ZOOTECHNICAL PERFORMANCE AND BLOOD BIOCHEMISTRY PROFILE OF ROSS 308 BROILER CHICKENS FED DIETS SUPPLEMENTED WITH DRIED Opuntia ficus-indica (prickly pear) FRUIT

INTRODUCTION

 Antibiotics have historically played a crucial role in modern broiler chickens production, serving as therapeutic agents, prophylactics and antimicrobial growth promoters (AGPs) to enhance growth performance and feed intake efficiency (El-Fateh et al., 2024). Since their introduction in the 1960s, antibiotic growth promoters (AGPs) have significantly improved poultry production outcomes by reducing mortality rates and controlling subclinical infections (Abudabos et al., 2016; Chand et al., 2016; Alhidary et al., 2017). However, increasing concerns over antibiotic resistance and the presence of antimicrobial residues in animal-derived food products led regulatory restrictions. In this context, the European commission implemented a complete ban on the use of AGPs in animal feed from January 1, 2006 (European Parliament and Council of the European Union, 2003). The withdrawal of AGPs has since been associated with a resurgence of pathogenic infections and decline in overall poultry performance (Aljumaah et al., 2020). This situation has encouraged the search for safer alternatives. Among the proposed options, phytobiotics have received increasing attention in poultry feeding (Aljumaah et al., 2020; Krauze et al., 2021; Zaikina et al., 2022). Phytobiotics are plant-derived bioactive compounds recognized for their multifaceted properties, including appetite stimulation, enhancement of digestive enzymes activity, antimicrobial and antioxidant actions, and improved nutriment utilization (Lee et al., 2015; Shahid et al., 2015; Urban et al., 2025).

 Opuntia ficus-indica (L.), commonly known as the prickly pear cactus is extensively distributed throughout the Mediterranean basin, Latin America and south of Africa. Renowned for its use in traditional medicine, this species has been extensively studied for its diverse bioactive properties, including antioxidant, anti-inflammatory, and antimicrobial activities. In addition, has shown neuroprotective, hepatoprotective, and cardioprotective effects, attributed to its rich content of phenolic compounds, flavonoids, betalains, and mucilaginous polysaccharides (Giraldo et al., 2023; Shoukat et al., 2023; Liu et al., 2025). Its fruit is particularly rich in bioactive compounds and has shown potential for enhancing animal productivity, including in poultry systems (Martins et al., 2023). Most studies have focused on specific extracts rather than the whole fruit, and data on its use in broiler diet remain limited. The whole fruit represents a complex matrix with potential synergistic interactions, and its inclusion in the diet may provide a practical and cost-effective alternative to purified extracts.

 Nevertheless, data on the effect of whole OFI fruit supplementation on growth performance and blood biochemical parameters in broiler chicken remain scarce.

 Therefore, this study aimed to assess the effect of dietary supplementation with dried Opuntia ficus-indica fruit on growth performance and blood biochemical parameters in Ross 308 broiler chicken. 

MATERIALS AND METHODS

Plant material preparation: Opuntia ficus-indica fruits were harvested from the rural commune of “Sbouya” in the Guelmim-Oued Noun region of Morocco in August 2023. Only intact fruits free from external defects were selected, washed, peeled, sliced and air-dried at ambient temperature (25-30 °C). To ensure uniform drying and minimize the risk of fungal contamination, the sliced fruits were turned manually once daily until constant weight was achieved.

 The dried fruits were ground into a fine powder using an attrition mill and passed through a 1-mm mesh sieve to ensure uniform particle size prior to diet formulation and incorporated into the basal diet at a fixed inclusion rate of 10% (w/w). This level was selected based on preliminary trials and previous reports indicating that 5-10% supplementation of OFI fruit can enhance growth performance without adverse effects on feed palatability or texture.  

Experimental diets: Throughout the experimental period, birds were fed a basal diet composed of soybean meal, corn and a premix of vitamin and mineral supplements. The composition and nutrient profile of the diet are summarized in Table 1.

Table 1. Formulation and chemical composition of the experimental diets

Ingredient composition (%)

Calculated chemical composition

Values of nutrient composition were calculated based in ingredient composition and standard feed composition tables (National Research Council, 1994; Aviagen, 2022). The diets were formulated to provide equal energy and nitrogen content.

ME: Metabolized energy; CP: Crude protein; CF Crude fiber; Ca: Calcium; AvP: Available phosphorus; Lys: Lysine; Met: Methionine.

The Vitamin–mineral premix provided per kg of diet: Vitamin A, 10,000 IU; Vitamin D₃, 2,000 IU; Vitamin E, 20 mg; vitamin K₃, 2 mg; Vitamin B₁, 2 mg; Vitamin B₂, 6 mg; Vitamin B₆, 4 mg; Vitamin B₁₂, 0.02 mg; Niacin, 40 mg; Pantothenic acid, 10 mg; Folic acid, 1 mg; Choline chloride, 500 mg; Iron, 60 mg; Zinc, 60 mg; Manganese, 80 mg; Copper, 8 mg; Iodine, 1 mg; Selenium, 0.3 mg.

Experimental design: For these experimental trials, a total of one-day-old male Ross 308 chicks were obtained from Had soualem commercial hatchery, located in the Casablanca region of Morocco. The birds were housed and fed in floor pens under standard commercial conditions. Stocking density was maintained at 10 birds/m² in accordance with recommended welfare guidelines for the Ross 308 breed. The poultry house was naturally ventilated and equipped with adequate air circulation to maintain appropriate temperature and humidity. Environmental conditions (temperature, ventilation and lighting programs) were managed uniformly for all experimental groups throughout the trial. During the first week, the ambient temperature was maintained at 32-34°C and then progressively lowered by about 2-3°C per week until it reached 22-24°C at the end of the experimental period. Lighting was provided continuously (23L: 1D) during the first week and then adjusted to a 20L: 4D photoperiod for the remainder of the trial.

 The chicks were randomly assigned to two experimental groups of 90 birds each. The control group (C) received a standard basal diet; while the treated group (E) was fed the same basal diet supplemented with 10% crushed dried OFI fruit. The trial lasted 48 days and was divided into two phases: a starter phase (days 1-22) and grower-finisher phase (days 23-48). During the starter phase, all broiler chickens were fed the same basal diet. From day 23 to day 48 (grower-finisher phase), birds in the treated group received the basal diet supplemented with 10% dried Opuntia ficus-indica fruit, while the control group continued to receive the basal diet without supplementation. Grower-finisher diets were provided in pellet form.

 Each treatment group (Control and OFI) was subdivided into three replicates (pens) of 30 birds each, making a total of 90 birds per group. All pens were managed under identical environmental and nutritional conditions. Feed intake and growth performance were recorded on a pen basis, which was considered to the experimental unit for statistical analysis. For Blood biochemical analysis, six birds were randomly selected from each replicate (n=18 per treatment) to represent the average physiological status of each group.

Mortality: Mortality was monitored daily throughout the experimental period.

Growth performances analysis: The parameters assessed in this study included body weight and weight gain progression, daily feed intake and Feed conversion efficiency.

Body weight and daily weight gain: Individual body weights were recorded every three days. At each interval, 6 birds were selected randomly from each experimental group for measurement. Growth performance was evaluated during the grower phase, spanning from day 22 to day 48 of the trial. At the end of this period, weight gain for each group was determined by calculating the average final body weight of the birds. The percentage of weight gain was then calculated using the following equation:

Weight gain (%) = [(Final weight – Initial weight)/Initial weight] x 100

Where the initial weight corresponds to the average body weight recorded at the beginning of the grower-finisher period (day 22). 

Average daily gain was calculated at the pen level using the following formula:

ADG = (final body weight – initial body weight) / number of experimental days

where the number of experimental days corresponded to the grower-finisher period from day 22 to day 48.

Feed intake (FI)

Daily feed intake was calculated at the pen level using the following formula:

Daily feed intake = Total feed consumed per pen during the experimental period /number of live birds/number of experimental days.

Feed Conversion Ratio (FCR): The feed conversion ratio, also referred to as the Consumption Index (CI), is a critical parameter in poultry production used to evaluate the efficiency with which animals convert feed into body weight. A lower FCR indicates better feed efficiency and improved growth performances. FCR is calculated as follows:

FCR=Total feed consumed per pen/total body weight gain per pen

Blood sampling and biochemical profile analysis:  At the end of the experimental period (day 48), blood samples were collected from 6 birds per replicate. Blood samples were collected during the morning hours to minimize circadian variation. Birds were sampled under normal feeding conditions without prior fasting, as commonly practiced in broiler production studies. Blood was obtained from live birds via the brachial (wing) vein using sterile needles, and no euthanasia was performed for blood collection. For serum biochemical analyses, blood was drawn into a plain BD Vacutainer® tube and then centrifuged at 3000 rpm for 15 minutes to separate the serum. The serum was stored at -20°C until further analysis. Biochemical indices measured included glucose, urea, creatinine, triglycerides, total cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), total protein (TP), albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT). These were analyzed using commercial kits (Roche Diagnostics “Elecsys BRAHMS, Germany) with an automated chemistry analyzer (Roche Hitachi, Cobas c 501 Chemistry Analyzer, Germany).

Ethical approval

 All procedures involving animals were conducted in accordance with internationally recognized guidelines for animal research and reporting, including the ARRIVE 2.0 guidelines and the Guide for the Care and Use of Laboratory Animals (8^th edition). The experiment involved routine poultry husbandry practices and non-invasive blood sampling performed by trained personnel, with efforts made to minimize stress and ensure animal welfare throughout the study.

Statistical analysis: The study was conducted as a completely randomized design with two dietary treatments and three replicate pens per treatment. Since dietary treatments were applied at the pen level, the pen was considered the experimental unit for all statistical analyses.

 Growth performance data were analyzed using pen-level means. Individual bird measurements were used only to calculate the mean value for each pen and were not considered independent experimental units. For serum biochemical parameters, six birds were sampled per pen, and individual values were averaged to obtain one representative value per pen. Thus, biochemical analyses were performed using replicate pen means, with n = 3 pens per treatment.

 Data were analyzed using SPSS (version 29, IBM). Treatment effects were evaluated by one-way ANOVA, and body weight over time was analyzed using repeated-leasures ANOVA. Results are presented as mean ± SD of pen means, and differences were considered significant at p < 0.05. Given the limited number of replicate pens, the reported p-values should be interpreted with caution. 

RESULTS AND DISCUSSION

Growth performance indicator analysis: Table 2 shows the values of body weight and daily weight gain, as daily feed consumption and efficiency indices at the end of experimental trials.

Table 2: Initial body weight, Final body weight, average daily gain, daily feed intake, and feed conversion ratio of broiler chickens during the grower-finisher period (mean ± SD)

 Significant differences in average body weight related to diet were observed around day 27. Although all chicks exhibited steady growth throughout the study, the growth rate differed significantly between the treated and control groups. By the end of breeding period, the final body weight of treated group was higher than that of the control’s, with an increase of 9.84%. Furthermore, a significant difference in average daily weight gain between the two groups became apparent from day 30 onwards. The treated chicks showed a higher average daily gain of 69.17 g/birds/day, corresponding to a 13.55% increase compared with 60.92 g/birds/day in the control group. Similar to growth patterns, FI increased progressively during experimental trials.

 Average daily feed intake was slightly higher in the OFI-supplemented group than in the control group. Birds fed the OFI-supplemented diet consumed 170.85 g/birds/day in the control group, corresponding to a 2.73% increase.

 The FCR values increased during starter phase (days 1-22), indicating lower feed efficiency in young birds, and then stabilized during the grower-finisher phase (days 23-45). After day 39, FCR values showed resurgence, particularly in the control chicks; whose FCR increased substantially until the end of the experiment. This increase in FCR among was attributed to a reduction in weight gain while FI remained constant. In contrast, the FCR in experimental chicks exhibited only a slight increase, reflecting a gradual decline in weight gain and resulting in lower FCR values by the study’s conclusion.

 From a practical standpoint, the lower feed conversion ratio observed in the OFI-supplemented group, decreasing from 2.73 in the control group to 2.47, indicates improved feed efficiency. This improvement may have economic relevance, as it could contribute to reducing feed cost per kilogram of live weight produced. The improvement in growth performance observed in the present study appears to be mainly associated with increased body weight gain and improved feed conversion efficiency, rather than a marked increase in feed intake.

 Mortality during the experimental period was 2.22 % (2/90 birds) in the control group and 3.33 % in the OFI-supplemented group (3/90 birds). The observed difference was minimal; however, mortality data were recorded descriptively only and were not subjected to statistical analysis.

 Growth performance is further influenced by nutrient digestibility and feed intake, both of which are closely linked to the physicochemical properties of the gastrointestinal tract (Alyileili et al., 2020). Several studies have explored relationship between phytobiotics and gut microbiota modulation, seeking to determine whether growth-promoting effects are mediated through microbial alterations (Windisch et al., 2008; Valenzuela et al., 2017; Ceylan et al., 2023). Although the effects of phytobiotics depend on their chemical nature, mode of administration, and dosage, numerous studies have demonstrated their ability to modulate gut microbiota composition, which often correlates with enhanced growth performance, even in cases where overall growth metrics are not significantly improved (Stanley et al., 2016; Liu et al., 2021; Chen et al., 2023; Liu et al., 2023). Discrepancies between in vivo and in vitro finding may be explained by multiple factors, including differences in phytobiotic concentrations and the complex biological environment of the digestive tract (Huyghebaert et al., 2011; Chodkowska et al., 2022). Moreover, certain dietary components, such as high level of lipids and proteins, may reduce the bioavailability of efficacy of bioactive compounds by forming protective barriers around bacterial cells (Alyileili et al., 2020).

 Although, the experimental diet contained a higher crude fiber level than the control diet (5.20 % vs 3.50 %), growth performance was improved in the OFI-supplemented group. This suggests that the fiber-rich fraction of Opuntia ficus-indica fruit, particularly its soluble polysaccharides and mucilaginous components, may have exerted a prebiotic-like effect that supported intestinal function and nutrient utilization.

 Regarding performance, the use of certain essential oils such as garlic or rosemary has been associated with up to a 10% increase in body weight gain (Salaheen et al., 2017; Y. Liu et al., 2021). It has also been demonstrated that phytobiotic influence feed intake: rosemary essential oil has been reported to reduce feed intake by 16%, whereas thyme may increase it by 11 % (Cross et al., 2007; Stanley et al., 2016). Variability in feed conversion ratio has also been documented; turmeric supplementation has been shown to reduce feed conversion by 16% while thyme and Zataria multiflora essential oil have led increase of 7% and 4%, respectively (Al-Sultan, 2003; Cross et al., 2007).

 The efficacy of phytobiotic remains inconsistent across studies, largely due to differences in experimental designs, animal species or strains, administration methods, and particularly the preparation and standardization of phytobiotic products (Salaheen et al., 2017; Stanley et al., 2016). Animal age and genetic background also contribute significantly to the observed effects.

Blood biochemical profile analysis: Table 3 summarized the values of blood parameters in chicken broilers at the end of experimental period.

Table 3. Serum biochemical parameters of broiler chickens fed a control of OFI-supplemented diet (48 days of age, mean ± SD, n = 3 replicates per treatment)

AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: Gamma-glutamyltransferase; HDL: High-density lipoprotein.

 As shown in table 3, dietary supplementation significantly (p < 0.05) influenced several serum biochemical parameters in broiler chickens at the end of the experimental period. Birds receiving the dietary additive exhibited higher levels of total protein (TP) and albumin compared to the control group (p < 0.05), indicating improved protein metabolism. Similarly, serum concentration of high density lipoproteins (HDL) were significantly higher in the treated group (p = 0.015), suggesting a beneficial modulation of lipid metabolism. Conversely, the activity of aspartate aminotransferase (AST) was significantly reduced (p < 0.05), potentially reflecting hepatoprotective effects of the additive. No significant differences (p >0.05) were found for glucose ([2.32 ± 0.07 vs 2.44 ± 0.01], p = 0.12), urea ([0.39 ± 0.03 vs 0.39 ± 0.01], p = 0.802), creatinine ([27.97 ± 1.11 vs 28.51 ± 1.01], p = 0.665), triglycerides ([1.56 ± 0.08 vs 1.29 ± 0.11], p = 0.073), total cholesterol ([1.21 ± 0.04 vs 1.07 ± 0.06], p = 0.063), alkaline phosphatase ([1790.37 ± 20.16 vs 1750.33 ± 39.82], p = 0.332), or alanine aminotransferase (45.82 ± 3.10 vs 44.17 ± 2.88], p = 0.055), indicating that renal and general lipid metabolism remained unaffected by supplementation.

 Liver marker enzymes are reliable indicators for evaluating hepatic integrity and functionality in poultry, with serum concentrations typically increasing under stress or hepatotoxic conditions (Tang et al., 2022). In the present study, the notably reduced serum AST levels in broilers receiving the supplemented diet suggest a hepatoprotective effect, aligning with finding by Odetola et al. (2019), who reported decreased AST activity following dietary inclusion of Petiveria alliacea root meal. Although serum AST activity was significantly reduced in broiler fed the OFI-supplemented diet, ALT values were not significantly affected (p = 0.055). This discrepancy should be interpreted cautiously, as the diagnostic relevance of these enzymes differs between mammals and birds. In avian species, AST is considered a more sensitive and early marker of hepatic stress, whereas ALT activity remains low and less specific to hepatocellular injury (Tang et al., 2022). Therefore, the observed reduction in AST, together with stable ALT and elevated GGT levels, may indicate improved hepatic metabolic efficiency rather than structural liver repair. Similar interpretations have been proposed by Odetola et al. (2019) and Bueno et al. (2017), who reported reduced AST but unchanged ALT following dietary supplementation with phytogenic additives exhibiting antioxidant and hepatoprotective effects. These patterns suggest that OFI fruit bioactives could enhance hepatic enzyme homeostasis and support liver function under normal physiological conditions.

 Serum albumin, synthesized exclusively by the liver, is the principal plasma protein with multiple physiological functions, including transport and antioxidant roles. An increase in serum albumin in birds fed rosemary leaves, rich in phenolic acids and flavonoids, had been previously documented (Ghozlan et al., 2017). Given that polyphenolic compounds bind extensively to serum albumin, this interaction is believed to influence their systemic distribution and metabolic clearance (Oso et al., 2023). The improved protein profile observed in broilers fed phytobiotic-supplemented diets may be partly attributed to increase body mass, which is associated with higher total protein content. Moreover, phytobiotics are known to enhance intestinal health by stimulating digestive enzyme secretion and improving nutrient absorption (Abudabos et al., 2016), contributing further to protein assimilation. Considering the rapid growth and protein accretion during the short broiler chickens production cycle, changes in serum protein levels are expected (Abdul Basit et al., 2020).

 These effects may be partly attributed to the high content of bioactive compounds in OFI fruit, particularly, betalains and polysaccharides. Betalains are known for their antioxidant and hepatoprotective properties, which may support hepatic protein synthesis, while polysaccharides can improve intestinal integrity and nutrient absorption, thereby contributing to enhanced systemic protein metabolism. Gamma glutamyltransferase (GGT) is present in several tissues but is mainly of hepatic origin in serum due to renal excretion (Bueno et al., 2017). It is involved in glutathione metabolism and serves as a marker of cholestasis and bile duct proliferation.

 However, it should be noted that an increase in serum GGT activity may reflect either enhanced hepatic metabolic and antioxidant activity or a mild adaptive response to metabolic stress. Therefore, this finding should be interpreted with caution. The increase in serum GGT observed in the OFI-fed group should be interpreted carefully, as GGT may reflect both enhanced metabolic activity and potential hepatic stress. In mammals and birds, this enzyme participates in glutathione metabolism and cellular antioxidant defense, and moderate elevations can therefore indicate increased hepatic metabolic turnover or adaptation to oxidative challenges (Bueno et al., 2017) However, markedly elevated GGT levels are also considered biomarkers of cholestasis or hepatobiliary dysfunction. In the present study, the concurrent decrease in AST and the lack of change in ALT suggest that the GGT increase is more likely associated with enhanced glutathione activity and improved hepatic metabolic function rather than liver injury. A similar pattern was reported by Oso et al. (2023), who observed mild GGT elevation alongside improved protein metabolism in broiler supplemented with phytogenic feed additives. Collectively, these findings imply that OFI fruit may stimulate hepatic detoxification and antioxidant systems within physiological limits.

 The present findings are in agreement with previous studies investigating OFI in poultry and related experimental models. Martins et al. (2023) reported that dietary inclusion of OFI-derived products improve nutrient digestibility and growth performance in broiler chickens, which was associated with enhanced protein utilization and metabolic efficiency. Similarly, Giraldo et al. (2023) highlighted the hepatoprotective and antioxidant properties of OFI, attributed mainly to its betalains and polysaccharide content, supporting the observed reduction in AST activity and improvement in serum protein profile in the present study.

 Notably, while most previous studies focused on OFI extracts or cladodes, the use of whole fruit in the current work suggests that similar biological effects can be achieved through a more practical and nutritionally integrated form, potentially allowing synergetic interactions among bioactive compounds.

 Variations reported in the literature concerning biochemical responses to dietary additives may stem from differences in birds genetics, diet composition, age and experimental conditions. 

 Overall, our results support the hypothesis that Opuntia ficus-indica fruit can serve as an effective natural feed additive in broiler chickens production, combining growth-promoting and hepatoprotective effects similar to other well-characterized cactus product. The use of the fruit fraction, however, represents a novel approach compared to most previous studies, which primarily focused on cladode or extract forms.

 Despite the promising results observed in the present study, certain limitations should be acknowledged. The evaluation was limited to zootechnical performance and serum biochemical parameters, without direct assessment of gut microbiota composition or intestinal and hepatic histological features. In addition, the use of three replicate pens per treatment provided adequate but limited statistical power; therefore, the present findings should be interpreted with caution and confirmed in larger-scale studies. Consequently, the underlying mechanisms by which Opuntia ficus-indica fruit exerts its metabolic and hepatoprotective effects cannot be fully elucidated. Future studies integrating microbiota profiling, oxidative stress markers, and tissue histopathology would provide deeper insight into biological pathways involved.

Conclusion: The inclusion of Opuntia ficus-indica fruit in broiler chickens diets improved body weight gain and feed conversion efficiency, with only a slight increase in feed intake. Moreover, this dietary incorporation induced changes in blood chemical parameters, particularly by improving serum protein levels, suggesting a potential hepatoprotective effects. This finding support the use of OFI fruit as functional feed additive in poultry nutrition. Future research should further investigate the effects of Opuntia ficus-indica fruit supplementation on gut health and microbiota composition, meat quality attributed, and optimal inclusion levels to maximize its benefits in broiler chickens production.

Authors' Contributions: Belghiti Ayoub and Mouslim Jamal designed experiments; Belghiti Ayoub and Rafik Oussama collected samples; Belghiti Ayoub, Rafik Oussama, Benidire Loubna and Mouslim Assia performed experiments; Bidah Sara conducted statistical analysis; El ouahabi Imane and Mohtadi Karima performed the biochemical tests; Belghiti Ayoub wrote the manuscript. All authors agreed to publish this article.

Conflicts of interest: The authors did not declare any conflict of interest in this research.

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