat stress. Total protein alterations likely reflect both changes in plasma volume and protein turnover. The reduction in ALP activity across all breeds may indicate modulation of enzymatic function to conserve energy and maintain homeostasis under stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplications for breeding and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physiological, hematological, and biochemical responses observed in North African sheep under heat stress carry direct implications for breeding and management. Animals with lower rectal temperatures, stable respiratory rates, and favorable blood profiles cope better with thermal stress. These traits can guide selective breeding. By choosing animals that maintain homeostasis under heat, producers can gradually improve flock resilience (Sejian et al., 2012; Mac\u0026iacute;as-Cruz et al., 2016).\u003c/p\u003e\n\u003cp\u003eLocal breeds display adaptations shaped by arid environments. Preserving these adaptive traits is crucial. Introducing them into breeding programs can enhance heat tolerance while maintaining productivity. Genetic selection should focus not only on production traits but also on indicators of thermotolerance. Such integration supports sustainable management in regions with seasonal extremes (Silva et al., 2016).\u003c/p\u003e\n\u003cp\u003eManagement practices complement breeding strategies. Providing shade, ventilation, or cooling reduces the thermal load on animals. Adequate water supply is essential. Without sufficient hydration, thermoregulation is impaired. Diet also plays a key role. Supplementing feed with antioxidants, electrolytes, or high-quality protein helps maintain metabolic balance and mitigate oxidative stress induced by heat (Nazifi et al., 2003; Sejian et al., 2012).\u003c/p\u003e\n\u003cp\u003eReproductive performance is sensitive to temperature. Heat stress can alter estrus expression, conception, and fetal development. Adjusting breeding schedules to cooler periods or applying targeted management during hot months can improve outcomes (Mac\u0026iacute;as-Cruz et al., 2016).\u003c/p\u003e\n\u003cp\u003eMonitoring is another critical tool. Recording rectal temperature, respiratory rate, and key metabolites allows early detection of stress. Timely interventions in feeding, watering, or housing conditions can preserve animal welfare and productivity (Silva et al., 2016; Mehaba et al., 2021).\u003c/p\u003e\n\u003cp\u003eIn practice, a multifaceted approach is most effective. Genetic selection, environmental adjustments, nutritional support, and reproductive planning must work together. Aligning management with the natural adaptive capacity of each breed ensures animals remain healthy and productive, even under intense thermal stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study comprehensively evaluated the impact of seasonal heat stress on three Tunisian sheep breeds. The results indicate that a higher temperature‒humidity index (THI) disrupts homeostasis, leading to significant physiological, hematological, biochemical, and hormonal changes. Key thermoregulatory measures, including the respiration rate, heart rate, and rectal temperature, increased notably under heat stress, highlighting activated heat dissipation mechanisms. Breed-specific responses were evident, with the NTB breed showing the highest rectal temperature, suggesting lower thermal tolerance than the BTR and QFO breeds.\u003c/p\u003e\n\u003cp\u003ePhysiological stress responses are linked to marked alterations in blood parameters, such as increased red blood cell count, hemoglobin, cholesterol, and total protein, in contrast with decreases in mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), glucose, and alkaline phosphatase (ALP) levels. These findings indicate metabolic reprioritization and potential hemoconcentration due to heat stress. Hormonal adaptations to chronic heat exposure were also apparent. All the breeds presented consistent decreases in both thyroxine (T4) and cortisol levels, indicating metabolic downregulation and a modified stress response, diverging from acute stress indicators. T3 and thyroid-stimulating hormone (TSH) levels remained stable, although significant TSH variation was noted in the NTB breed, indicating complex endocrine adjustments.\u003c/p\u003e\n\u003cp\u003eIn summary, this study identified distinct adaptive mechanisms and vulnerabilities of Tunisian sheep breeds under heat stress. The findings on breed-specific differences in rectal temperature and hormonal responses provide critical biomarkers for assessing heat tolerance. This research emphasizes the need for breed-specific management strategies and the selection of resilient genotypes, contributing to sustainable sheep production in Tunisia and analogous Mediterranean and arid regions facing climate change challenges \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; .\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was made possible thanks to generous financial support from ADIPARA Research Laboratory. The authors gratefully acknowledge the technical staff of the Higher School of Agriculture of Mateur for their assistance with blood sample collection. We also sincerely thank all members of the ADIPARA Research Laboratory for their valuable technical support and assistance throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was conducted within the research activities of the ADIPARA Research Laboratory \u0026ldquo;Integrated Improvement and Development of Animal Productivity and Feed Resources\u0026rdquo;-LR13AGR02), IRESA\u0026ndash;University of Carthage, Higher School of Agriculture of Mateur (Tunisia), as part of the research project \u0026ldquo;Management of Animal Genetic Resources (GRGA)\u0026rdquo;, funded by the Ministry of Higher Education and Scientific Research of Tunisia. The funding body had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWalid Ma\u0026acirc;oui, Bayrem Jemmali, and Abderrahmen Ben Gara conceived and designed the study. Material preparation, data collection, and primary data analysis were carried out by Walid Ma\u0026acirc;oui and Jihen Toumi. Mohamed Amine Ferchichi conducted the statistical analyses and contributed to the interpretation of the results. The first draft of the manuscript was prepared by Walid Ma\u0026acirc;oui and Zahran Khaldi. Bayrem Jemmali, Mounir Nafti, and Zahran Khaldi provided technical input and critical suggestions during the revision of the manuscript. Hamadi Rouissi and Abderrahmen Ben Gara ensured scientific supervision and overall guidance of the study. All authors critically reviewed the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Official Committee of Protection and Use of Animals of the Tunisian National Institute of Agronomy (protocol n\u0026deg;05/15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbilay TA, Johnson HD, Madan M (1975) Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. \u003cem\u003eJ Dairy Sci\u003c/em\u003e 58(12):1836\u0026ndash;1840. https://doi.org/10.3168/jds.s0022-0302(75)84795-3.\u003c/li\u003e\n\u003cli\u003eAdham A, Wesseling JG, Abed R, Riksen M, Ouessar M, Ritsema CJ (2019) Assessing the impact of climate change on rainwater harvesting in the Oum Zessar watershed in southeastern Tunisia. \u003cem\u003eAgric Water Manag\u003c/em\u003e 221:131\u0026ndash;140. https://doi.org/10.1016/j.agwat.2019.05.006.\u003c/li\u003e\n\u003cli\u003eAl-Haidary AA (2004) Physiological responses of Niamey sheep to heat stress challenge under semiarid environment. \u003cem\u003eInt J Agric Biol\u003c/em\u003e 6(2):307\u0026ndash;309.\u003c/li\u003e\n\u003cli\u003eChristison GI, Johnson HD (1972) Cortisol turnover in heat-stressed cows. \u003cem\u003eJ Anim Sci\u003c/em\u003e 35(5):1005\u0026ndash;1010. https://doi.org/10.2527/jas1972.3551005x.\u003c/li\u003e\n\u003cli\u003eCollier RJ, Beede DK, Thatcher WW, Israel LA, Wilcox CJ (1982) Influence of environment and its modification on dairy animal health and production. \u003cem\u003eJ Dairy Sci\u003c/em\u003e 65(11):2213\u0026ndash;2227. https://doi.org/10.3168/jds.s0022-0302(82)82484-3.\u003c/li\u003e\n\u003cli\u003eDhanda OP, Kundu RL (2001) Effect of climate on the seasonal endocrine profile of native and crossbred sheep under semiarid conditions. \u003cem\u003eTrop Anim Health Prod\u003c/em\u003e 33:241\u0026ndash;252. https://doi.org/10.1023/A:1010318922445.\u003c/li\u003e\n\u003cli\u003eGiorgi G, Lionello P (2008) Climate change projections for the Mediterranean region. \u003cem\u003eGlob Planet Change\u003c/em\u003e 63(2\u0026ndash;3):90\u0026ndash;104. https://doi.org/10.1016/j.gloplacha.2007.09.005.\u003c/li\u003e\n\u003cli\u003eIPCC (2013) \u003cem\u003eClimate Change 2013: The Physical Science Basis\u003c/em\u003e. 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Teubner, Stuttgart.\u003c/li\u003e\n\u003cli\u003eWise ME, Armstrong DV, Huber JT, Hunter R, Wiersma F (1988) Hormonal alterations in lactating dairy cows in response to thermal stress. \u003cem\u003eJ Dairy Sci\u003c/em\u003e 71(9):2480\u0026ndash;2485. https://doi.org/10.3168/jds.s0022-0302(88)79834-3.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Heat stress, Sheep breeds, Physiological responses, Hematological and biochemical parameters, Hormonal changes, Tunisia","lastPublishedDoi":"10.21203/rs.3.rs-8610819/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8610819/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat stress is a major environmental challenge for small ruminant production in Mediterranean and arid areas. This study investigated the effects of seasonal heat stress on physiological, hematological, hormonal, and biochemical parameters in Tunisian sheep raised in northern Tunisia. Thirty multiparous ewes (10 per breed) from White Fat-tailed Tunisian Barbarin (BTR), Fine-tailed Tunisian Western (QFO), and Fine-tailed Thibar Black (NTB) breeds were evaluated during a thermo-neutral period and a heat stress period characterized by elevated temperature\u0026ndash;humidity index (THI) values.\u003c/p\u003e \u003cp\u003eRectal temperature, respiration rate, heart rate, and skin temperature were recorded, with skin temperature measured at selected anatomical sites. Blood samples collected during both periods were analyzed for hematological indices, plasma concentrations of thyroxine, triiodothyronine, cortisol, and thyroid-stimulating hormone, as well as biochemical parameters including glucose, cholesterol, total proteins, and alkaline phosphatase. Data were analyzed using analysis of variance.\u003c/p\u003e \u003cp\u003eHeat stress significantly increased rectal temperature, respiration rate, and heart rate in all breeds, indicating activation of thermoregulatory mechanisms. The NTB breed exhibited the greatest increase in rectal temperature, suggesting higher sensitivity to heat stress. Hematological and biochemical responses to increasing THI included elevated red blood cell count, hemoglobin, cholesterol, and total protein concentrations, along with decreases in mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, glucose, and alkaline phosphatase. Heat stress significantly reduced thyroxine and cortisol concentrations, whereas triiodothyronine remained unaffected. Thyroid-stimulating hormone was significantly altered only in NTB ewes.\u003c/p\u003e \u003cp\u003eThese findings demonstrate breed-specific adaptive responses to heat stress and provide relevant information for sheep management and selection under Mediterranean climatic conditions.\u003c/p\u003e","manuscriptTitle":"Seasonal Heat Stress Effects on the Physiological, Hematological, Hormonal, and Biochemical Responses of Tunisian Sheep Breeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 17:07:25","doi":"10.21203/rs.3.rs-8610819/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-17T12:17:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-17T12:09:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T06:41:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2026-01-16T03:29:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"636f0ec5-ac13-42b9-8f48-c4f843399dc2","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T17:07:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 17:07:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8610819","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8610819","identity":"rs-8610819","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}