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.