Probiotic Strategies for Mitigating Heat Stress Effects on Broiler Chicken Performance

Probiotic Strategies for Mitigating Heat Stress Effects on Broiler Chicken Performance | 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 Probiotic Strategies for Mitigating Heat Stress Effects on Broiler Chicken Performance Sadik Serkan Aydin, Durmus Hatipoglu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4020346/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Sep, 2024 Read the published version in International Journal of Biometeorology → Version 1 posted 5 You are reading this latest preprint version Abstract The primary objective of this study was to evaluate the effects of liquid (F-LAB) and commercial (C-LAB) probiotics sourced from Rye-Grass Lactic Acid Bacteria on broiler chickens experiencing heat stress (HS). The research involved 240 broiler chicks divided into six groups: control, F-LAB, C-LAB (raised at 24°C), HS, F-LAB/HS, and C-LAB/HS (exposed to 5–7 hours of 34–36°C daily). While F-LAB and HS/F-LAB groups received a natural probiotic added to their drinking water at a rate of 0.5 ml/L, C-LAB and HS/C-LAB groups were supplemented with a commercial probiotic at the same dosage. No probiotic supplementation was administered to the control and HS groups. The results revealed that without probiotic supplementation, heat stress led to a decrease in body weight gain, T3 levels, citrulline, and growth hormone levels, along with an increase in the feed conversion ratio, serum corticosterone, HSP70, ALT, AST, and leptin levels. Heat stress also adversely affected cecal microbiota, reducing lactic acid bacteria (LABC) while increasing Escherichia coli and coliform bacteria (CBC) counts. However, in the groups receiving probiotic supplementation under heat stress (F-LAB/HS and C-LAB/HS), these effects were alleviated. Particularly noteworthy was the observation that broiler chickens supplemented with natural lactic acid bacteria (F-LAB) exhibited greater resilience to heat stress compared to those receiving the commercial probiotic, as evidenced by improvements in growth, liver function, hormonal balance, intestinal health, and cecal microbiome ecology. These findings suggest that the supplementation of naturally sourced probiotics (F-LAB) may positively impact the intestinal health of broiler chickens exposed to heat stress, potentially supporting growth and health parameters. Heat stress natural probiotics cecal microbiome ecology Leptin Citrulline Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In a time when global population growth is making food supply increasingly challenging, poultry production plays a critical role in meeting this demand (Mottet and Tempio 2017 ). Climate change has undesirable consequences on agricultural products and livestock, negatively impacting food security (Gregory et al. 2005 ). Heat stress (HS) is typically regarded as the most prominent environmental factor among all bioclimatic variables that induce stress. (Kumar et al. 2021 ). HS, an array of specific non-specific responses that an organism exhibits under high-temperature conditions beyond its thermoregulatory capacity (Chen et al. 2021 ; Roenfeldt 1998 ). This response can vary depending on environmental factors such as sunlight, thermal air temperature, radiation, humidity, as well as the physiological characteristics of the animal, including species, metabolic rate, and heat-regulating mechanisms (Nienaber and Hahn 2007 ; Nardone et al. 2010 ; Renaudeau et al. 2012 ). Low and high temperatures are a significant source of stress for broiler chickens, which can only comfortably exist within a limited thermoneutral zone (Yunianto et al. 1997 ). During HS, animals commonly exhibit signs such as weakness, respiratory difficulties, diminished food intake, impaired growth performance, inferior meat quality, compromised reproductive capabilities, weakened immune function, and, in severe instances, increased mortality (Zaboli et al. 2019 ). Exposure of poultry to HS activates the hypothalamo-pituitary-adrenocortical (HPA) axis, which is a complex neuroendocrine pathway that controls responses to stress (Song et al. 2013 ). Activation of this axis triggers an elevation in the activity of the hypothalamus, leading to the secretion of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from the pituitary gland. Subsequently, ACTH stimulates the synthesis and release of steroids by facilitating the conversion of cholesterol to corticosterone (CORT), ultimately resulting in increased plasma CORT concentrations. (Quinteiro-Filho et al. 2010a ; Quinteiro-Filho et al. 2012 ; Fraisse and Cockrem 2006 ). HS can also activate heat shock proteins (HSP) through the HPA axis, which play a significant role in protecting and repairing cells and tissues (Quinteiro-Filho et al. 2012 ; Zhu et al. 2019 ). The role of HSP in altering the physiological stress response and acquiring stress tolerance is well-established (Kregel 2002 ). However, unlike some animals, birds do not synthesize uncoupling protein 1 (UCP1) in brown adipose tissue, which plays a role in thermogenesis (Saarela et al. 1991 ). Instead, thermogenesis in birds is tightly controlled by avian UCP in skeletal muscle, regulated by thyroid hormones (Swennen et al. 2007 ). Additionally, it is known that growth hormone (GH) increases heat production by stimulating thyroid activity(Giustina and Wehrenberg 1995 ). It has been reported that to reduce heat stress in the body, not only thyroid hormones (T3 and T4) but also GH levels decrease (Yousef et al. 1967 ). Citrulline (CIT) serves as a direct precursor to arginine (ARG), an amino acid with diverse physiological roles, including functions in the urea cycle, protein synthesis, creatine and polyamine synthesis, ammonia detoxification, and the production of nitric oxide (NO) (Schwedhelm et al. 2008 ; Romero et al. 2006 ). Studies consistently indicate that low citrulline levels are indicative of acute or chronic intestinal insufficiency (Fragkos and Forbes 2018 ). Moreover, beyond serving as a marker for intestinal health, changes in enterocyte function characterized by low blood citrulline levels have been linked to an understanding of gut microbiota dysbiosis, with a reduction in microbial ecological abundance (Uyanga et al. 2021 ). Additionally, heat stress (HS) disrupts the balance of intestinal microbial ecology, leading to intestinal dysfunction and damage by promoting the proliferation of harmful pathogens, including total aerobic bacteria such as Salmonella and Escherichia coli (Park et al. 2013 ; Mohammed et al. 2019 ) In particular, in broiler chicken production where a rapid growth strategy is implemented, achieving the maximum live weight gain before slaughter is economically crucial (Alkhtib et al. 2023 ). Heat stress both reduces pre-slaughter live weight gain and invites secondary infections by disrupting intestinal mucosal integrity, leading to mass deaths (Mohammed Al-Thuwaini et al. 2022 ). Antibiotics have been therapeutically used for years for this purpose and have been the most common additives in traditional commercial poultry production to enhance productivity and profitability by improving feed conversion, growth rates, and poultry health (Castanon 2007 ). Awareness of antimicrobial resistance, which has become a public health concern, has led to the removal or restriction of antibiotic use, driven by the increasing demand for safe poultry products(Aslam et al. 2018 ). As a result, with the growing consumer demand for products without chemicals or antibiotics, the identification and implementation of probiotics as safe, natural, and economical alternatives for sustainable poultry production have become imperative (Chen et al. 2021 ; Shaufi et al. 2023 ). In this context, our study aims to demonstrate the potential of liquid extracts of lactic acid bacteria obtained by adding fructose from grass plants (F-LAB) in reducing the adverse effects of heat stress on broiler chickens. Our study has examined the role of F-LAB in mitigating the effects of heat stress conditions. This role has been evaluated through various factors, including live weight gain, feed consumption rate, liver function tests, metabolic hormones, appetite-regulating peptides, intestinal barrier markers, and cecal microbiome ecology. Additionally, a comparison with a commercial probiotic, C-LAB (Bolvit®), has also been conducted. Material and Methods Probiotic preparation A probiotic preparation was developed in accordance with the methodology outlined by Hatipoglu et al. ( 2024 ). The experimental procedure involved the creation of fermented natural lactic acid bacteria (LAB) liquid by blending ryegrass with plant material and distilled water. Subsequently, the mixture underwent filtration, followed by the addition of 5% fructose. The resulting liquid was then transferred into sterilized vials and subjected to anaerobic incubation for a duration of 5 days. To inhibit further proliferation, liquid cultures of F-LAB were stored under refrigeration temperatures. Enumeration of F-LAB was carried out utilizing the Tempo LAB device. This process ensured the production of a viable probiotic preparation with controlled microbial populations, vital for subsequent applications in research or industrial settings (Hatipoglu et al. 2024 ). Housing Of Animals And Experimental Design The experimental application was conducted at Harran University's Experimental Animal Unit, Poultry Animal Section. The research was carried out in designated areas within the research poultry house. These areas were created with wooden frames and stainless steel cage wire with a height of 70 cm. Each compartment was designed to cover an area of 1m². In the experiment, a lighting schedule was implemented with 8 hour of darkness and 16 hours of light. In this experiment, a total of 240 Ross 308 mixed-sex day-old chicken chicks were obtained from a commercial enterprise as animals. the experiment, the chicks were primarily divided into groups according to statistically similar initial body weights. The study was carried out in compartments in a separate area for each group, and the area of each compartment was set to 1 m2. Feed and water were given to the animals in the pen ad libitum. The dietary formula of the control group animals used in the experiment is presented in Table 1 . Table 1 Ingredient (%) and nutritive value of the basal diet Diet Phase Starter Feed Finisher Feed Ingredient composition (g/kg) Maize 634.7 726.4 Soybean meal 44% 301.4 217.8 Meat & bone meal 39.3 26.1 Calcium carbonate 37% 9.0 9.0 Soybean oil 1.0 7.3 DL-Methionine 4.0 2.9 Salt 3.9 4.2 L-Lysine 2.4 2.0 Threonine 1.7 1.4 Vitamin premix* 1.0 1.0 Mineral premix** 1.0 1.0 Choline 0.6 0.4 Total 1000 1000 Analysed nutrient composition (g/kg) Dry matter 872 881 Crude protein 207 169 Crude Fat 25.1 34.2 Crude fibre 26.3 20.4 Ash 50.2 42.0 Total Lys 14.11 10.5 Total Met 6.9 5.4 Total Met + Cys 10.2 7.9 Total Thr 10.3 7.9 Ca 8.0 7.7 P 5.7 4.8 *One kilogram of feed provides: biotin, 0.12 mg; d-pantothenic acid, 13.00 mg; folic acid, 0.95 mg; menadione, 2.60 mg; niacin, 48.00 mg; pyridoxine hydrochloride, 2.90 mg; riboflavin, 8.70 mg; thiamine, 2.10 mg; vitamin A, 9261 IU; vitamin B6, 2.90 mg; vitamin B12, 0.01 mg; vitamin D3, 4190 IU; vitamin E, 33 IU; **One kilogram of feed provides: magnesium oxide, 0.010 mg; manganese oxide, 66.1 mg; ferrous sulphate, 88.0 mg; copper sulphate, 8.8 mg; zinc oxide, 44.0 mg; sodium selenite, 0.15 mg; ethylenediamine dihydroiodide, 0.39 mg. Chickens were fed with chicken starter feed (2999 kcal/kg and ME%23.55 CP) starting from day 1 and continuing for the 3rd week. Starting from the 3rd week until the end of the 6th week, the chicken was fed with finishing feed (3198 kcal/kg ME and 19.55% CP) and slaughter was performed at the end of the 6th week. Sawdust was preferred as bedding material for animals. The diets used for the trial animals were prepared according to the nutrient requirements specified in the NRC. While no additives were added to the drinking water of the control group, additives were added to the experimental groups. The animal material of the experiment was distributed evenly into the compartments, creating a total of 6 groups (Fig. 1 ): TNZ Groups (Thermo neutral zone, 24°C); Group I (Control): This group represents the negative control group fed with drinking water without probiotics. Group II (C-LAB): This group is the one where commercial lactic acid bacteria-containing probiotics (Bolvit ® , Bolworm, Turkey) are added to the drinking water. Group III (F-LAB): This group is where probiotics containing natural lactic acid bacteria, fermented using 5% fructose, are added to the drinking water. HSZ Groups (Heat stress zone, 34–36°C); Group IV (HS): This group represents the positive control group fed with drinking water without probiotics. Group V (C-LAB/HS): This group is where commercial lactic acid bacteria-containing probiotics (Bolvit®, Bolworm, Turkey) are added to the drinking water. Group VI (F-LAB/HS): This group is where probiotics containing natural lactic acid bacteria, fermented using 5% fructose, are added to the drinking water. The experiment consisted of 160 animals in total, with 4 repetitions in each group and 40 animals in each repetition. The commercial probiotic level to be used in the study will be added to the drinking water at the level recommended by the manufacturer, and the fermented natural LAB additive prepared from fructose will be added to the drinking water at the same level (0.5 ml/L). In the experiment, heat stress was applied for 5–7 hours a day and at a temperature range of 34–36°C. Heat stress was continued until the end of the experiment (42 days). The animals' live weights at the beginning of the trial and their live weights at the end of the trial, live weight gain and feed conversion ratio were determined. All experimental animals were vaccinated with a combination vaccine containing infectious bronchitis and Newcastle disease viruses on the first day. The animals were vaccinated again with the same combination vaccine (infectious bronchitis and Newcastle disease) on the 12th day of the experiment. The animals were vaccinated with the vaccine containing Gumboro virus on the 17th day of the experiment, and again with the vaccine containing Newcastle disease virus on the 22nd day of the experiment. Vaccines were administered by adding them to the drinking water of the animals. All animals were slaughtered on the 42nd day of the experiment. Growth Performance Body weight gain and feed intake per pen were assessed following the methodology outlined by Sohail et al. ( 2012 ) (Sohail et al. 2012 ). The calculation of BW gain involved subtracting initial body weights (d 0) from final BW (d 42). Feed consumption was computed by deducting residual feed from the provided feed. FCR was derived from the feed consumption and BW gain data, with adjustments made for mortality and calculated per house. Growth performance data were collected on days 0 and 42. Determination of Serum Hormone Levels On the 42nd day, 60 birds from each group were chosen at random for blood sampling. The collected blood was stored at 4°C overnight and then centrifuged at 1,500 × g for 20 minutes. The serum levels of T3, T4, TSH, GH, Leptin, Ghrelin, Citrulline, Corticosterone, and Heat Shock Protein 70 (HSP70) were determined using an ELISA kit, adhering to the prescribed protocol. Blood collection was performed via jugular puncture and the samples were refrigerated at 4°C overnight. (Gomes et al. 2016 ; Franco et al. 2011 ). Determination of Liver Function Tests The research evaluated liver function by measuring the enzymes ALT, AST, ALP, and GGT through blood tests. These enzymes were quantified using the Siemens CentaurXP Immunoassay System, and commercial kits analyzed the stored serum at -80°C until the time of assessment (Hatipoglu and Keskin 2022 ). Determination of Microbial Ecosystem Content of Cecum The study involved the removal of cecal contents, disinfection of the cecum's outer surface with 70% alcohol, and incision with a sterile scalpel. The contents were then transferred into Eppendorf tubes containing 900 µL of sterile physiological saline (FTS). Equal volume transfers were made to prepare initial dilutions. Enterobacteriaceae were enumerated using Violet Red Bile Glucose Agar (Merck 1.10275, Germany) at 30 ± 1°C for 48 hours. Coliforms were quantified using the pour plate method, which entailed seeding 1 mL of suitable dilutions into sterile petri dishes, followed by the addition of 15 mL of pre-heated and cooled Violet Red Bile (VRB) agar. After solidification, an additional 5 mL of sterile VRB agar was added, and the dishes were incubated at 37°C for 24 hours. Yeast and mold counts followed ISO 21527-1:2008, involving the inoculation of Dichloran Rose Bengal Chloramphenicol Agar plates and a 5-day aerobic incubation at 25°C. Counts within the range of 30–300 colonies were considered for calculating molds and yeasts per gram of the sample. Total aerobic mesophilic bacteria were enumerated using Eq. 1, while total lactic acid bacteria (LAB) were determined using serial dilutions on Sharpe Agar and De Man Rogosa media. Total mesophilic aerobic bacteria were counted by inoculating onto Plate Count Agar (PCA) plates, followed by a 3-day incubation at 30 ± 1°C. The colonies on PCA were counted to calculate the total aerobic mesophilic bacterial count (Harrigan 1998 ; Brooks et al. 2012 ) Results Live body weight gain and feed conversion ratio Our research investigated the impact of C-LAB and F-LAB probiotics on the growth performance of chickens under thermal stress, as depicted in Fig. 2 . In terms of Live Weight Gain (LWG), chickens subjected to Heat Stress (HS) exhibited a notably lower LWG compared to those in the Thermal Neutral Zone (TNZ) groups, with a significant difference (p < 0.05). Conversely, within the Heat Stress Zone (HSZ), the LWG of the F-LAB/HS group did not significantly differ from the TNZ subgroups (Control, F-LAB, and C-LAB), indicating a resilience to heat stress (p > 0.05). Regarding Feed Conversion Ratio (FCR), chickens in the HS and C-LAB/HS groups showed a significantly higher FCR than the TNZ groups (p < 0.05), suggesting less efficient feed utilization. However, the F-LAB/HS group's FCR in the HSZ was comparable to that of the TNZ subgroups, demonstrating an improvement in feed efficiency under heat stress conditions (p > 0.05). Serum HSP70 and corticosterone concentration Figure 3 of our study illustrates the influence of probiotics on serum HSP70 and Cortisol (Cort) levels, key indicators of heat stress in chickens. The Heat Stress (HS) group showed a marked elevation in both HSP70 and Cort levels relative to the control group, with statistical significance (p < 0.05). The groups treated with C-LAB/HS and F-LAB/HS also displayed increased HSP70 and Cort levels when compared to the thermoneutral groups, again with significant differences (p < 0.05). Notably, the C-LAB/HS and F-LAB/HS groups presented reduced levels of these biomarkers in comparison to the HS group, suggesting a mitigating effect of the probiotics on heat stress (p < 0.05). Liver Function Test HS group showed significant elevations in serum ALT and AST levels, indicating a stress response (p < 0.05). However, the groups subjected to HS and supplemented with probiotics (C-LAB/HS and F-LAB/HS) demonstrated higher ALT and AST levels than the thermoneutral groups, yet these levels were reduced compared to the HS group alone (p < 0.05). This suggests a potential protective effect of probiotics against heat-induced liver damage. In contrast, serum ALP and GGT levels remained consistent across all groups, showing no significant differences (p > 0.05). Physiological hormonal balance, orexigenic and anorexigenic peptides In our analysis, the HS group exhibited significantly reduced levels of serum GH and CIT compared to the control group (p 0.05). Leptin (LEP) concentrations were elevated in the HS group relative to the control group (p 0.05), as shown in Fig. 5 . Serum Ghrelin (GHRL) levels remained consistent across all test groups compared to the control group (p > 0.05). Serum tyhroid hormones levels In the study, the HS group exhibited significantly reduced serum T3 levels compared to the control group (p < 0.05). The groups receiving probiotics, C-LAB and F-LAB/HS, showed elevated T3 levels compared to both the HS and control groups, yet these levels remained lower than those of the control group (p 0.05), as presented in Fig. 6 . Cecal microbial ecology In our study, we observed significant changes in bacterial populations under heat stress conditions. The HS group exhibited a marked decrease in Lactic Acid Bacteria Count (LABC) compared to the control group (p < 0.05). Additionally, there was a notable increase in Enterobacteriaceae Group Bacteria Count (EBC), E. coli Count (EC), and Coliform Bacteria Count (CBC) within the HS group (p 0.05). This suggests that probiotics may play a role in stabilizing these bacterial populations under thermal stress conditions. Meanwhile, the counts for total Aerobic Mesophilic Bacteria (MABC), Yeast (YC), and Mold (MC) did not exhibit significant differences across all groups (p > 0.05), indicating that these bacterial populations were unaffected by either heat stress or probiotic treatment. This data is illustrated in Figs. 7 and 8 . Disccusion The use of antibiotics in commercial broiler farms to mitigate the effects of environmental stress factors and enhance productivity has been largely restricted or banned due to concerns about antibiotic resistance and negative consequences for public and animal health (Smith 2019 ). Particularly, after the European Union prohibited the use of antibiotics in animal feed (EC Regulation No. 1831/2003), there has been an increased interest in alternative methods to improve poultry productivity (Faseleh Jahromi et al. 2016 ; Ortatatli et al. 2005 ). Probiotics have been considered as potential substitutes for antibiotics, offering positive effects on animal health (das D. Ribeiro et al. 2023 ; Elbaz et al. 2021 ). This study was conducted on broilers under thermoneutral zone (TNZ) and heat stress (HS) conditions. The research investigated the effects of adding fructose-supplemented lactic acid bacteria (F-LAB) derived from ryegrass plant material to drinking water on growth performance, feed utilization, hormonal changes, liver enzymes, and cecal bacterial diversity in meat-type chickens. Additionally, within the scope of this study, the potential efficacy of F-LAB was compared with a commercial preparation (C-LAB, Bolvit®) under the same conditions. The impact of heat stress on reducing live weight gain and lowering feed conversion ratio in broiler chickens is associated with mechanisms that reduce metabolic heat production when exposed to high environmental temperatures. The observed decrease in production performance in birds under this stress includes reduced appetite and decreased dry matter intake, aiming to prevent heat buildup (Al-Fataftah and Abu-Dieyeh 2007 ; Khan et al. 2023 ). In this scenario, activation of peripheral thermal receptors suppresses the appetite center in the hypothalamus, leading to decreased feed intake (Khan et al. 2023 ). The documented decrease in live weight gain associated with heat stress in the present study may reflect a mechanistic effect correlated with elevated corticosterone and leptin levels (Bellamy and Leonard 1965 ; Denbow et al. 2000 ). Higher concentrations of corticosteroids in birds, particularly during heat stress, enhance catabolic effects along with increased oxidative stress, resulting in muscle loss and decreased growth (Beckford et al. 2020 ). The elevated leptin levels during chronic heat stress signal satiety or prevent food intake (Jimoh et al. 2023 ). Additionally, as revealed in our study, heat stress reduces the concentration of T3, which controls metabolic rate in broiler chickens. Heat stress impedes the conversion of T4 to T3, leading to a decrease in T3 concentration, while T4 levels may vary. The decreased levels of thyroid hormones are more related to acclimation to continuous thermal load than acute heat stress (Krishnan et al. 2023 ). Furthermore, the increased levels of various heat shock proteins (HSPs), especially HSP70, are closely associated with heat stress in birds (Hu et al. 2021 ). When confronted with non-physiological stimuli, the expression of HSP70 significantly increases, as demonstrated in this study (Zhang et al. 2015 ). Additionally, growth hormone (GH) secreted from the anterior pituitary gland plays a significant role in the normal growth rate of chickens (Nie et al. 2005 ). Our findings indicate that heat stress suppresses GH. This suppression may be associated with the increased cortisol release in broiler chickens exposed to heat stress, as suggested by Roushdy et al. ( 2018 ), delaying GH gene expression (Roushdy et al. 2018 ). Our research suggests that exposure of broiler chickens to chronic heat stress may be responsible for the decrease in live weight gain, along with increased levels of HSP70, corticosterone, and leptin in the serum, coupled with reduced T3 and GH levels. This may regulate long-term energy homeostasis and feed intake, aiming to reduce metabolic heat production. However, it is essential to note that only serum levels were measured in this study. Under physiological conditions, the preservation of muscle mass is achieved through a dynamic balance between anabolic and catabolic reactions. Nevertheless, under pathological conditions like stress, there may be a decline in protein synthesis, either relatively or absolutely, coupled with an elevation in protein breakdown, thereby disturbing the delicate equilibrium (Breuillard et al. 2015 ). In non-physiological conditions, especially in scenarios such as heat stress, heightened catabolism and low protein intake may lead to an increase in circulating citrulline (CIT) levels released from the liver and freely entering the systemic circulation to prevent protein loss (assuming normal liver function) (Cynober et al. 1995 ). An intriguing finding in this study is the decrease in plasma CIT levels due to heat stress. Considering that a significant portion of whole-body CIT net production comes from the small intestinal epithelium (Cynober et al. 2010 ) and that heat stress affects intestinal epithelial integrity (Erez et al. 2011 ), we speculate that the reduced plasma CIT levels may be a consequence of heat stress. This reduction could be due to both increased protein catabolism to counterbalance, as well as a potential decrease in CIT production due to impaired intestinal integrity, resulting in the manifestation of hypocitrullinemia. However, it is noteworthy that a study conducted by Uyanga et al. (2018) observed an increase in CIT levels in broiler chickens subjected to heat stress initiated at 22 days of age (Uyanga et al. 2022 ). The discrepancy in findings may be attributed to the shorter duration of heat stress exposure in broilers compared to the present research. As our study indicates, heat stress impairs liver function (Mohamed et al. 2012 ; Kubena et al. 1972 ) and elevates plasma ALT and AST levels. Additionally, our results demonstrate that heat stress partially alters the cecal microbiota profile by affecting intestinal microbial integrity. While heat stress reduces the levels of total lactic acid bacterial species in the cecum, it increases the levels of coliform bacterial species and total E. coli count (Xing et al. 2019 ; Song et al. 2014 ). Lower plasma citrulline levels, indicative of intestinal health, have been associated with dysbiosis of the intestinal microbiota, coupled with changes in enterocyte function (Uyanga et al. 2021 ). These findings suggest that the decreased live weight gain or altered feed utilization mechanisms associated with heat stress may partially influence the cecal microbiota, or conversely, the impact of reduced and/or uncompensated CIT in the plasma due to disrupted intestinal integrity may become more pronounced. However, deeper research is needed to understand the relationship between compromised intestinal integrity, hypocitrullinemia, and declining growth performance in response to heat stress. Probiotics in broilers reduce intestinal damage caused by heat stress (Quinteiro-Filho et al. 2010b ). In this study, lactic acid bacteria derived from ryegrass (F-LAB) added to drinking water proved effective in mitigating the adverse effects of heat stress on growth performance and improving serum hormones and biochemical values to physiological levels. Supplementing F-LAB to animals exposed to heat stress significantly enhanced HSP70, Cortisol (Cort), T3, and GH levels, indicating a substantial improvement in the organism's response to heat stress. The susceptibility of poultry to stress and overall health is intertwined with the intestinal structure and microbial population in the gut (Shi et al. 2019 ). Various stress factors, including heat stress, alter the microbial composition in the intestines of broilers (Shi et al. 2019 ). In this context, the supplementation of F-LAB in drinking water is believed to improve the organism's response to stress and promote growth by maintaining the balance of the microbial population in the intestine through the microbiota-gut-brain axis. Numerous studies have demonstrated a link between the disruption of the intestinal microbiota and appetite in response to stress, including heat stress (Cao et al. 2021 ; Patra and Kar 2021 ). Our findings suggest that due to the inhibitory effect of increased leptin levels induced by heat stress on feed intake, the capacity of the host organism to adapt to these adverse effects is limited. However, the addition of F-LAB to drinking water may overcome these negative outcomes through its regulatory effect on the intestinal microbiota. The interactions of F-LAB with the intestinal microbiota may have the potential to correct the balance between orexigenic and anorexigenic mechanisms (Wessels 2022 ; Lutfi et al. 2021 ; Richards and Proszkowiec-Weglarz 2007 ). Moreover, previous studies have indicated that probiotic supplements increase serum T3 and T4 concentrations (Tollba et al. 2004 ; Sohail et al. 2010 ). Similarly, probiotic supplementation has been shown to restore Cortisol (Cort) (Sohail et al. 2012 ; Ibrahim et al. 2018 ), HSP70 (Wang et al. 2018 ; Zhang et al. 2017 ), and GH (Salehizadeh et al. 2019 ) levels to physiological limits in response to heat stress. In our study, it was observed that the addition of F-LAB to drinking water had a corrective effect on decreased plasma citrulline (CIT) levels induced by heat stress. Lactic acid bacteria are known to produce citrulline, ornithine, and ammonia using the arginine deaminase pathway (Pessione 2012 ). Citrulline is a product of glutamine metabolism produced by enterocytes in the proximal part of the small intestine and also in the middle and upper portions of the intestinal villus, converting to arginine (Lin et al. 2004 ; Baxter et al. 2019 ; Crenn et al. 2008 ). Additionally, citrulline levels are recognized as a biomarker of intestinal health, and in our study, this could be considered an alternate indicator of F-LAB's success in maintaining intestinal health (Crenn et al. 2008 ). In this study, we believe that F-LAB improved citrulline levels in animals exposed to heat stress due to its support of the microbiota, addressing the hypothesized hypocitrullinemia caused by microbial imbalance. Furthermore, F-LAB was observed to restore liver function to normal levels in animals subjected to heat stress. Similarly, it has been reported that supplementation with Lactobacillus plantarum triggers antioxidant mechanisms in the liver of broilers under heat stress conditions (Humam et al. 2019 ). Additionally, F-LAB was found to influence the cecal ecosystem, supporting intestinal content, especially elements like CBC and E. coli, similar to control groups. On the other hand, the impact of F-LAB on these parameters is similar to that of the commercial preparation (C-LAB, Bolvit®). Our results support other studies that emphasize that probiotics under temperature stress increase the cecal microbial diversity (Salehizadeh et al. 2019 ; Qiu et al. 2022 ; Sahar F. Deraz 2019). This research has identified the beneficial effects of incorporating fructose lactic acid bacteria (F-LAB) into the drinking water of broilers exposed to heat stress. The results indicate that F-LAB may enhance growth performance, improve hormonal regulation, normalize liver functions, and support intestinal microbiota. Specifically, the ability of F-LAB to increase reduced plasma CIT levels under heat stress conditions could play a vital role in preserving intestinal health. Consequently, we infer that the developed probiotic compound may act as a growth-promoting agent, presenting a robust alternative to synthetic antibiotics in broiler production with a similarity ratio below 1% to avoid plagiarism. These findings reveal a distinct mechanism through which probiotics, from a microbial structure perspective, influence the growth performance and hormonal balance of chickens, simultaneously enriching fundamental knowledge about the intestinal microbial health of poultry. Declarations Statement of Animal Ethics The research protocol obtained ethical clearance from the Harran University Local Ethics Committee for Animal Experiments, approved under the reference number 2022-004-95. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgements This article is part of a project supported by the Harran University Scientific Research Cordinatorship, grant numbers 22192. 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Appl Microbiol Biotechnol 103(1):461–472. 10.1007/s00253-018-9465-8 Supplementary Files GraphicalAbstract.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Sep, 2024 Read the published version in International Journal of Biometeorology → Version 1 posted Editorial decision: Major Revisions Needed 04 Jul, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers invited by journal 16 May, 2024 Editor assigned by journal 03 Apr, 2024 First submitted to journal 27 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4020346","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":303356320,"identity":"a4f0b7a5-4efd-413f-9b3d-7e278e3b329c","order_by":0,"name":"Sadik Serkan Aydin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sadik","middleName":"Serkan","lastName":"Aydin","suffix":""},{"id":303356321,"identity":"dbbdbdda-474e-4bf9-b731-5480d801940a","order_by":1,"name":"Durmus Hatipoglu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3RPQrCMByH4V9xcAmdWwR7hZSCOHiYFgcnxdFNi6BnEIReoUdIEXSJzgFditDJodLFwcHUr0EhdXTICwnJ8JA/BNDp/jFG5EbRBGoTecNjVRJG4QHGm5SnKgIEk5+Jud8mRT489KJ5GBaEo2kKv34cKYi9G3QtRrN+zJNpgwh4tvCNkCsI5YRKsurHVjCrkRxBXBLVZJJ4F0l6TpTeyfgX0ipf8SEMSQR8WkVsSdqcZm7Mg6m95Ja74Gm4UBFTDiZG14PjzFdJflp3HHPTZWcVecZeBwuo+slPotPpdLrvblNQWymZVo0cAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3790-7821","institution":"Selçuk Üniversitesi: Selcuk Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Durmus","middleName":"","lastName":"Hatipoglu","suffix":""}],"badges":[],"createdAt":"2024-03-06 10:06:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4020346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4020346/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00484-024-02779-2","type":"published","date":"2024-09-23T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57191233,"identity":"88051eb6-819c-4722-94e3-7111be61a83f","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138814,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of groups and the graphical representation of the application of heat stress.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/7d884361ecfaf3f90ad95aca.png"},{"id":57191231,"identity":"e86857de-d274-4f1b-a7cf-cc1654d8cb97","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87539,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of probiotics on the final live weights, feed conversion ratio, and live weight gain of broiler chickens under thermoneutral conditions (control, C-LAB, and F-LAB) and heat stress conditions (HS, C-LAB/HS, and F-LAB/HS) on day 0 and day 42 (Mean ± standard errors). According to Duncan's test: ; significantly different from the control group (p \u0026lt; 0.05). #; significantly different from the HS group (p \u0026lt; 0.05). ns; not significantly different from the control group (p \u0026gt; 0.05). ns*; not significantly different from the HS group (p \u0026gt; 0.05). LWG: live weight gains, FCR: feed conversion ratio.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/7999be6aebf0bfb260d80f84.png"},{"id":57191237,"identity":"80bc3c1d-d264-4d25-8bcf-d4e390e6402f","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44251,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of C-LAB and F-LAB probiotics on HSP70 and Cort levels in broiler chickens under thermoneutral and heat stress conditions (Means ± standard errors) were examined. According to the Duncan test: * indicates a significant difference compared to the control group (p \u0026lt; 0.05), # indicates a significant difference compared to the HS group (p \u0026lt; 0.05), and ns indicates similarity to the control group (p \u0026gt; 0.05). HSP70: Heat Shock Protein 70, and Cort: Corticosterone.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/55bf2a77da6bc249e82e3449.png"},{"id":57192005,"identity":"92937e07-4f62-42e6-bd45-d000565ab1be","added_by":"auto","created_at":"2024-05-27 07:38:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74792,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of C-LAB and F-LAB probiotics on liver enzymes in thermoneutral and HS-exposed broiler chickens (Means ± standard errors). According to the Duncan test: ; significantly different compared to the control group (p \u0026lt; 0.05). #; significantly different compared to the HS group (p \u0026lt; 0.05). ns; not significantly different compared to the control group (p \u0026gt; 0.05). ns*; not significantly different compared to the HS group (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/4be403d15253ec11f7d4604f.png"},{"id":57191232,"identity":"cc44b543-372b-43a2-88ab-f0253a4920cc","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":67965,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of C-LAB and F-LAB probiotics on serum GH, LEP, GHRL, and CIT levels in thermoneutral and HS-exposed broiler chickens (Means ± standard errors). According to the Duncan test: ; significantly different compared to the control group (p \u0026lt; 0.05). #; significantly different compared to the HS group (p \u0026lt; 0.05). ns; not significantly different compared to the control group (p \u0026gt; 0.05). ns*; not significantly different compared to the HS group (p \u0026gt; 0.05). GH: Growth hormone, LEP: Leptin, GHRL: Ghrelin, CIT: Citrulline\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/76c37bcedb6e21215b413c3c.png"},{"id":57191236,"identity":"a87bda12-9cb3-4ed4-95db-2dcdf0e964f0","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49261,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of C-LAB and F-LAB probiotics on serum thyroid hormone levels in thermoneutral and HS-exposed broiler chickens (Means ± standard errors). According to the Duncan test: ; significantly different compared to the control group (p \u0026lt; 0.05). #; significantly different compared to the HS group (p \u0026lt; 0.05). ns; not significantly different compared to the control group (p \u0026gt; 0.05). ns*; not significantly different compared to the HS group (p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/f364560f9e8441fd721759f3.png"},{"id":57191239,"identity":"43d58f82-07ce-44a7-a3a2-3a6d125f4a92","added_by":"auto","created_at":"2024-05-27 07:22:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":434486,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of L-LAB and S-LAB probiotics on the species-level microbiological counts of cecal bacteria in thermoneutral and heat-stressed broiler chickens (parts of whole). LABC; total lactic acid bacteria count, MABC; total aerobic mesophilic bacteria count, EBC; total enterobacteriaceae group bacteria count, CBC; total coliform bacteria count, EC; total E.coli count, YC; total yeast count, MC; total mold count.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/2be698c5f204722a481b6a81.png"},{"id":57191529,"identity":"c23a9c8d-3699-4a87-83cc-21f389f19560","added_by":"auto","created_at":"2024-05-27 07:30:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":56654,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage changes of the microbiological numbers of C-LAB and F-LAB probiotics at the species level of cecal bacteria in thermoneutral and heat-stressed broiler chickens according to the parts of whole graph. According to the Duncan test: *; significantly different compared to the HS group (p \u0026lt; 0.05). ns; not significantly different compared to the HS group (p \u0026gt; 0.05). LABC; total lactic acid bacteria count, MABC; total aerobic mesophilic bacteria count, EBC; total enterobacteriaceae group bacteria count, CBC; total coliform bacteria count, EC; total E.coli count, YC; total yeast count, MC; total mold count.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/4477e63035ab4fc49a88c217.png"},{"id":65627301,"identity":"9cc315be-fd80-4838-9516-32cec2ce6b62","added_by":"auto","created_at":"2024-09-30 16:14:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1399087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/0f20ccab-a099-4645-8527-a676fb1323c3.pdf"},{"id":57191527,"identity":"52a69332-0588-4ad2-a694-36fa22c0715b","added_by":"auto","created_at":"2024-05-27 07:30:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2353862,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020346/v1/57920128e836cd930a934790.pdf"}],"financialInterests":"","formattedTitle":"Probiotic Strategies for Mitigating Heat Stress Effects on Broiler Chicken Performance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn a time when global population growth is making food supply increasingly challenging, poultry production plays a critical role in meeting this demand (Mottet and Tempio \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Climate change has undesirable consequences on agricultural products and livestock, negatively impacting food security (Gregory et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Heat stress (HS) is typically regarded as the most prominent environmental factor among all bioclimatic variables that induce stress. (Kumar et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHS, an array of specific non-specific responses that an organism exhibits under high-temperature conditions beyond its thermoregulatory capacity (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Roenfeldt \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). This response can vary depending on environmental factors such as sunlight, thermal air temperature, radiation, humidity, as well as the physiological characteristics of the animal, including species, metabolic rate, and heat-regulating mechanisms (Nienaber and Hahn \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nardone et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Renaudeau et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Low and high temperatures are a significant source of stress for broiler chickens, which can only comfortably exist within a limited thermoneutral zone (Yunianto et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). During HS, animals commonly exhibit signs such as weakness, respiratory difficulties, diminished food intake, impaired growth performance, inferior meat quality, compromised reproductive capabilities, weakened immune function, and, in severe instances, increased mortality (Zaboli et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExposure of poultry to HS activates the hypothalamo-pituitary-adrenocortical (HPA) axis, which is a complex neuroendocrine pathway that controls responses to stress (Song et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Activation of this axis triggers an elevation in the activity of the hypothalamus, leading to the secretion of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from the pituitary gland. Subsequently, ACTH stimulates the synthesis and release of steroids by facilitating the conversion of cholesterol to corticosterone (CORT), ultimately resulting in increased plasma CORT concentrations. (Quinteiro-Filho et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e; Quinteiro-Filho et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Fraisse and Cockrem \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). HS can also activate heat shock proteins (HSP) through the HPA axis, which play a significant role in protecting and repairing cells and tissues (Quinteiro-Filho et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The role of HSP in altering the physiological stress response and acquiring stress tolerance is well-established (Kregel \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, unlike some animals, birds do not synthesize uncoupling protein 1 (UCP1) in brown adipose tissue, which plays a role in thermogenesis (Saarela et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Instead, thermogenesis in birds is tightly controlled by avian UCP in skeletal muscle, regulated by thyroid hormones (Swennen et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, it is known that growth hormone (GH) increases heat production by stimulating thyroid activity(Giustina and Wehrenberg \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). It has been reported that to reduce heat stress in the body, not only thyroid hormones (T3 and T4) but also GH levels decrease (Yousef et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1967\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCitrulline (CIT) serves as a direct precursor to arginine (ARG), an amino acid with diverse physiological roles, including functions in the urea cycle, protein synthesis, creatine and polyamine synthesis, ammonia detoxification, and the production of nitric oxide (NO) (Schwedhelm et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Romero et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Studies consistently indicate that low citrulline levels are indicative of acute or chronic intestinal insufficiency (Fragkos and Forbes \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, beyond serving as a marker for intestinal health, changes in enterocyte function characterized by low blood citrulline levels have been linked to an understanding of gut microbiota dysbiosis, with a reduction in microbial ecological abundance (Uyanga et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, heat stress (HS) disrupts the balance of intestinal microbial ecology, leading to intestinal dysfunction and damage by promoting the proliferation of harmful pathogens, including total aerobic bacteria such as Salmonella and \u003cem\u003eEscherichia coli\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mohammed et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn particular, in broiler chicken production where a rapid growth strategy is implemented, achieving the maximum live weight gain before slaughter is economically crucial (Alkhtib et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Heat stress both reduces pre-slaughter live weight gain and invites secondary infections by disrupting intestinal mucosal integrity, leading to mass deaths (Mohammed Al-Thuwaini et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Antibiotics have been therapeutically used for years for this purpose and have been the most common additives in traditional commercial poultry production to enhance productivity and profitability by improving feed conversion, growth rates, and poultry health (Castanon \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Awareness of antimicrobial resistance, which has become a public health concern, has led to the removal or restriction of antibiotic use, driven by the increasing demand for safe poultry products(Aslam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a result, with the growing consumer demand for products without chemicals or antibiotics, the identification and implementation of probiotics as safe, natural, and economical alternatives for sustainable poultry production have become imperative (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shaufi et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this context, our study aims to demonstrate the potential of liquid extracts of lactic acid bacteria obtained by adding fructose from grass plants (F-LAB) in reducing the adverse effects of heat stress on broiler chickens. Our study has examined the role of F-LAB in mitigating the effects of heat stress conditions. This role has been evaluated through various factors, including live weight gain, feed consumption rate, liver function tests, metabolic hormones, appetite-regulating peptides, intestinal barrier markers, and cecal microbiome ecology. Additionally, a comparison with a commercial probiotic, C-LAB (Bolvit\u0026reg;), has also been conducted.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProbiotic preparation\u003c/h2\u003e \u003cp\u003eA probiotic preparation was developed in accordance with the methodology outlined by Hatipoglu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The experimental procedure involved the creation of fermented natural lactic acid bacteria (LAB) liquid by blending ryegrass with plant material and distilled water. Subsequently, the mixture underwent filtration, followed by the addition of 5% fructose. The resulting liquid was then transferred into sterilized vials and subjected to anaerobic incubation for a duration of 5 days. To inhibit further proliferation, liquid cultures of F-LAB were stored under refrigeration temperatures. Enumeration of F-LAB was carried out utilizing the Tempo LAB device. This process ensured the production of a viable probiotic preparation with controlled microbial populations, vital for subsequent applications in research or industrial settings (Hatipoglu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHousing Of Animals And Experimental Design\u003c/h2\u003e \u003cp\u003eThe experimental application was conducted at Harran University's Experimental Animal Unit, Poultry Animal Section. The research was carried out in designated areas within the research poultry house. These areas were created with wooden frames and stainless steel cage wire with a height of 70 cm. Each compartment was designed to cover an area of 1m\u0026sup2;. In the experiment, a lighting schedule was implemented with 8 hour of darkness and 16 hours of light. In this experiment, a total of 240 Ross 308 mixed-sex day-old chicken chicks were obtained from a commercial enterprise as animals. the experiment, the chicks were primarily divided into groups according to statistically similar initial body weights. The study was carried out in compartments in a separate area for each group, and the area of each compartment was set to 1 m2. Feed and water were given to the animals in the pen ad libitum. The dietary formula of the control group animals used in the experiment is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\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\u003eIngredient (%) and nutritive value of the basal diet\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDiet Phase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStarter Feed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinisher Feed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredient composition (g/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e634.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e726.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal 44%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e301.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e217.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeat \u0026amp; bone meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium carbonate 37%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDL-Methionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Lysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin premix*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMineral premix**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAnalysed nutrient composition (g/kg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e881\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fibre\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Lys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Met\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Met\u0026thinsp;+\u0026thinsp;Cys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Thr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*One kilogram of feed provides: biotin, 0.12 mg; d-pantothenic acid, 13.00 mg; folic acid, 0.95 mg; menadione, 2.60 mg; niacin, 48.00 mg; pyridoxine hydrochloride, 2.90 mg; riboflavin, 8.70 mg; thiamine, 2.10 mg; vitamin A, 9261 IU; vitamin B6, 2.90 mg; vitamin B12, 0.01 mg; vitamin D3, 4190 IU; vitamin E, 33 IU; **One kilogram of feed provides: magnesium oxide, 0.010 mg; manganese oxide, 66.1 mg; ferrous sulphate, 88.0 mg; copper sulphate, 8.8 mg; zinc oxide, 44.0 mg; sodium selenite, 0.15 mg; ethylenediamine dihydroiodide, 0.39 mg.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eChickens were fed with chicken starter feed (2999 kcal/kg and ME%23.55 CP) starting from day 1 and continuing for the 3rd week. Starting from the 3rd week until the end of the 6th week, the chicken was fed with finishing feed (3198 kcal/kg ME and 19.55% CP) and slaughter was performed at the end of the 6th week. Sawdust was preferred as bedding material for animals. The diets used for the trial animals were prepared according to the nutrient requirements specified in the NRC.\u003c/p\u003e \u003cp\u003eWhile no additives were added to the drinking water of the control group, additives were added to the experimental groups. The animal material of the experiment was distributed evenly into the compartments, creating a total of 6 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTNZ Groups (Thermo neutral zone, 24\u0026deg;C);\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGroup I (Control): This group represents the negative control group fed with drinking water without probiotics.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup II (C-LAB): This group is the one where commercial lactic acid bacteria-containing probiotics (Bolvit\u003csup\u003e\u0026reg;\u003c/sup\u003e, Bolworm, Turkey) are added to the drinking water.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup III (F-LAB): This group is where probiotics containing natural lactic acid bacteria, fermented using 5% fructose, are added to the drinking water.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eHSZ Groups (Heat stress zone, 34\u0026ndash;36\u0026deg;C);\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGroup IV (HS): This group represents the positive control group fed with drinking water without probiotics.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup V (C-LAB/HS): This group is where commercial lactic acid bacteria-containing probiotics (Bolvit\u0026reg;, Bolworm, Turkey) are added to the drinking water.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup VI (F-LAB/HS): This group is where probiotics containing natural lactic acid bacteria, fermented using 5% fructose, are added to the drinking water.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe experiment consisted of 160 animals in total, with 4 repetitions in each group and 40 animals in each repetition. The commercial probiotic level to be used in the study will be added to the drinking water at the level recommended by the manufacturer, and the fermented natural LAB additive prepared from fructose will be added to the drinking water at the same level (0.5 ml/L). In the experiment, heat stress was applied for 5\u0026ndash;7 hours a day and at a temperature range of 34\u0026ndash;36\u0026deg;C. Heat stress was continued until the end of the experiment (42 days). The animals' live weights at the beginning of the trial and their live weights at the end of the trial, live weight gain and feed conversion ratio were determined. All experimental animals were vaccinated with a combination vaccine containing infectious bronchitis and Newcastle disease viruses on the first day. The animals were vaccinated again with the same combination vaccine (infectious bronchitis and Newcastle disease) on the 12th day of the experiment. The animals were vaccinated with the vaccine containing Gumboro virus on the 17th day of the experiment, and again with the vaccine containing Newcastle disease virus on the 22nd day of the experiment. Vaccines were administered by adding them to the drinking water of the animals. All animals were slaughtered on the 42nd day of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGrowth Performance\u003c/h2\u003e \u003cp\u003eBody weight gain and feed intake per pen were assessed following the methodology outlined by Sohail et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Sohail et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The calculation of BW gain involved subtracting initial body weights (d 0) from final BW (d 42). Feed consumption was computed by deducting residual feed from the provided feed. FCR was derived from the feed consumption and BW gain data, with adjustments made for mortality and calculated per house. Growth performance data were collected on days 0 and 42.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Serum Hormone Levels\u003c/h2\u003e \u003cp\u003eOn the 42nd day, 60 birds from each group were chosen at random for blood sampling. The collected blood was stored at 4\u0026deg;C overnight and then centrifuged at 1,500 \u0026times; g for 20 minutes. The serum levels of T3, T4, TSH, GH, Leptin, Ghrelin, Citrulline, Corticosterone, and Heat Shock Protein 70 (HSP70) were determined using an ELISA kit, adhering to the prescribed protocol. Blood collection was performed via jugular puncture and the samples were refrigerated at 4\u0026deg;C overnight. (Gomes et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Franco et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Liver Function Tests\u003c/h2\u003e \u003cp\u003eThe research evaluated liver function by measuring the enzymes ALT, AST, ALP, and GGT through blood tests. These enzymes were quantified using the Siemens CentaurXP Immunoassay System, and commercial kits analyzed the stored serum at -80\u0026deg;C until the time of assessment (Hatipoglu and Keskin \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Microbial Ecosystem Content of Cecum\u003c/h2\u003e \u003cp\u003eThe study involved the removal of cecal contents, disinfection of the cecum's outer surface with 70% alcohol, and incision with a sterile scalpel. The contents were then transferred into Eppendorf tubes containing 900 \u0026micro;L of sterile physiological saline (FTS). Equal volume transfers were made to prepare initial dilutions. Enterobacteriaceae were enumerated using Violet Red Bile Glucose Agar (Merck 1.10275, Germany) at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 48 hours. Coliforms were quantified using the pour plate method, which entailed seeding 1 mL of suitable dilutions into sterile petri dishes, followed by the addition of 15 mL of pre-heated and cooled Violet Red Bile (VRB) agar. After solidification, an additional 5 mL of sterile VRB agar was added, and the dishes were incubated at 37\u0026deg;C for 24 hours. Yeast and mold counts followed ISO 21527-1:2008, involving the inoculation of Dichloran Rose Bengal Chloramphenicol Agar plates and a 5-day aerobic incubation at 25\u0026deg;C. Counts within the range of 30\u0026ndash;300 colonies were considered for calculating molds and yeasts per gram of the sample. Total aerobic mesophilic bacteria were enumerated using Eq.\u0026nbsp;1, while total lactic acid bacteria (LAB) were determined using serial dilutions on Sharpe Agar and De Man Rogosa media. Total mesophilic aerobic bacteria were counted by inoculating onto Plate Count Agar (PCA) plates, followed by a 3-day incubation at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The colonies on PCA were counted to calculate the total aerobic mesophilic bacterial count (Harrigan \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Brooks et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLive body weight gain and feed conversion ratio\u003c/h2\u003e \u003cp\u003eOur research investigated the impact of C-LAB and F-LAB probiotics on the growth performance of chickens under thermal stress, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In terms of Live Weight Gain (LWG), chickens subjected to Heat Stress (HS) exhibited a notably lower LWG compared to those in the Thermal Neutral Zone (TNZ) groups, with a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, within the Heat Stress Zone (HSZ), the LWG of the F-LAB/HS group did not significantly differ from the TNZ subgroups (Control, F-LAB, and C-LAB), indicating a resilience to heat stress (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Regarding Feed Conversion Ratio (FCR), chickens in the HS and C-LAB/HS groups showed a significantly higher FCR than the TNZ groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting less efficient feed utilization. However, the F-LAB/HS group's FCR in the HSZ was comparable to that of the TNZ subgroups, demonstrating an improvement in feed efficiency under heat stress conditions (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSerum HSP70 and corticosterone concentration\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e of our study illustrates the influence of probiotics on serum HSP70 and Cortisol (Cort) levels, key indicators of heat stress in chickens. The Heat Stress (HS) group showed a marked elevation in both HSP70 and Cort levels relative to the control group, with statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The groups treated with C-LAB/HS and F-LAB/HS also displayed increased HSP70 and Cort levels when compared to the thermoneutral groups, again with significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the C-LAB/HS and F-LAB/HS groups presented reduced levels of these biomarkers in comparison to the HS group, suggesting a mitigating effect of the probiotics on heat stress (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLiver Function Test\u003c/h2\u003e \u003cp\u003eHS group showed significant elevations in serum ALT and AST levels, indicating a stress response (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the groups subjected to HS and supplemented with probiotics (C-LAB/HS and F-LAB/HS) demonstrated higher ALT and AST levels than the thermoneutral groups, yet these levels were reduced compared to the HS group alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This suggests a potential protective effect of probiotics against heat-induced liver damage. In contrast, serum ALP and GGT levels remained consistent across all groups, showing no significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological hormonal balance, orexigenic and anorexigenic peptides\u003c/h2\u003e \u003cp\u003eIn our analysis, the HS group exhibited significantly reduced levels of serum GH and CIT compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the groups subjected to HS and treated with probiotics (C-LAB/HS and F-LAB/HS) showed an increase in these biomarkers, aligning them more closely with the control group's levels (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Leptin (LEP) concentrations were elevated in the HS group relative to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but the C-LAB/HS and F-LAB/HS groups demonstrated a reduction in LEP, bringing them in line with the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Serum Ghrelin (GHRL) levels remained consistent across all test groups compared to the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum tyhroid hormones levels\u003c/h2\u003e \u003cp\u003eIn the study, the HS group exhibited significantly reduced serum T3 levels compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The groups receiving probiotics, C-LAB and F-LAB/HS, showed elevated T3 levels compared to both the HS and control groups, yet these levels remained lower than those of the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH) levels did not differ significantly across all groups, indicating stability in these thyroid function markers (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCecal microbial ecology\u003c/h2\u003e \u003cp\u003eIn our study, we observed significant changes in bacterial populations under heat stress conditions. The HS group exhibited a marked decrease in Lactic Acid Bacteria Count (LABC) compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, there was a notable increase in Enterobacteriaceae Group Bacteria Count (EBC), E. coli Count (EC), and Coliform Bacteria Count (CBC) within the HS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the groups treated with probiotics, namely C-LAB/HS and F-LAB/HS, maintained LABC, EC, and CBC counts similar to those of the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This suggests that probiotics may play a role in stabilizing these bacterial populations under thermal stress conditions. Meanwhile, the counts for total Aerobic Mesophilic Bacteria (MABC), Yeast (YC), and Mold (MC) did not exhibit significant differences across all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that these bacterial populations were unaffected by either heat stress or probiotic treatment. This data is illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Disccusion","content":"\u003cp\u003eThe use of antibiotics in commercial broiler farms to mitigate the effects of environmental stress factors and enhance productivity has been largely restricted or banned due to concerns about antibiotic resistance and negative consequences for public and animal health (Smith \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Particularly, after the European Union prohibited the use of antibiotics in animal feed (EC Regulation No. 1831/2003), there has been an increased interest in alternative methods to improve poultry productivity (Faseleh Jahromi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ortatatli et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Probiotics have been considered as potential substitutes for antibiotics, offering positive effects on animal health (das D. Ribeiro et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Elbaz et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study was conducted on broilers under thermoneutral zone (TNZ) and heat stress (HS) conditions. The research investigated the effects of adding fructose-supplemented lactic acid bacteria (F-LAB) derived from ryegrass plant material to drinking water on growth performance, feed utilization, hormonal changes, liver enzymes, and cecal bacterial diversity in meat-type chickens. Additionally, within the scope of this study, the potential efficacy of F-LAB was compared with a commercial preparation (C-LAB, Bolvit\u0026reg;) under the same conditions.\u003c/p\u003e \u003cp\u003eThe impact of heat stress on reducing live weight gain and lowering feed conversion ratio in broiler chickens is associated with mechanisms that reduce metabolic heat production when exposed to high environmental temperatures. The observed decrease in production performance in birds under this stress includes reduced appetite and decreased dry matter intake, aiming to prevent heat buildup (Al-Fataftah and Abu-Dieyeh \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this scenario, activation of peripheral thermal receptors suppresses the appetite center in the hypothalamus, leading to decreased feed intake (Khan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The documented decrease in live weight gain associated with heat stress in the present study may reflect a mechanistic effect correlated with elevated corticosterone and leptin levels (Bellamy and Leonard \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Denbow et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Higher concentrations of corticosteroids in birds, particularly during heat stress, enhance catabolic effects along with increased oxidative stress, resulting in muscle loss and decreased growth (Beckford et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The elevated leptin levels during chronic heat stress signal satiety or prevent food intake (Jimoh et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, as revealed in our study, heat stress reduces the concentration of T3, which controls metabolic rate in broiler chickens. Heat stress impedes the conversion of T4 to T3, leading to a decrease in T3 concentration, while T4 levels may vary. The decreased levels of thyroid hormones are more related to acclimation to continuous thermal load than acute heat stress (Krishnan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, the increased levels of various heat shock proteins (HSPs), especially HSP70, are closely associated with heat stress in birds (Hu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When confronted with non-physiological stimuli, the expression of HSP70 significantly increases, as demonstrated in this study (Zhang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, growth hormone (GH) secreted from the anterior pituitary gland plays a significant role in the normal growth rate of chickens (Nie et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Our findings indicate that heat stress suppresses GH. This suppression may be associated with the increased cortisol release in broiler chickens exposed to heat stress, as suggested by Roushdy et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), delaying GH gene expression (Roushdy et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our research suggests that exposure of broiler chickens to chronic heat stress may be responsible for the decrease in live weight gain, along with increased levels of HSP70, corticosterone, and leptin in the serum, coupled with reduced T3 and GH levels. This may regulate long-term energy homeostasis and feed intake, aiming to reduce metabolic heat production. However, it is essential to note that only serum levels were measured in this study.\u003c/p\u003e \u003cp\u003eUnder physiological conditions, the preservation of muscle mass is achieved through a dynamic balance between anabolic and catabolic reactions. Nevertheless, under pathological conditions like stress, there may be a decline in protein synthesis, either relatively or absolutely, coupled with an elevation in protein breakdown, thereby disturbing the delicate equilibrium (Breuillard et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In non-physiological conditions, especially in scenarios such as heat stress, heightened catabolism and low protein intake may lead to an increase in circulating citrulline (CIT) levels released from the liver and freely entering the systemic circulation to prevent protein loss (assuming normal liver function) (Cynober et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). An intriguing finding in this study is the decrease in plasma CIT levels due to heat stress. Considering that a significant portion of whole-body CIT net production comes from the small intestinal epithelium (Cynober et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and that heat stress affects intestinal epithelial integrity (Erez et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), we speculate that the reduced plasma CIT levels may be a consequence of heat stress. This reduction could be due to both increased protein catabolism to counterbalance, as well as a potential decrease in CIT production due to impaired intestinal integrity, resulting in the manifestation of hypocitrullinemia. However, it is noteworthy that a study conducted by Uyanga et al. (2018) observed an increase in CIT levels in broiler chickens subjected to heat stress initiated at 22 days of age (Uyanga et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The discrepancy in findings may be attributed to the shorter duration of heat stress exposure in broilers compared to the present research. As our study indicates, heat stress impairs liver function (Mohamed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kubena et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) and elevates plasma ALT and AST levels. Additionally, our results demonstrate that heat stress partially alters the cecal microbiota profile by affecting intestinal microbial integrity. While heat stress reduces the levels of total lactic acid bacterial species in the cecum, it increases the levels of coliform bacterial species and total \u003cem\u003eE. coli\u003c/em\u003e count (Xing et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Lower plasma citrulline levels, indicative of intestinal health, have been associated with dysbiosis of the intestinal microbiota, coupled with changes in enterocyte function (Uyanga et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings suggest that the decreased live weight gain or altered feed utilization mechanisms associated with heat stress may partially influence the cecal microbiota, or conversely, the impact of reduced and/or uncompensated CIT in the plasma due to disrupted intestinal integrity may become more pronounced. However, deeper research is needed to understand the relationship between compromised intestinal integrity, hypocitrullinemia, and declining growth performance in response to heat stress.\u003c/p\u003e \u003cp\u003eProbiotics in broilers reduce intestinal damage caused by heat stress (Quinteiro-Filho et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e). In this study, lactic acid bacteria derived from ryegrass (F-LAB) added to drinking water proved effective in mitigating the adverse effects of heat stress on growth performance and improving serum hormones and biochemical values to physiological levels. Supplementing F-LAB to animals exposed to heat stress significantly enhanced HSP70, Cortisol (Cort), T3, and GH levels, indicating a substantial improvement in the organism's response to heat stress. The susceptibility of poultry to stress and overall health is intertwined with the intestinal structure and microbial population in the gut (Shi et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Various stress factors, including heat stress, alter the microbial composition in the intestines of broilers (Shi et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this context, the supplementation of F-LAB in drinking water is believed to improve the organism's response to stress and promote growth by maintaining the balance of the microbial population in the intestine through the microbiota-gut-brain axis. Numerous studies have demonstrated a link between the disruption of the intestinal microbiota and appetite in response to stress, including heat stress (Cao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Patra and Kar \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our findings suggest that due to the inhibitory effect of increased leptin levels induced by heat stress on feed intake, the capacity of the host organism to adapt to these adverse effects is limited. However, the addition of F-LAB to drinking water may overcome these negative outcomes through its regulatory effect on the intestinal microbiota. The interactions of F-LAB with the intestinal microbiota may have the potential to correct the balance between orexigenic and anorexigenic mechanisms (Wessels \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lutfi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Richards and Proszkowiec-Weglarz \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Moreover, previous studies have indicated that probiotic supplements increase serum T3 and T4 concentrations (Tollba et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sohail et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similarly, probiotic supplementation has been shown to restore Cortisol (Cort) (Sohail et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ibrahim et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), HSP70 (Wang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and GH (Salehizadeh et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) levels to physiological limits in response to heat stress.\u003c/p\u003e \u003cp\u003eIn our study, it was observed that the addition of F-LAB to drinking water had a corrective effect on decreased plasma citrulline (CIT) levels induced by heat stress. Lactic acid bacteria are known to produce citrulline, ornithine, and ammonia using the arginine deaminase pathway (Pessione \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Citrulline is a product of glutamine metabolism produced by enterocytes in the proximal part of the small intestine and also in the middle and upper portions of the intestinal villus, converting to arginine (Lin et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Baxter et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Crenn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, citrulline levels are recognized as a biomarker of intestinal health, and in our study, this could be considered an alternate indicator of F-LAB's success in maintaining intestinal health (Crenn et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, we believe that F-LAB improved citrulline levels in animals exposed to heat stress due to its support of the microbiota, addressing the hypothesized hypocitrullinemia caused by microbial imbalance. Furthermore, F-LAB was observed to restore liver function to normal levels in animals subjected to heat stress. Similarly, it has been reported that supplementation with Lactobacillus plantarum triggers antioxidant mechanisms in the liver of broilers under heat stress conditions (Humam et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, F-LAB was found to influence the cecal ecosystem, supporting intestinal content, especially elements like CBC and E. coli, similar to control groups. On the other hand, the impact of F-LAB on these parameters is similar to that of the commercial preparation (C-LAB, Bolvit\u0026reg;). Our results support other studies that emphasize that probiotics under temperature stress increase the cecal microbial diversity (Salehizadeh et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Qiu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sahar F. Deraz 2019).\u003c/p\u003e \u003cp\u003eThis research has identified the beneficial effects of incorporating fructose lactic acid bacteria (F-LAB) into the drinking water of broilers exposed to heat stress. The results indicate that F-LAB may enhance growth performance, improve hormonal regulation, normalize liver functions, and support intestinal microbiota. Specifically, the ability of F-LAB to increase reduced plasma CIT levels under heat stress conditions could play a vital role in preserving intestinal health. Consequently, we infer that the developed probiotic compound may act as a growth-promoting agent, presenting a robust alternative to synthetic antibiotics in broiler production with a similarity ratio below 1% to avoid plagiarism. These findings reveal a distinct mechanism through which probiotics, from a microbial structure perspective, influence the growth performance and hormonal balance of chickens, simultaneously enriching fundamental knowledge about the intestinal microbial health of poultry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eStatement of Animal Ethics\u003c/h2\u003e \u003cp\u003eThe research protocol obtained ethical clearance from the Harran University Local Ethics Committee for Animal Experiments, approved under the reference number 2022-004-95.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis article is part of a project supported by the Harran University Scientific Research Cordinatorship, grant numbers 22192.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Fataftah A, Abu-Dieyeh Z (2007) Effect of chronic heat stress on broiler performance in Jordan. 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Appl Microbiol Biotechnol 103(1):461\u0026ndash;472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00253-018-9465-8\u003c/span\u003e\u003cspan address=\"10.1007/s00253-018-9465-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-biometeorology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijbm","sideBox":"Learn more about [International Journal of Biometeorology](http://link.springer.com/journal/484)","snPcode":"484","submissionUrl":"https://www.editorialmanager.com/ijbm/default2.aspx","title":"International Journal of Biometeorology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Heat stress, natural probiotics, cecal microbiome ecology, Leptin, Citrulline","lastPublishedDoi":"10.21203/rs.3.rs-4020346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4020346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe primary objective of this study was to evaluate the effects of liquid (F-LAB) and commercial (C-LAB) probiotics sourced from Rye-Grass Lactic Acid Bacteria on broiler chickens experiencing heat stress (HS). The research involved 240 broiler chicks divided into six groups: control, F-LAB, C-LAB (raised at 24\u0026deg;C), HS, F-LAB/HS, and C-LAB/HS (exposed to 5\u0026ndash;7 hours of 34\u0026ndash;36\u0026deg;C daily). While F-LAB and HS/F-LAB groups received a natural probiotic added to their drinking water at a rate of 0.5 ml/L, C-LAB and HS/C-LAB groups were supplemented with a commercial probiotic at the same dosage. No probiotic supplementation was administered to the control and HS groups. The results revealed that without probiotic supplementation, heat stress led to a decrease in body weight gain, T3 levels, citrulline, and growth hormone levels, along with an increase in the feed conversion ratio, serum corticosterone, HSP70, ALT, AST, and leptin levels. Heat stress also adversely affected cecal microbiota, reducing lactic acid bacteria (LABC) while increasing Escherichia coli and coliform bacteria (CBC) counts. However, in the groups receiving probiotic supplementation under heat stress (F-LAB/HS and C-LAB/HS), these effects were alleviated. Particularly noteworthy was the observation that broiler chickens supplemented with natural lactic acid bacteria (F-LAB) exhibited greater resilience to heat stress compared to those receiving the commercial probiotic, as evidenced by improvements in growth, liver function, hormonal balance, intestinal health, and cecal microbiome ecology. These findings suggest that the supplementation of naturally sourced probiotics (F-LAB) may positively impact the intestinal health of broiler chickens exposed to heat stress, potentially supporting growth and health parameters.\u003c/p\u003e","manuscriptTitle":"Probiotic Strategies for Mitigating Heat Stress Effects on Broiler Chicken Performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-27 07:22:18","doi":"10.21203/rs.3.rs-4020346/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-07-04T08:39:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-14T08:57:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-16T16:52:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-03T21:19:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Biometeorology","date":"2024-03-27T08:54:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-biometeorology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijbm","sideBox":"Learn more about [International Journal of Biometeorology](http://link.springer.com/journal/484)","snPcode":"484","submissionUrl":"https://www.editorialmanager.com/ijbm/default2.aspx","title":"International Journal of Biometeorology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"48d0073a-3528-42b5-a3fa-df5fd8d8a548","owner":[],"postedDate":"May 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:04:37+00:00","versionOfRecord":{"articleIdentity":"rs-4020346","link":"https://doi.org/10.1007/s00484-024-02779-2","journal":{"identity":"international-journal-of-biometeorology","isVorOnly":false,"title":"International Journal of Biometeorology"},"publishedOn":"2024-09-23 15:57:48","publishedOnDateReadable":"September 23rd, 2024"},"versionCreatedAt":"2024-05-27 07:22:18","video":"","vorDoi":"10.1007/s00484-024-02779-2","vorDoiUrl":"https://doi.org/10.1007/s00484-024-02779-2","workflowStages":[]},"version":"v1","identity":"rs-4020346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4020346","identity":"rs-4020346","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}