Ameliorative Effects of Antibiotic-, Probiotic- and Phytobiotic-Supplemented Diets on the Performance, Intestinal Health, Carcass Traits, and Meat Quality of Clostridium perfringens-Infected Broilers.

Simple Summary Necrotic enteritis is considered the most important economic problem for the poultry industry due to the sudden death rates of up to 50%. However, there is limited information concerning the ameliorative role of probiotic and/or phytobiotic compounds in the prevention of Clostridium perfringens infections in broilers. Hence, this trial is conducted to evaluate the influence of some antibiotic, probiotic and phytobiotic compounds (Maxus, CloStat, Sangrovit Extra, CloStat + Sangrovit Extra, and Gallipro Tect) on the growth performance, carcass traits, intestinal health, and meat quality of broiler chicks. The obtained in vivo results highlight that a probiotic- and/or phytobiotic-supplemented diet has many positive effects on the performance, organ weight, and meat quality of broilers. Besides, a notable reduction in the lesion score is observed with a combined probiotic and phytobiotic diet. Abstract The poultry industry needs efficient antibiotic alternatives to prevent necrotic enteritis (NE) infections. Here, we evaluate the effects of probiotic and/or prebiotic dietary supplementation on performance, meat quality and carcass traits, using only an NE coinfection model, in broiler chickens. Three hundred and twenty-four healthy Ross 308 broiler chicks are allocated into six groups. Taking a 35 d feeding trial, the chicks are fed a basal diet with 0.0, 0.1, 0.5, 0.12, 0.5 + 0.12, and 0.2 g Kg−1 for the control (T1), Avilamycin (Maxus; T2), live probiotic (CloStat (Bacillus subtilis);T3), natural phytobiotic compounds (Sangrovit Extra (sanguinarine and protopine); T4), CloStat + Sangrovit Extra (T5), and spore probiotic strain (Gallipro Tect (Bacillus subtilis spores); T6) treatments, respectively. Occurring at 15 days-old, chicks are inoculated with Clostridium perfringens. The obtained results reveal that all feed additives improve the performance, feed efficiency, and survival rate, and reduces the intestinal lesions score compared with the control group. The T6 followed by T3 groups show a significant (p < 0.05) increase in some carcass traits, such as dressing, spleen, and thymus percentages compared with other treatments. Also, T5 and T6 have significantly recorded the lowest temperature and pHu values and the highest hardness and chewiness texture values compared to the other treated groups. To conclude, probiotics combined with prebiotic supplementation improves the growth, meat quality, carcass characterization and survival rate of NE-infected broiler chickens by modulating gut health conditions and decreasing lesion scores. Moreover, it could be useful as an ameliorated NE disease alternative to antibiotics in C. perfringens coinfected poultry.

informed that the prebiotic supplemented (Sanguinarine) diet could be an effective treatment alternative to antibiotics for controlling necrotic enteritis diseases in broilers. Diverse types of additives include phytobiotics (primary or secondary components of plants that contain bioactive compounds that exert a positive effect on the growth and health of animals) which could be a beneficial strategy that regulates the gastro-intestinal microbial community and improve broiler health [34].
Found in the literature, the positive effects of both B. subtilis and phytobiotic compounds have been reported. However, their synergistic effects have not been described yet. The present study aims to evaluate the ameliorative effects of probiotic and phytobiotic compounds alone or in a combined form of two different types of B. subtilis on the performance, carcass traits, meat measurements, and intestinal health of C. perfringens-infected broilers during the starter and finisher phases.

Materials and Methods
The experiment was performed in cage pens under similar managerial and hygienic conditions in an environmentally controlled poultry unit at the Animal Production Department, College of Food Science and Agriculture Science, King Saud University. All protocols were chosen according to the experimentation guidelines of the Animal Use Ethics in Research Committee of King Saud University (approval number: SE-19-150).

Experimental Design and Feeding Regime
Altogether, 324 day-old broiler chicks (Ross 308 strain) were randomly distributed into six groups. Each group contained nine replicates, with six birds per replicate, and were used for a 35 d feeding trial period. Each group was assigned to one of the following dietary treatments: T 1 ; basal diet (control), T 2 ; diet supplemented with 0.1 g kg −1 of Maxus (antibiotic), T 3 ; diet supplemented with 0.5 g kg −1 of Clostat (natural probiotic strain), T 4 ; diet supplemented with 0.12 g kg −1 of Sangrovit (phytobiotic), T 5 ; diet supplemented with 0.5 g kg −1 of Clostat combined with 0.12 g kg −1 of Sangrovit, and T 6 ; diet supplemented with 0.2 g kg −1 of Gallipro Tect (spore probiotic). The chicks were fed a starter diet between days 0-14, which was then switched to a grower diet between days 15-35 (Table 1). After 14 d, all treated groups were inoculated with C. perfringens bacteria. Birds were fed ad libitum, and water was available at all times during the experimental period.

Challenge with Clostridium perfringens Bacteria
The C. perfringens challenge model was performed as described by Prescott [35]. All treated groups received a C. perfringens challenge at a rate of 4 × 10 8 CFU g −1 via oral gavages on day 14, as recommended by Olkowski et al. [36], using the defined B positive bacteria C. perfringens isolated from a local farm. The identified bacterium was previously confirmed to be sensitive to antibiotics (Avilamycin) using the broth dilution method (MIC testing) [37]. An inoculum was equipped to contain nearly 10 8 cells of C. perfringens per ml and was processed at a ratio of 1:1.5 ration-to-broth [11]. The challenge feed was mixed with the treatment diet. After the administration of the challenged material, dead birds were counted as study mortalities. These birds underwent a necropsy evaluation to estimate the cause of death/disease.

Growth Performance Parameters
During the starter (0-14-days-old) and finisher (15-35-days-old) periods, the growth and feed efficiency parameters of the broiler chicks were estimated. The daily feed intake was calculated by subtracting the quantity of feed rejected from the feed offered. Additionally, live body weight was estimated at biweekly intervals, while the final body weight and total feed consumption were determined at the end of each trial period. The body weight gain (BWG) was measured by calculating the difference between the live body weight and final body weight for each trial period. The feed conversion ratio (FCR) was computed for each group, as mentioned by Abudabos et al. [32], using the following formula: FCR = Feed intake/Weight gain (1) Meanwhile, the production efficiency factor (PEF) was calculated as suggested by Griffin [38], using the following formula: During the feeding trial, the number of deaths was counted to calculate the survival rate, as mentioned by Hussein et al. [39], using the following formula: Survival rate (SR)% = (the number of the surviving broilers / the initial number of broilers) × 100 (3)

Carcass Measurements and Lesion Scores
Ten broiler chicks were randomly collected from each group to estimate the organ weights (heart, gizzard, liver, bursa, spleen, thymus, small and large intestine, and ceca). Chicks were weighed before sacrifice. All internal organs were weighed immediately after slaughter. The gizzard was weighed after its content was removed. The small intestine was measured by determining the distance between the site of the duodenum emergence from the gizzard and the beginning of the ceca. The organ weight was calculated relative to the live body weight.
According to the Hofacre et al. [40] procedure, two birds per pen were examined for gross intestinal lesions. The characteristic of necrotic enteritis was defined using the description of the Long et al. [41] study. The lesion scores were outlined as follows: 0 = none, 1 = mild, 2 = moderate and 3 = marked (severe) [42].

Meat Characteristics
At the end of the finisher period (35-days-old chicks), three birds were randomly selected per pen to estimate the meat characteristics followed by the Chen et al. [43] method. After euthanasia, the jugular vein was cut, the feathers, heads, and shanks were removed, and the remaining carcasses were dissected. The left and right breast from each bird were used for the quality measurements. The breast samples were stored at −80°C until further analysis. At the time of the analysis, frozen muscles were thawed overnight in a chiller at 4°C.
The pH of the breast muscle was measured twice (after slaughtering and 24 h postmortem), using a microprocessor pH-Meter (Model PH 211, Hanna Instruments, Padova, Italy). The core temperature in the breast muscle was measured after slaughtering with a portable digital thermocouple (Eco Scan Series, Temp JKT, Eu tech Instruments, 7 Gul Circle, level 2M, Keppel Logistic Building, Singapore 629563). The colour values of the CIELAB Color System (L*(lightness), a* (redness), and b* (yellowness)) were determined for the breast muscles 15 min and 24 h after slaughtering, using a Chroma meter (Konica Minolta, CR-400-Japan) following the method used by Castellini et al. [44].
The myofibril fragmentation index (MFI) of the breast muscle was determined by multiplying the absorbance value at 540 nm, as described by Culler et al. [45]. The water-holding capacity (WHC) was determined based on the technique described by Hamm [46] and following the modification performed by Wilhelm et al. [47], using the following equation: where, Wi and Wf are the initial and final sample weights, respectively. The drip loss (DL) was determined as a percentage based on the initial sample weight. The cooking loss (CL) was determined as the difference between the initial and final weights. Then, cooked samples were used to evaluate the shear force according to the procedure described by Wheeler et al. [48], under a 200 mm/min crosshead speed. The texture profile analysis (TPA) values were estimated using a Texture Analyzer (TA-HD-Stable MicroSystems, Golborne, Warrington WA3 3GR, England) equipped with a compression-platen attachment. The variables determined included the hardness (maximum force needed to compress the sample), cohesiveness (ratio between the total energy required for the first and second compression), springiness (the ability of a sample to recover to its original form after the removal of the compressing force), and chewiness (a resultant of springiness × hardness × cohesiveness).

Statistical Analysis
The data underwent a one-way ANOVA using a completely randomized design. Before analysis, the data were examined for normality of distributions and homogeneity of variance. Percentage data were subjected to arcsine transformation before analysis. SPSS 22 analysis software was used for all statistical analyses. Data are expressed as the mean ± SEM, with a statistical significance level of p ≤ 0.05. Significant differences between means were determined using Duncan's Multiple Range [49].

Growth Performance and Intestinal Lesions Score
The effects of some feed additives on the total feed intake (TFI), body weight gain (BWG), feed conversion ratio (FCR), and protein efficiency ratio (PEF) of broilers in the starter and finisher periods are shown in Table 2. During the starter period, our results revealed no significant differences between all treatment and non-treatment groups. T 4 produced the lowest TFI values, while T 3 and T 1 exhibited the highest. Regarding the FCR values, both the T 4 and T 5 treatments had lower values than those of the other treatments. The dietary supplementation of probiotic bacteria increased the PEF (except for T 3 ) compared with the control group. During the finisher period, all experimental additives significantly increased (p < 0.01) the FBW, BWG, FCR, PEF, survival rate (SR), and decreased the intestinal lesions score compared with those of the control group (Table 2). However, the TFI was not significantly affected by the supplemented diets. The T 3 , T 4 and T 5 groups had higher BWG values compared with those of the other groups. Additionally, the T 3 -treated group exhibited the best FCR values, while the different treatment groups produced intermediate FCR values. Regarding the PEF values, compared with the control, all experimental additives were significantly enhanced (p < 0.05). The SR (%) and lesions score were enhanced considerably by the dietary treatments at 15-35 days of age (Table 2).

Carcass Measurements
The effects of supplemented diets on carcass traits after a 35-day feeding trial are presented in Table 3. Regarding the T 6 treatment group, the dressing percentage showed significant (p < 0.05) increases. Also, the T 1 and T 3 treatment groups recorded the highest significant thymus values, while the lowest values were recorded for T 4 . Meanwhile, the highest significant (p < 0.05) spleen weight percentage value was recorded in the T 5 group.

Meat Characteristics
The effects of feed additives on broiler meat quality parameters are presented in Table 4. The T 3 and T 5 treatments resulted in the lowest pHi values, with the highest values observed in T 2 . Conversely, the pHu was significantly reduced by the treatments, with the lowest values recorded in T 5 and T 6 . Furthermore, the Temperature was significantly reduced in T 6 followed by T 5 -treated groups compared with other treated groups. However, no significant differences were demonstrated among the other treatment groups concerning the colour component after slaughter (L15 and b15) and 24 h (L24, a24, and b24) at 35 days of age. Table 4. Effects of some feed additives on the meat quality of broilers challenged with C. perfringens during a feeding trial period. The results presented in Table 5 reveal the effects of feed additives on meat characteristics, such as the Cooking Loss (CL), Water Holding Capacity (WHC), Myofiber Fragmentation Index (MFI), Shear Force (SF) and texture profile analysis (TPA) at 35 days of age under a C. perfringens challenge test. All treatment groups showed no significant differences concerning the CL, WHC, MFI, and SF. The T 6 and T 5 treatments exhibited significantly increased (p < 0.001) hardness and chewiness values compared with those of other treatment groups, respectively. Although the dietary supplementation showed no significant differences in Springiness and Cohesiveness values between all treated groups.

Discussion
Necrotic enteritis (NE) has become one of the most critical problems in the poultry industry [50]. Feed additives, including antibiotics, prebiotics, and probiotics, have frequently been used for improving the health, growth, and feed efficiency parameters of animals [51]. Recently, interest in incorporating probiotics and antibiotics into broiler treatments has been rapidly increasing [52]. Several studies have shown that the addition of probiotics has positive effects on the growth rate, feed utilization, feed efficiency, and mortality rate [53,54]. However, the efficacy of probiotics depends upon the selection of more efficient strains, manipulation of genes, a combination of several strains, and the combination of probiotics and synergistically=acting components [55]. The use of multi-strain probiotics seems to be the best way of potentiating the efficacy of probiotics, as it beneficially affects the host by improving growth-promoting bacteria with competitive antagonism against pathogenic bacteria in the gastrointestinal tract [56].

Growth Performance
The performance results obtained in the starter period and the finisher period are presented in Table 2. The IBW, TFI, BWG, and FCR of all groups receiving feed-supplemented diets and basal diets had no significant differences among them. However, the BWG, FBW, PEF, FCR, and SR during the finisher phase, at 15-35 days-old, were significantly (p < 0.05) improved in all supplemented diets compared to the control group. These results are similar to those of Khaksefidi and Ghoorchi [57], who found that the BWG of birds fed a diet supplemented with 50 mg/kg of Bacillus subtilis was significantly higher during the finisher period (22-42 d) than birds fed the control diets. Additionally, the feed conversion ratio of birds fed a diet supplemented with probiotics significantly reduced from 22 to 42 d compared with birds fed the control diets. Consequently, Patel et al. [55] indicated that the dietary supplementation of combined probiotics and prebiotics (Protexin) at 100 g/ton of feed significantly enhanced the BWG, along with an improved FCR, and benefitted without any adverse effects on the feed intake, mortality, or carcass characteristics. Also, Anjum et al. [58] and Singh et al. [59] observed similar results. The improvement of all performance parameters may be due to the biological role of probiotics in altering the intestinal pH, which modifies both the microbial population and nutrient absorption, ultimately improving the efficiency of feed utilization [60]. Moreover, increased feed intake and digestive enzyme secretions also are detected in animals' fortified phytobiotic-supplemented feed [61]. Growth enhancement through the use of phytobiotics probably depends on the synergistic effects among complex active molecules present in phytobiotics [39,62].
The mortality rate in this study was low (finisher period), representing the positive effect of feed additives on the mortality rate (Table 2). These findings were similar to those found by Abdel-Hafeez et al. [63] and Riad et al. [64], who indicated that the addition of probiotics as feed additives decreased the mortality rate. The reduction in mortality was attributed to the inhibitory effects of these additives toward enteric microorganisms via modifying the intestinal pH [63]. Compared to a wide range of antibiotics (including Avilamycin), a significant decrease in mortality was seen for treated broilers with 2 g/kg Bio-Mannan-oligosaccharides (Bio-MOS) as a source of prebiotic, depending on the age of birds [65,66]. Moreover, there are major modes of action by which broiler performance is improved by proposed oligosaccharide prebiotics, such as control of type-1fimbriae pathogenic bacteria (mannose-sensitive lectin), immune modulation effects, and modulation of intestinal morphology and expression of mucin and brush border enzymes [67].

Carcass Traits
The mean values for the carcass characteristics, dressing percentage, and organ weight (%) relative to the bodyweight of the broilers are presented in Table 3. The dressing percentage was significantly (p < 0.05) increased in the T 6 group compared with those of the other treatments. However, non-significant differences were observed in the other carcass measurements amongst the treatment groups. Some organ weight (spleen and thymus) significantly increased in probiotic supplemented diets alone or in combination with prebiotics compared with other treated groups. Generally, the inclusion of probiotics in broiler rations had no extra additional benefits for the organ weight or carcass yield [55]. Also, Salamkhan et al. [68], Panda et al. [69], and Patel et al. [55] reported that the dressing percentage did not differ significantly (p ≤ 0.05) between the probiotics-fed broilers and control broilers. To contrast, Banday and Risam [70] and Kabir et al. [71] observed a significant (p ≤ 0.05) improvement in the dressing percentage in groups supplemented with probiotics. Nevertheless, Sarangi et al. [72] reported a highly significant increment in breast yield in birds supplemented with prebiotics. Jin et al. [73] attributed the lower fat deposition in birds treated with probiotics to how the product could interfere in the availability of fat for lipogenesis in the birds.

Meat Quality Parameters
The effects of antibiotic-, probiotic-, and phytobiotic-supplemented diets on the measurements of meat characteristics under C. perfringens infection in broilers are presented in Table 4. The change in pH is one of the most significant changes that can occur during rigor mortis and can affect meat quality characteristics, such as texture, colour, and WHC [74]. Fletcher et al. [75] reported that post-mortem pH measurements are a good indicator of meat characteristics. Generally, a rapid post-mortem pH decline in breast meat can lead to protein denaturation, which may result in a pale colour and low WHC [76]. During this trial, the temperature and pH values obtained after slaughter and 24 h post-slaughter showed significant (p ≤ 0.05) enhancements for all supplemented diets compared with the group fed the basal diet. These results are similar to those found by Battula et al. [77] and Corzo et al. [78] where the bacterial challenge had negative impacts on the temperature and pH of the breast muscle.
Additionally, the cooking loses (CL) is a measure of the percentage of water lost during cooking as a result of shrinkage. Consequently, the degree of shrinkage upon cooking is directly correlated with a loss of juiciness. During this trial, the CL, WHC, and SF showed non-significant differences between the control and treatment groups. During a subsequent study, Pelicano et al. [79] examined the effects of Lactobacillusand Bacillus subtilis-based probiotics on SF and reported non-significant values compared with the control group. However, the MFI of the breast muscle was influenced by the treatments (p ≤ 0.05), as shown in Table 4. The MFI decreased in T 2 and T 3 compared with the other treatment groups. Myofibrillar fragmentation is the extent of myofibril destruction caused by homogenization [80]. Olson and Stromer [81] reported that MFI values are correlated with other muscle indices, such as SF and tenderness. Therefore, it can be concluded that probiotic supplementation causes less damage to the myofibrils [76].
TPA is an instrumental measurement of the sensory attributes of chicken breasts which imitates the conditions to which food is subjected to in the mouth [76]. The TPA, which includes hardness, cohesiveness, springiness, and chewiness, is shown in Table 5. The treatments had no significant effects on any TPA variable (p ≤ 0.05). All tested groups were very tender, except for the T 6 and T 5 supplemented groups, which had hardness values that were significantly (p ≤ 0.001) higher than those of the other treatment groups. The data presented in this trial aligns with that of Angelovicova et al. [82] who noticed that probiotics (CloStat) moderately affect the hardness, cohesiveness, springiness, and chewiness of cooked breast meat. Also, Lisowski et al. [83] investigated a beneficial effect of the prebiotic on breast muscle weight and carcass percentage. It could be supposed on the one hand, that using prebiotics in broiler diets has a positive influence on muscle weight [84].

Conclusions
To conclude, fortified feed diets with 0.2, 0.5, 0.12 g kg −1 of Bacillus subtilis spores (Gallipro Tect), live Bacillus strain (CloStat) and phytobiotic natural compounds (Sangrovit Extra) respectively, alone or in combined form, could promote growth and reduce the NE mortality rates. Additionally, using these probiotic and phytobiotic compounds as an alternative for antibiotics in feed diets could be a useful strategy to ameliorate the harmful effects of C. perfringens bacterium on broilers, in terms of the performance, feed efficiency, meat quality and carcass characteristics regarding necrotic enteritis infections.