1. Introduction
Global warming and climate change adversely affect livestock and poultry production sectors under tropical and subtropical conditions. Chickens are particularly vulnerable to high ambient temperature because of their higher inner body temperature and the lack of sweat glands. Therefore, several physiological disturbances, such as hyperpnea, electrolyte imbalance, rapid pulse, vascular dilatation, endocrine disorders, systemic immune dysregulation, muscle tremors, weakness, and collapse, occur in heat-stressed birds [
1,
2] that impair their welfare and productivity. Furthermore, high ambient temperature leads to elevated body temperature and, consequently, results in serious tissue damage induced by excessive reactive oxygen species (ROS) [
3]. Increased production of ROS is potentially detrimental to the maintenance of homeostasis [
4,
5]. Moreover, variations in the response of broiler strains and their ability to withstand heat stress have been documented. In general, the heat stress resistance of broiler chickens is low owing to breeding programs constantly selecting for higher growth performance; however, some broiler strains exhibit higher performance and/or higher resistance to climatic changes than others [
6,
7,
8,
9]. Therefore, it is essential to implement mitigation strategies that can promote the physiological antioxidant system of these broiler strains to neutralize the excess amounts of generated ROS. Environmental strategies, for example, establishing an intermittent light schedule, increasing ventilation, lowering stocking density, and implementing early feed restriction [
10], and nutritional manipulations, for example, use of probiotics [
11,
12], vitamins [
3,
13], trace elements [
10,
13,
14,
15], and herbal extracts and products [
16,
17,
18], have been considered as common approaches in poultry production. It has been established that the inclusion of herbs and their extracts in poultry diets is generally effective in maintaining their health, welfare, and performance as well as the oxidative stability of their eggs and meat products [
16,
19,
20,
21,
22].
Boldo (
Peumus boldus molina) is an abundant native Chilean tree that has been widely used as a medicinal herb to improve hepatic and digestive complaints [
23]. Boldo has been used in numerous pharmacopeias as a herbal remedy owing to its high content of polyphenols (flavonoids), alkaloids (boldine), and essential oils [
24,
25,
26,
27]. Polyphenols account for as much as approximately 12%–36% of the total solids in the aqueous extract of boldo leaves [
23]. Free radical scavenging and lipid peroxidation inhibition activities of the water extract of boldo leaves have been demonstrated [
27]. These established characteristics of boldo suggest it as one of the promising herbal plants that could be used to counteract various stressors affecting broiler chicks, especially heat stress. Therefore, the aim of the present study was to investigate the effect of administration of boldo leaf extract in drinking water to mitigate the adverse impacts of heat stress in two broiler strains.
2. Materials and Methods
Bird management followed the regulations prescribed by the Animal Care and Ethics Committee (DMU/VetMed-2019-/0040) at the Animal Husbandry and Animal Wealth Development Department, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt.
2.1. Experimental Birds, Design, and Management
A total of 120 broiler chicks at 14 days of age from two different strains (sixty Arbor Acres (AA) and sixty Avian-48 (AV)) with nearly the same starting body weight (343.3 ± 2.2 g) were used in this study. Each strain was subdivided equally into two experimental groups; one was considered as the control (thermoneutral group, TN), and the other was subjected to high temperature to induce heat stress (HS). Heat-stressed birds were subjected to a temperature of 34 ± 2 °C for 12 h (from 9:00 to 18:00 and then 25 ± 2 °C) for three successive days a week, whereas birds of the TN group were maintained at the common housing temperature (25 ± 2 °C). Relative humidity under both conditions was maintained at 45% ± 3%. The temperature was elevated and maintained constant using electric heaters. Each group was further split into two groups, the control group and the boldo extract treatment group that received 1 g boldo leaf extract/4 L drinking water during the period of heat stress previously described. Three replicates of five birds each were used for the control and boldo-treated groups. Birds were caged in stainless steel batteries with dimensions of 100 × 90 × 45 cm; length × width × height/pen/replicate; equipped with nipple drinkers and manual feeders. During the period of the boldo extract administration, nipple lines were turned upward to prevent birds from consuming water through them and making the birds rely only on manual drinkers that were supplemented with the extract of boldo leaves. Water intake during a certain period was measured by adding the amount of water consumed via nipple drinkers to that consumed via manual drinkers during the same period. Water and feed were offered ad libitum, and all birds were reared under the same managerial and vaccination programs. Manual thermometers and hygrometers placed at the center of the house were used to record and ensure the stability of the indoor temperature and relative humidity four times a day (at 9:00, 12:00, 15:00, and 18:00). The temperature humidity index (THI) was calculated according to the equation described by Tao [
2] (THI = 0.85 t
db + 0.15 t
wb, where t
db and t
wb are dry bulb and wet bulb temperatures, respectively), as depicted in
Figure 1. Birds were fed starter and grower diets (
Table 1), which were formulated to meet the requirements of the NRC [
28]. Arbor Acres and Avian-48 chicks were obtained from commercial hatcheries in Egypt. Boldo extract (the extract of leaves of
Peumus boldus from the Monimiaceae family) was obtained from Hongda Shaanxi Company in China (Hongda Park, Xizhang Village, Dacheng Town, Sanyuan County, Xianyang City, Shaanxi Province, China).
2.2. Growth Performance
Starting from the second week of age, body weight (BW) and feed intake (FI) per replicate were recorded weekly until the 6th week of age. Body weight gain (BWG) was calculated weekly by subtracting the initial body weight at a certain week from the final body weight at the next week. Feed conversion ratio (FCR) was calculated as g feed/g gain per period. Prior to slaughtering, birds were deprived of feed for 12 h and then weighed. Two birds from each replicate were slaughtered, scalded, wet-plucked, and eviscerated and inner organs (liver, heart, spleen, and gizzard) were weighed separately to determine the dressed weight and the dressing percentage. The blood, viscera, lungs, limbs, head, and neck were termed as the offal and discarded. The abdominal fats in the pelvic and abdominal cavity were completely collected from the carcass and then weighed. The carcass was separated into cuts including the breast (breast muscles with the sternum), thigh (average of two thighs weight), shoulder (average of two shoulders weight), and left fillet (the de-skinned left breast muscle on the left side of the sternum), and each was weighed.
2.3. Blood Hematology and Biochemistry
Two blood samples were collected in separate labeled centrifuge tubes from the wing vein at 42 days of age from two birds within each replicate. The first tube contained 3.2% sodium citrate solution in order to determine hemoglobin and hematocrit values, white blood cells, red blood cells, and differential leucocytic counts. Phagocytic activity and index were estimated according to the method described by Kawahara et al. [
29]. The other tube was left to clot and then centrifuged at 4500×
g for 15 min. The serum samples were collected and preserved in a deep freezer at (−20 °C) until the time of analysis. Total protein (TP), albumin (ALB), total lipids (TL), triglycerides (TG), and total cholesterol (TC) were spectrophotometrically determined (Spectronic 1201; Milton Roy, Ivyland, PA, USA) using commercial kits from Bio-diagnostic Co., Egypt, according to the manufacturer’s instructions.
Activities of oxidative stress biomarkers, including malondialdehyde (MDA), glutathione peroxidase (GPx), and superoxide dismutase (SOD), were assessed using the ELISA Kit from Quanti ChromTM, BioAssay Systems, USA and Cayman Chemical Company, USA.
2.4. Economic Efficiency
Feed cost (US $/kg feed) was calculated by multiplying the total feed intake per bird by the cost of one kg feed (0.36 US $/kg feed). The average cost of feed/kg body weight (US $/kg gain) was calculated as feed cost (US $/kg feed) × feed intake per bird (kg)/weight gain per bird (kg). The cost of the boldo extract supplementation (0.02 US $/bird) was included in the total costs of the treatment groups. The other expenditure including the cost of day-old chicks, housing, labor, drugs, disinfectant, vaccines, veterinary supervision, and depreciation cost were common to all groups. Total costs were calculated by summing all fixed costs and variable costs. The net return was obtained by the difference between the total return, considering the average value of the bird (1.45 US $/kg live body weight) and the feeding cost for each group. Benefit/cost ratio (B/C ratio) was calculated as total return/total costs.
2.5. Statistical Analysis
Data were analyzed using the Statistical Analysis System (SAS, 2002), three-way analysis of variance, and the general linear model procedure. The effect of fixed factors, broiler strain, heat stress, and administration of boldo extract, and their interaction were estimated. Tukey’s multiple comparison test was used to identify the presence of significance (
p < 0.05) among multiple means. To ensure that the results from the three replicates used were sufficient and the dispersion of the data around the means was unique and within the accepted limits, the coefficient of variation (C.V.%) was calculated as
where SD is the standard deviation, and
is the sample mean. C.V.% values are listed in all tables; it was noted that all C.V.% values were within the acceptable limits.
4. Discussion
Results of the present investigation revealed that the administration of boldo extract in drinking water was effective in promoting growth of birds reared under the TN condition and partially enhanced the ability to recover weight loss in HS birds. Growth of AA birds was less affected than that of AV birds by HS. Furthermore, the exposure to HS significantly reduced FI of birds regardless of the treatment with boldo extract; whereas FCR was not significantly affected. Broiler strain affected FI; AV birds consumed less feed than AA birds in general. Water intake was not significantly altered by the interaction among broiler strain × heat stress × boldo treatments with an increase in water consumption by heat-stressed birds of both strains. Our results are consistent with those of previous studies [
30,
31,
32]. The adverse effect of HS on broiler growth performance can be attributed to the mucosal epithelial damage caused by HS that impairs nutrients absorption. In addition, heat-stressed birds expend more energy to adapt to HS conditions, leading to the reduction in their growth performance as less energy is used for growth [
31]. Another possible explanation for the reduction in growth and FI is that under the HS condition, poor appetite and lower FI were observed; these are considered as a defense mechanism to reduce the increment in body heat of broilers [
31]. Furthermore, the impaired growth rate in heat-stressed birds might also be attributed to changes in the blood circulation pattern [
33]. Under high ambient temperature, blood supply to the peripheral organs increases to promote the dissipation of inner heat to the environment. Consequently, blood flow to the gut is reduced, leading to a leaky gut, greatly deprived in nutrients and oxygen. Under such circumstances, disturbance in the intestinal ecosystem and reduction in bacterial substances production occurred [
33], which in turn reduced the growth of birds. Moreover, the decreased amount of bacterial substances such as lipopolysaccharides stimulates the production of pro-inflammatory cytokines, for example, TNF-Y, IL-1α, IL-1β, and IL-6, which activates T and B lymphocytes in order to eliminate the damaged tissue. The increase in water consumption of heat-stressed birds can be attributed to the important role of water in maintaining thermoregulatory balance, as birds under heat stress conditions lose a high amount of water through evaporative cooling via the respiratory tract as a means of achieving effective thermal regulation. Supplementation of phytogenic additives (FA), such as boldo extract, is able to reverse the deleterious impacts of heat stress through promoting the growth of healthier microbes [
34], reducing the creation of growth-depressing microbial substances, such as biogenic amines and ammonia [
35,
36], and increasing the availability of nutrients to the host [
37]. Furthermore, it has been reported that essential oils of FA might be efficient in controlling the intestinal mucosa inflammation evidenced by the reduction in the number of intra-epithelial leucocytes of the intestinal mucosa of pigs [
38]. The control of intestinal inflammation should spare nutrients for absorption [
39,
40]. Additionally, FA can enhance the production of digestive enzymes and manipulate the intestinal microflora [
41,
42]. These previously demonstrated cumulative effects of boldo may explain the improvement in the growth performance of heat-stressed birds treated with boldo leaf extract. Moreover, AA chicks were better able to withstand heat stress as these birds showed higher BW, BWG, and FI than AV chickens. Heat-stressed boldo-treated AA chicks exhibited a higher growth rate than even heat-stressed AV birds treated or not with boldo extract. Our results are consistent with those reported by Hascik et al. [
6] and Abdo et al. [
9].
Heat stress decreased dressing percentage and relative weights of the liver and breast in AV birds compared to those of AA birds. Boldo treatment restored values of these parameters near to the values in TN birds and increased their values in the TN birds. Consistent with our results, Shim et al. [
43] and Plavnik and Yahav [
44] reported that the relative liver weight was lower in heat-stressed chicks as a result of decreased metabolic needs. Song et al. [
31] reported that the proportion of breast muscle as a percentage of BW was reduced by cyclic heat stress. Relative weight of the pectoralis major of birds reared under 32 °C was lower than that of those reared under 22 °C [
45]. The authors attributed this reduction in muscle weight to the elevation of protein synthesis susceptibility than to proteolysis at 32 °C, which thereby lowered the deposition of muscle protein. The amelioration effects of FA on carcass traits of heat-stressed birds have been reported [
32,
46,
47,
48]. Possible explanations for the heat stress mitigation effect of FA may be their ability to boost the antioxidant defense system, immune response, digestive enzyme secretion as well as stimulate the appetite and feed intake [
32,
49]. Moreover, the effects of heat exposure on abdominal fat have been controversial among researchers for a long time. Consistent with our results, Smith and Teeter [
50] and Fisher [
51] reported a significant reduction in fat content in heat-stressed birds, whereas an elevation in fat deposition was observed by Baziz et al. [
52] and Geraert et al. [
53]. The reduced fat pad size observed in boldo-treated birds compared to that in heat-stressed birds suggested the reduction in fat deposition through increasing lipolysis and/or inhibition of the lipogenesis process [
54].
Regarding hematological observations, our results indicated no interaction effect among heat stress, boldo treatment, and chicken strain on values of blood erythrocytes, leucocytes, hemoglobin, and hematocrit. These results are in agreement with those of Tamzil et al. [
55], who observed that these parameters were not altered in normal kampong, Arabic, and commercial chickens compared to that in birds exposed to acute heat stress. However, heat stress significantly affected the phagocytic index and activity and proportional count of leucocytes. It has been reported that heat stress led to increased counts of basophils, heterophils, and lymphocytes but did not affect the eosinophil count [
55,
56]. These disturbances in leucocytes counts may be because of the adverse effect of heat burden on lipid peroxidation, inflammatory response, and tissue damage that trigger the production of phagocytic leucocytes to engulf damaged cells and foreign pathogens [
57]. Moreover, the increased heterophil percentage and depressed lymphocyte count may be attributed to the elevation in glucocorticoid and corticosterone secretion under high ambient temperature conditions [
55]. Administration of boldo extract in drinking water was able to balance the heat burden and restore the leucocytes count close to the values of TN birds because of the ability of its bioactive substances to control intestinal mucosa inflammation and inhibit enteric pathogens, which subsequently down-regulated leukocyte synthesis.
Serum TP, A/G ratio, and TG levels were not affected by the fixed factors or their interactions. However, the ALB level increased in both strains treated with boldo either under TN or HS conditions but was not affected by the other fixed factors or their interactions. Our results agree with the previous investigations, which used menthol, anethol, and eugenol sources of FA [
58,
59,
60]. In contrast to our findings, Reis et al. [
61] reported a significant reduction in serum concentration of TP and GLO in broilers fed FA containing diets. The authors linked this reduction in serum protein levels to the lower immune response evidenced by lower lymphocyte count. Moreover, serum concentrations of TL and TC were significantly lower in chicks exposed to heat stress than in TN birds. These results are consistent with those of Shim et al. [
43] and Takahashi and Jensen [
62]. However, the serum level of cholesterol tended to increase, especially in AA-stressed birds. Increase in the cholesterol level may be caused by the lipolysis of body lipids in order to compensate for the energy required resulting from the decrease in feed intake [
63]. However, administration of boldo extract in drinking water induced a reversal of these factors to levels of the control, thereby implying that boldo extract increased lipogenesis in the liver of heat-stressed chickens.
It is well-documented that heat stress augments the generation of free radicals, which increases lipid peroxidation [
1,
5,
8,
12]. As an indicator of lipid peroxidation, MDA concentration increased in the blood of heat-stressed birds concomitantly with an elevation in the activities of antioxidant enzymes (SOD and GPx). These increases in antioxidant enzyme activities during the period of non-damaging exercise have been considered as a physiological response to protect living cells from oxidative stress [
64,
65,
66]. In the present study, both broiler strains exposed to high ambient temperature showed higher serum levels of MDA, GPx, and SOD. However, the treatment with boldo reduced the values of these parameters. The similar response of the antioxidant defense system of both strains to heat stress may be attributed to the large differences in gene expression of these enzymes. Abdo et al. [
9] reported that gene expressions of SOD and catalase in the liver of heat-stressed Ross and Cobb chickens were not significantly altered. Furthermore, the strong free radical scavenging and antioxidant activity of boldo leaves could be attributed to their high content of boldine and catechin [
67]. Boldine has been reported as the principal antioxidant alkaloid fraction [
68], whereas, the total antioxidant activity of catechin, the main flavonoid compound, is estimated to be 60.9% [
23,
69]. These active compounds compensate in scavenging and neutralizing the excess of free radicals that subsequently boosts the antioxidant system of heat-stressed birds and reduces the levels of the studied oxidative stress biomarkers to the normal level [
70,
71,
72,
73,
74].
The enhancement of economic efficiency in boldo-treated groups may be attributed to the ability of the boldo leaf extract to increase broiler growth performance, improve feed efficiency, and reduce bird mortality by stimulating their immune system. Moreover, AA chicks were more responsive to the utilization of boldo extract and exhibited higher profitability after treatment than AV birds. Previous investigations attributed the alterations in profitability in broiler production to the variations in growth rate and the cost of one-day-old chicks, drugs, labor, feed, veterinary services, and the sale price of birds [
75,
76]. Our results are consistent with those reported in earlier studies [
32,
77,
78].