Effects of Superdose phytase and acidifier supplementation in a phosphorus-deficient diet on growth performance, carcass characteristics, Cecal microbiota, and intestinal and liver morphology in broiler chickens

Abstract

This study evaluated the effects of super dose phytase and acidifier supplementation in a phosphorus-deficient diet on growth performance, carcass characteristics, cecal microflora, and intestinal and liver morphology in broiler chickens. A total of 400 one-day-old male broiler chickens of the Ross 308 strain was used in a completely randomized design, with 5 dietary treatments and 4 replications, each containing 20 birds. The treatments were as follows: 1. Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg). The study assessed daily weight gain (DWG), daily feed intake (DFI), Feed conversion Ratio (FCR), live body weight (LBW), carcass characteristics, tibia morphometric and mechanical traits, cecal microbial populations, and jejunal and hepatic histomorphology. Broilers fed PHAC showed higher DWG, improved FCR, and greater LBW during the starter period (1–10 d), while LPHAC increased DWG in the grower phase (11–24 d) and resulted in the highest overall DWG over 1–42 d compared with ACI (P < 0.05).The relative weight of the gastrointestinal tract (GIT) was significantly higher in birds fed with ACI diet compared to those receiving PHY (P < 0.05). Additionally, villus height (VH) was significantly greater in broilers fed with ACI diet than in those receiving either with LPHAC or PHY diets (P < 0.05). Crypt depth (CD) was significantly increased in broilers fed with LPHAC diet compared to those receiving the control diet (P < 0.05). Overall, combined supplementation of phytase (2000 FTU/kg) and acidifier (3 g/kg) improved growth performance, whereas dietary inclusion of 3 g/kg acidifier enhanced intestinal morphology by increasing villus height and width under reduced-phosphorus conditions. These findings suggest that phytase and acidifier supplementation may support early intestinal development and mineral utilization without inducing adverse hepatic histomorphological changes or altering cecal microbial populations in broiler chickens.

Introduction

The contemporary poultry industry demands high efficiency in both production and feed utilization, which can be partially supported through the careful use of targeted feed additives (Bist et al., 2024). Enhancing nutrient absorption and supporting gut health are essential strategies to boost growth rates, improve carcass traits, and promote overall well-being in broilers. Most poultry diets rely on plant-derived ingredients, such as corn and soybean meal, where nearly 67% of the total phosphorus is present as phytate-bound phosphorus (Kairallaet al., 2025; Rizwanuddin et al. 2023). Since this form of phosphorus is poorly available to birds, supplementation with expensive inorganic phosphorus is often required, which also raises environmental concerns due to increased phosphorus excretion (Abudabos, 2012; Alshelmani et al., 2024).

While phosphorus is abundant in plant-derived feedstuffs, it is largely indigestible for poultry unless hydrolyzed by intrinsic, microbial, or supplemented phytase enzymes (Khattak et al., 2023). This indigestibility contributes to inefficient phosphorus utilization and elevated phosphorus excretion into the environment. Consequently, strategies to enhance phosphorus utilization are crucial for both economic viability and environmental sustainability in poultry production (Zhang et al., 2026). Exogenous phytase supplementation has been widely adopted to improve phosphorus digestibility, enhance growth performance, and support bone mineralization (Selim et al., 2022). For example, Akter et al. (2016) demonstrated that phytase supplementation at 500 FTU/kg significantly improved feed intake and body weight gain in broiler chickens. Recent developments in enzyme technology have led to the practice of "super-dosing" using doses significantly above the nutritional requirement which has been shown to further enhance nutrient digestibility and modulate intestinal morphology. Shi et al. (2024) reported that super-dosing phytase increased villus height and the villus height to crypt depth ratio, thereby improving nutrient absorption efficiency. Similarly, Leyva-Jimenez et al. (2019) found that super-dosing phytase (1,500-2,000 FTU/kg) significantly improved body weight, weight gain, bone mineralization, and ileal digestibility of energy. These benefits may be mediated through improvements in gut microbial balance, including an increase in beneficial bacterial populations and suppression of pathogenic species, which are influenced by factors such as pH and the presence of other dietary additives (Abd El-Hack et al., 2018; Moita et al., 2021).

Parallel to enzyme supplementation, considerable attention has been directed toward identifying effective alternatives to antibiotic growth promoters. Organic acids (OAs), particularly acidifier, have shown promise due to their antimicrobial and growth-promoting effects (Abudabos et al., 2017; Choi et al., 2022). Acidifier can lower gastrointestinal pH, enhance nutrient digestibility, and modulate intestinal microflora, potentially augmenting the efficacy of phytase (Dibner and Buttin, 2002). The antimicrobial activity of acidifier contributes to a more balanced cecal microbiota by reducing pathogenic bacteria and supporting beneficial populations (Galgano et al., 2023). Rehman et al. (2016) reported that 0.5% acidifier supplementation significantly improved daily weight gain, feed conversion ratio (FCR), and intestinal villus height, while reducing cecal pH and pathogenic bacterial counts. Likewise, Martinez et al. (2021) and Waghmare et al. (2025) found that supplementation with 0.2 mL/L in drinking water or 1 kg/MT in feed enhanced growth performance, increased feed intake, reduced cecal pH, and promoted beneficial bacterial populations.

Emerging evidence suggests that the combined use of phytase and organic acids may not only enhance growth performance and carcass characteristics but also positively influence the morphology of the intestinal tract and liver organs essential for digestion, absorption, and metabolic regulation (Jalal et al., 2025). Recent studies have also suggested a potential immunomodulatory effect of this combination, helping to strengthen mucosal barriers and reduce systemic inflammation in poultry (Phillips et al., 2023). Given the challenges of phosphorus deficiency in practical poultry diets, the potential of combining super-dose phytase with acidifiers represents a promising strategy to overcome these limitations and improve overall poultry health and productivity (De Léo et al., 2025). However, studies evaluating the concurrent effects of phytase and acidifier in broiler chickens remain limited, particularly for histo-morphological outcomes and microbial ecology (Microbiota) in the cecum.

While the individual effects of phytase and organic acids have been well documented, research investigating their potential synergistic action under conditions of phosphorus restriction remains limited. A better understanding of this interaction is essential for promoting sustainable, antibiotic-free poultry production. We hypothesize that the combined supplementation of phytase and acidifiers may exert synergistic effects by enhancing nutrient utilization, improving gut health, and supporting microbial balance. Therefore, the present study evaluated the effects of dietary phytase and acidifier supplementation on growth performance, carcass characteristics, cecal microbial populations, and the histological architecture of the intestine and liver in broiler chickens.

Materials and methods

Animal ethics statement

All experimental protocols were reviewed and approved by the Animal Welfare Committee of the Department of Animal Science, University of Tehran (approval number No. 99183277, Date: September 15, 2023).

Bird management and treatments

This trial was carried out in the research poultry unit of the University of Tehran under uniform environmental conditions. A total of 400 one-day-old Ross 308 male broiler chicks (average initial body weight 43 ± 1 g) were allocated to five dietary treatments following a completely randomized design with four replicates and 20 birds per replicate. The treatments were as follows: 1. Control (control); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg). Commercial phytase (Persizyme-P, 10,000 FTU/g; Pardis Roshd Mehregan Co., Tehran, Iran) and acidifier (Selacid; Rooyandarou Co., Tehran, Iran) were added according to the treatment plan. Since prior research had already demonstrated the independent benefits of phytase and organic acids in phosphorus-deficient diets, single-additive low-phosphorus groups were excluded. The experimental setup therefore focused on determining their combined or individual roles under standard and phosphorus-restricted conditions. All birds were reared in floor pens (2 × 1 m) bedded with fresh wood shavings and equipped with individual feeders and manual drinkers. Feed and water were provided ad libitum throughout the 42-day period. Lighting was continuous during the first week and then adjusted to 23 h light: 1 h dark (23L: 1D). Light intensity was initially 30 lx and reduced to 20 lx after day 3 (TES 1335 lux meter). The lighting program (23L: 1D) was applied in accordance with the Ross 308 management guidelines (Aviagen, 2019) under controlled experimental conditions and was reviewed and approved by the Institutional Animal Ethics Committee of the University of Tehran; birds were monitored daily, and no adverse welfare or health effects were observed. The ambient temperature started at 33°C and was reduced by 3°C weekly until reaching 21°C by week 5. Relative humidity was maintained at approximately 60%.

Diets were formulated according to the 2019 Ross 308 nutrient requirement guidelines (Aviagen, 2019), which were adopted in our research facility to ensure consistency with previous experimental trials and comparability of results; all diets were formulated to meet or exceed the recommended nutrient specifications, except for the intentionally reduced phosphorus level in the low-phosphorus treatment (Table 1) (Aviagen, 2019). Feed was provided in mash form, and the chicks’ body weights were recorded at 1, 10, 24, and 42 days of age. Multiple hanging feeders were used to ensure equal feeding opportunity among all birds.

Table 1.

Ingredient and chemical composition of basal diets (%, as fed basis).

Ingredients (%)	Starter (1 to 10 d)		Grower (11 to 24 d)		Finisher (25 to 42 d)
	Control	Phosphorus- deficient5	Control	Phosphorus- deficient	control	Phosphorus-deficient
Yellow corn (CP=7.20%)	57.13	58.09	61.01	61.02	65.73	66.32
Soybean meal1 (CP=48.00%)	34.43	35.00	31.93	32.88	28.68	28.57
Corn gluten meal	3.00	1.85	1.31	0.70	0.00	0.00
Soybean oil	1.07	1.06	1.50	1.50	2.00	1.81
Mono Calcium Phosphate	1.34	0.74	1.28	0.69	1.09	0.57
Oyster shell	1.46	1.75	1.37	1.62	1.24	1.47
Vitamin premix2	0.25	0.25	0.35	0.35	0.25	0.25
Mineral premix3	0.25	0.25	0.35	0.35	0.25	0.25
Common salt	0.30	0.30	0.30	0.30	0.25	0.25
DL-Methionine	0.32	0.32	0.27	0.28	0.26	0.26
L-Lysine-HCl	0.35	0.29	0.23	0.21	0.15	0.15
L-Threonine	0.10	0.10	0.10	0.10	0.10	0.10
Total	100	100	100	100	100	100
Calculated Nutrients
AMEn4 (kcal/kg)	2870	2870	2935	2935	3000	3000
Crude protein (%)	22.00	22.00	20.20	20.22	18.10	18.15
Ca (%)	0.91	0.91	0.82	0.82	0.71	0.71
Ava. P (%)	0.45	0.36	0.42	0.32	0.36	0.26
Dig. Lys (%)	1.19	1.19	1.08	1.08	0.93	0.93
Dig. Met (%)	0.65	0.65	0.56	0.57	0.52	0.54
Dig. Met + Cys (%)	0.90	0.90	0.83	0.85	0.74	0.77
Na (%)	0.14	0.14	0.15	0.15	0.17	0.17
Analyzed Nutrients
Crude protein (%)	21.84	21.91	20.05	20.11	17.96	18.08
Ca (%)	0.93	0.92	0.81	0.82	0.70	0.72
Total P (%)	0.81	0.74	0.75	0.73	0.66	0.62
Non-phytate P (%)	0.463	0.357	0.417	0.331	0.352	0.254

Growth performance

Feed intake (FI), daily weight gain (DWG), and mortality were recorded daily during the early period and weekly thereafter for starter (1–10 d), grower (11–24 d), and finisher (25–42 d) phases. Feed consumption was determined by subtracting the remaining feed from the total feed offered in each replicate. Daily feed intake (DFI) was calculated by dividing total feed consumed by the number of days in each phase. Feed conversion ratio (FCR) was expressed as grams of feed intake per gram of weight gain. Live body weight (LBW) at 42 days and cumulative performance indices were adjusted for mortality using live bird-days.

Physical specification, calcium and phosphorus content parameters

At 42 d of age, five birds per replicate (n = 20 per treatment), with body weights close to the replicate mean, were randomly selected for tibia evaluation. The left tibia was excised, cleaned of adhering tissues, and the periosteum was removed.

Tibia morphometric traits, including length, width, and weight, were recorded. Bone length (cm) was measured from the proximal to distal epiphysis, whereas width was determined at the mid-diaphysis using a digital vernier caliper. Tibia volume (cm³) was estimated using the water displacement method based on Archimedes’ principle (Fayeye et al., 2006). Mechanical strength was evaluated by measuring tibia breaking strength (N) using a universal testing machine (Shim et al., 2012).

For mineral analysis, tibiae were dried at 105°C for 24 h and subsequently fat-extracted using a Soxhlet apparatus with petroleum ether for 48 h. The fat-free bones were then ashed in a muffle furnace at 550°C for 12 h to determine total ash percentage. The resulting ash was dissolved in 6 N hydrochloric acid (HCl). Calcium concentration was quantified using an atomic absorption spectrophotometer (Shimadzu AA-7000), whereas phosphorus concentration was determined by the molybdo-vanadate colorimetric method using a UV–Vis spectrophotometer at 400 nm (Yan et al., 2005).

Carcass characteristics

At day 42, five birds with body weights close to the group mean were randomly selected per pen for carcass evaluation. Birds were subjected to a 12 h fasting period prior to slaughter. Thereafter, birds were euthanized by cervical dislocation following Underwood and Anthony (2020). Carcasses were then eviscerated and the liver and GIT were excised and weighed individually. For the GIT weight determination, the entire tract from the proventriculus to the cloaca was excised, gently cleared of residual digesta, and weighed to obtain the empty tract weight. Carcass yield and relative organ weights were expressed as a percentage of live body weight (Celik et al., 2014), with carcass yield calculated based on skinned eviscerated carcasses with the head and feet removed.

Cecal microbial populations

On day 42, one cecum from four birds per replicate was aseptically collected and immediately chilled for microbial analysis. One gram of cecal content was diluted in 9 mL of sterile saline, serially diluted, and cultured on de Man–Rogosa–Sharpe (MRS) agar to enumerate Lactobacillus spp. Plates were incubated at 37°C for 48 h under anaerobic conditions and 24 h aerobically. Each sample was cultured in duplicate (Masouri et al., 2017).

Intestinal and liver morphological assay

Histology was performed as described by Uni et al. (2001). Eight birds per treatment were euthanized at day 42, and 3 cm samples from the mid-jejunum and liver were collected, rinsed in phosphate-buffered saline (PBS), and fixed in 10% neutral-buffered formalin (0.1 M phosphate buffer, pH 7.3). Fixed tissues were dehydrated, embedded in paraffin, and sectioned (5 μm) for hematoxylin and eosin staining. Slides were examined under a light microscope (BX51, Olympus, Japan) at 200 × magnification with a digital camera (Discover Echo, USA). Villus height (VH), villus width (VW), and crypt depth (CD) were measured using Motic Images Plus 2.0 software. The VH: CD ratio was calculated as an index of mucosal architecture (Berg et al., 2001).

Statistical analysis

Data were analyzed using one-way ANOVA in the General Linear Model (GLM) procedure of SAS (2009). The normality of residuals and homogeneity of variances were verified using the UNIVARIATE and HOVTEST options, respectively. Microbial counts were log-transformed (log10) prior to analysis. The pen (20 birds per pen) was considered the experimental unit for all analyses, and data were subjected to one-way ANOVA using a completely randomized design. The model used was: Y_ij = μ + T_j + e_ij, where Y_ij is the observed value, μ the overall mean, T_j the dietary treatment effect, and e_ij the random error. Treatment means were compared using Tukey’s test, with statistical significance declared at P < 0.05.

Results and discussion

Growth performance

Growth performance parameters of broiler chickens as affected by dietary phytase and acidifier supplementation are presented in Table 2. During the starter phase, dietary treatments significantly affected DWG, FCR, and LBW at 10 d (P < 0.05), whereas DFI remained unchanged. In the same period, broilers, chicks fed with PHAC diet showed significantly higher DWG and lower FCR compared to those receiving the LPHAC diet (P < 0.05). Moreover, LBW at 10 d was significantly greater in chicks fed PHAC compared with those fed LPHAC (P < 0.05). However, DWG and FCR did not differ between the LPHAC and control groups during the starter period, whereas LBW at 10 d was significantly higher in the control group compared with LPHAC.

Table 2.

Effects of dietary phytase and acidifier supplementation on growth performance of broiler chickens during starter (1–10 d), grower (11–24 d), finisher (25–42 d), and total (1–42 d) periods.

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
DWG2 (g/bird per d)
1-10 d	20.95ab	21.05ab	20.93ab	21.07a	20.87b	0.041	0.018
11-24 d	41.71b	45.25ab	43.72ab	45.94ab	46.60a	1.116	0.049
25-42 d	67.07	62.95	62.42	67.45	69.65	2.583	0.267
1-42 d	46.55ab	46.37ab	45.69b	48.58ab	49.23a	0.737	0.016
DFI3 (g/bird per d)
1-10 d	23.14	23.15	23.16	23.22	23.25	0.048	0.503
11-24 d	61.51	61.90	62.50	60.99	63.31	0.600	0.114
25-42 d	126.27	122.32	123.10	125.57	126.15	3.158	0.847
1-42 d	77.87	76.70	77.55	78.13	78.12	0.980	0.828
FCR4 (g feed/g gain)
1-10 d	1.11ab	1.10ab	1.11ab	1.09b	1.12a	0.003	0.001
11-24 d	1.48a	1.37ab	1.43ab	1.33b	1.36ab	0.032	0.025
25-42 d	1.90	1.95	1.97	1.86	1.81	0.060	0.370
1-42 d	1.67	1.65	1.70	1.60	1.59	0.028	0.086
LBW5 (g)
10 d	249.69b	250.32ab	249.09bc	250.44a	248.86c	0.285	0.002
24 d	837.57	883.82	866.56	896.49	902.42	15.510	0.062
42 d	2102.42	2055.82	2015.37	2174.17	2240.22	57.130	0.086

a–d Means with different superscripts within a row are different at P<0.05.

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg).

2 Daily Weight gain = Weight gain per experimental unit (g) / number of hen days

3 Daily feed intake = The amount of feed given week (g) - The amount of feed left at the end of the week (g) / number of hen days.

4 Feed conversion ratio = (g FI/ g WG)

5 Live body weight= End live body weight / number of hens.

During the grower phase (11–24 d), broilers fed the LPHAC diet showed significantly higher DWG compared with those receiving the control diet (P < 0.05). Chicks fed the PHAC diet exhibited a significantly lower FCR than those fed the control diet (P < 0.05). However, FCR did not differ between the LPHAC and control groups during the grower period. Moreover, LBW at 24 d was not significantly affected by dietary treatments.

During the finisher phase (25–42 d), no significant differences were observed among dietary treatments for DWG, DFI, or FCR. Moreover, LBW at 42 d was not significantly affected by dietary supplementation during this period. Although final body weights were lower than commercial performance targets, this outcome may be attributed to mash feeding and controlled experimental conditions; importantly, all birds remained clinically healthy, and the uniform management across treatments ensured the validity of the comparative results.

Over the entire experimental period (1–42 d), broilers fed the LPHAC diet exhibited significantly higher DWG compared with those receiving the ACI diet (P < 0.05). However, DFI and overall FCR were not significantly influenced by dietary treatments throughout the total period. In addition, final LBW at 42 d did not differ significantly among experimental groups.

Overall, phytase and acidifier co-supplementation, particularly under low-phosphorus conditions, improved growth performance primarily during the early growth phases, with limited effects during the finisher and overall periods. These results suggest that phytase-mediated hydrolysis of phytate-bound phosphorus, together with acidifier-induced modulation of the gastrointestinal environment, may partially compensate for reduced dietary phosphorus without adversely affecting growth performance (Ahmad et al., 2023).

Over the entire rearing period (1–42 days), broilers fed the reduced-phosphorus diet supplemented with phytase and acidifier showed a significant improvement in DWG compared to those on the control diet with acidifier alone. This sustained enhancement in feed efficiency under phosphorus-restricted conditions highlights the pivotal role of phytase in conjunction with acidifier in optimizing nutrient utilization throughout the production cycle. While phytase directly enhances phosphorus bioavailability via phytate hydrolysis (Selim et al., 2022), the acidifier likely contributes to this enhanced digestive efficiency by modulating gut microbial populations (Bedford and Apajalahti, 2022; Dersjant‐Li et al., 2015), further facilitating the absorption of phosphorus and other essential nutrients.

The economic and environmental benefits of this approach are also noteworthy. Replacing costly inorganic phosphorus sources like di-calcium phosphate (DCP) with phytase not only reduces feed costs (Pieniazek et al., 2017) but also significantly contributes to environmental sustainability by lowering phosphorus excretion (Scholey et al., 2018). Beyond direct phosphorus release, phytase supplementation exerts multiple physiological effects that enhance broiler performance. It improves sodium reabsorption in the small intestine, indirectly facilitating overall nutrient absorption (Truong et al., 2017), and mitigates the anti-nutritional effects of phytate, boosting the bioavailability of various nutrients (Beeson et al., 2017). This broader improvement in nutrient availability, coupled with increased digestibility of amino acids and proteins (Gehring et al., 2013) and modulation of endogenous digestive enzymes (Liu et al., 2010), collectively supports enhanced production performance, especially crucial when dietary phosphorus is limited. The dose-dependent benefits of phytase on BWG, as reported by Pirgozliev et al. (2008), further underscore its capacity to meet broiler phosphorus requirements.

The observed improvements in growth performance during the starter and grower phases may be partly attributed to changes in the gastrointestinal environment. Organic acids have been reported to reduce gastrointestinal pH, thereby enhancing the activity of pepsin and other digestive enzymes (Omogbenigun & Nyachoti, 2003). This acidified condition, together with potential improvements in intestinal morphology, may provide a more efficient absorptive surface for mineral and protein utilization. Consequently, these mechanisms could help explain the increased weight gain observed between days 1 and 24, in agreement with previous reports (Al-Kassi and Mohssen, 2009; Al-Tarazi and Alshawabkeh, 2003).

Physical specification, calcium and phosphorus content Parameters

Table 3 shows the effects of diets containing different phytase enzyme and acidifier supplementation on tibia bone characteristics (length, width, volume, and strength), calcium (Ca) and P content of tibia morphometric parameters in broiler chicken on d 42. There were no significant differences in the on-bone parameters among the treatment groups. These findings suggest that, under the conditions of this study, neither phytase nor acidifier supplementation individually or in combination exerted a measurable influence on bone mineralization. This result contrasts with several previous studies which reported improved bone mineral content and strength in response to phytase supplementation in P -deficient diets. This result contrasts with several previous studies that reported improved bone mineral content and strength with phytase supplementation in P-deficient diets (Adeola and Sands, 2004; Sebastian et al., 1996). The observed lack of response in the present study may be attributed to sufficient baseline dietary P levels across all treatment groups, which could have potentially masked the osteogenic benefits of phytase and acidifier supplementation. Moreover, while OA such as acidifier can lower gut pH and potentially improve mineral solubility and absorption (Khan and Iqbal, 2016), their effect on skeletal development remains inconsistent across studies. For instance, while Adil et al. (2011) found improvements in tibia ash with citric acid inclusion, others such as Gunal et al. (2006) observed no significant differences with acidifier use. The absence of significant changes in bone parameters in the current study may also be related to the birds' age, strain, or the duration of the trial. It is also possible that improvements in mineral retention were functionally redirected toward soft tissue growth and metabolic needs rather than skeletal deposition. Although phytase and acidifier are often beneficial for nutrient digestibility and performance, their impact on bone mineralization in broilers appears to be influenced by dietary composition and other interacting factors. Further research with varying P levels and extended observation periods may help clarify their role in skeletal development. It should be emphasized that both the specific bone evaluated and the genetic line of the broilers greatly influence the response of bone morphology and composition to phytase supplementation (Mohammed et al., 2021). Variations in skeletal architecture among different broiler strains have been documented (Burton et al., 2020). According to previous findings, the femur appears to be a more suitable bone for examination at 42 days of age than the tibia, as the femur continues to mineralize during this period, while the tibia tends to exhibit a more mature and stabilized structure (Scholey and Burton, 2017).

Table 3.

Effect of diets containing different phytase enzyme and acidifier supplementation on physical specification, calcium and phosphorus content of tibia bone in broiler chicken on d 42.

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
Length (cm)	8.94	8.78	8.92	8.84	8.90	0.231	0.346
Width (cm)	0.84	0.86	0.83	0.87	0.91	0.121	0.243
Strength (N)	25.66	25.97	25.66	27.66	26.14	1.145	0.097
volume (cm3)	9.68	9.05	9.96	10.01	9.45	0.765	0.543
Calcium (%)	33.12	33.15	33.26	33.95	33.31	0.644	0.149
Phosphor (%)	17.08	17.12	17.09	17.26	17.04	0.613	0.721
Ash (%)	54.01	54.34	55.07	54.61	53.09	0.836	0.187

a–d Means with different superscripts within a row are different at P<0.05

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); Control + Phytase (PHY; 2000 FTU/kg); Control + Acidifier (ACI; 3 gr/ kg); Control + Phytase + acidifier (PHAC), and Low-phosphorus diet + Phytase + Acidifier (LPHAC).

Carcass yield and weight internal of organs

The effects of dietary supplementation with phytase and acidifier on carcass characteristics and the relative weight of internal organs (expressed as a percentage of carcass weight) in broiler chickens at day 42 are presented in Table 4. The relative weight of the GIT was significantly higher in birds fed with ACI diet compared to those receiving the PHY diet (P < 0.05). However, supplementation with phytase and/or acidifier had no significant effect on carcass yield, or on the relative weights of the liver, abdominal fat, and ceca. The evaluation of carcass traits and internal organ weights at day 42 indicated minimal detrimental effects of phytase and acidifier supplementation on broiler physiological parameters, even in the context of reduced dietary phosphorus. Specifically, the relative weights of carcass yield, liver, abdominal fat, and ceca were largely unaffected by dietary treatments. This suggests that the combined inclusion of these additives effectively supported broiler development without negatively altering major anatomical indices, implying efficient nutrient partitioning despite phosphorus constraints. However, broilers fed the control diet supplemented with acidifier showed a significant increase in the relative weight of the GIT compared to those receiving the control diet with phytase alone. This finding suggests acidifier's potential to influence gut development and digestive capacity (Martinez et al., 2021). This increased gut tissue mass could be a physiological adaptation to enhanced mucosal activity or shifts in the intestinal microbiota, contributing to an environment more conducive to nutrient absorption, including phosphorus, from a deficient diet. Despite this localized effect, the stability in overall carcass yield across dietary treatments supports the notion that the primary benefits of phytase and organic acid supplementation are more likely reflected in improved nutrient digestibility and feed efficiency rather than significant alterations to carcass composition (Rahman, 2018). While phytase and acidifier generally maintained carcass quality under phosphorus limitation, phytase supplementation alone numerically improved eviscerated carcass yield and leg muscle ratio. This positive influence on specific carcass components may stem from enhanced nutrient availability due to phytate hydrolysis, supporting muscle development (Chisato et al., 2003). However, consistent with other findings (Yonemochi et al., 2003), phytase did not significantly affect breast muscle ratio or abdominal fat deposition. These observations collectively suggest that while the combination effectively addresses phosphorus deficiency, the individual components, particularly phytase, can selectively optimize certain carcass traits by enhancing overall nutrient efficiency.

Table 4.

Effect of diets containing different phytase enzyme and acidifier supplementation on carcass yield and relative weight of internal organs (% of carcass) in broiler chicken on d 42.

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
Carcass yield (%)	63.36	63.20	61.98	63.88	63.21	0.734	0.482
Gut (%)	11.69ab	11.15b	13.33a	11.17ab	11.18ab	0.504	0.035
Liver (%)	2.160	2.375	2.105	1.938	1.954	0.106	0.065
Abdominal fat (%)	0.873	1.259	1.019	0.672	1.348	0.168	0.071
Ceca (%)	31.36	26.34	24.09	25.89	34.50	2.849	0.105

a–d Means with different superscripts within a row are different at P<0.05.

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg).

Cecal microbial population

Table 5 presents the effects of diets containing phytase and acidifier on the cecal microbial population of broiler chickens at day 42. No significant differences were observed among treatment groups in the counts of Lactobacillus spp., coliforms, or total bacteria. The analysis of cecal microbiota at day 42 revealed no statistically significant changes in the populations of Lactobacillus spp., coliforms, or total bacteria following dietary supplementation with phytase and acidifier. This indicates that, under the study conditions, these feed additives did not markedly alter the overall microbial balance within the cecum in later growth stages. Several factors may contribute to this observed microbial stability. By day 42, the cecal microbial ecosystem likely reached a relatively mature and stable state, potentially limiting its responsiveness to dietary interventions (Dittoe et al., 2018; Künzel et al., 2021). The inherent buffering capacity of the cecum and the specific dosage or combination of phytase and acidifier used might also have been insufficient to induce measurable shifts in total microbial counts (Dittoe et al., 2022). Nonetheless, the absence of significant detrimental alterations in cecal microbiota is noteworthy, as it indicates that these additives effectively mitigated phosphorus deficiency and supported performance without disrupting gut microbial homeostasis a key consideration for intestinal health. While OA are known for their ability to lower gut pH and exert antimicrobial properties, potentially suppressing pathogenic bacteria and improving nutrient availability (Ji et al., 2023; Peh et al., 2020; Saleem et al., 2016), these effects might be more pronounced in earlier growth phases or when targeting specific pathogenic taxa not measured here. The improved growth performance observed in this study, despite stable total microbial counts, suggests that the benefits of the combined supplementation particularly in overcoming phosphorus deficiency may stem more from direct effects on nutrient digestion, absorption, and overall gut integrity rather than broad shifts in the cecal microbial population. For instance, the acidified environment created by OA not only supports digestive enzyme secretion (amylase, lipase, and protease) but also enhances the solubility and absorption of key minerals, including calcium and phosphorus (Liu et al., 2014). This direct action on nutrient availability and absorption, with phytase's phosphorus-releasing capabilities, may explain the observed performance improvements in phosphorus-deficient diets without necessarily inducing major changes in the overall measured cecal microbiota. Future research should consider investigating the effects of various types of organic acids, higher supplementation levels, and earlier sampling time points to gain a more comprehensive understanding of the dynamic interactions between dietary additives and gut microbiota composition and function in broiler chickens.

Table 5.

Effect of diets containing different phytase enzyme and acidifier supplementation on ileal microflora of broiler chicken on d 42 (Log10).

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
Lactobacillus (cfu/g-1)	4.41	4.42	4.42	4.42	4.42	4.42	4.42
Coliform (cfu/g-1)	7.59	7.64	7.64	7.64	7.64	7.64	7.64
Total bacteria count (cfu/g-1)	8.34	8.41	8.41	8.41	8.41	8.41	8.41

a–d Means with different superscripts within a row are different at P<0.05.

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg).

Morphology of intestine and liver

Table 6 shows the effects of dietary phytase and acidifier on jejunal morphology in broiler chickens at day 42. The VH was significantly greater in birds fed with ACI diet compared to those receiving either the LPHAC or PHY diets (P < 0.05). Similarly, VW was significantly higher in birds fed ACI diet than in those fed the LPHAC or control diets (P < 0.05). In contrast, CD was significantly increased in birds receiving LPHAC diet compared to those fed the control diet (P < 0.05).

Table 6.

Effect of diets containing different phytase enzyme and acidifier supplementation on morphology of the jejunum in broiler chicken on d 42.

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
Villus height (μm)	905.59bc	835.73c	1057.94a	944.17bc	988.39b	12.910	<0.0001
Villus width (μm)	161.91c	205.11ab	228.47a	181.69bc	201.29b	7.951	0.0004
Crypt depth (μm)	147.80b	152.33ab	190.37ab	150.80ab	267.94a	26.503	0.027
Crypt diameter (μm)	11.43	13.92	16.79	14.97	14.99	1.940	0.426
Villus height/ Crypt depth ratio	6.30	5.87	5.58	6.46	4.28	0.847	0.261

a–d Means with different superscripts within a row are different at P<0.05.

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg).

No significant differences were observed among the treatment groups in terms of hepatocyte nucleus diameter or hepatocyte cell diameter (Table 7).

Table 7.

Effect of diets containing different phytase enzyme and acidifier supplementation on morphology of liver in broiler chicken on d 42.

Items	Dietary treatments1					SEM	P-value
	Control	PHY	ACI	PHAC	LPHAC
Hepatocyte nucleus diameter (μm)	7.44	7.94	7.97	8.09	7.16	0.416	0.428
Hepatocyte cell diameter (μm)	19.74	18.78	18.25	20.98	19.99	1.109	0.475

a–d Means with different superscripts within a row are different at P<0.05.

SEM = Standard Error of means.

1 The experimental groups included: Control (basal diet without additives); 2. Control + Phytase: (PHY; 2000 FTU/kg); 3. Control + acidifier (ACI; 3 gr/ kg); Control + phytase + acidifier (PHAC; 2000 FTU/kg + 3 g/kg); and 5. Low-phosphorus diet + phytase + acidifier (LPHAC; 2000 FTU/kg + 3 g/kg).

This study's results demonstrated that phytase and acidifier supplementation significantly influenced jejunal morphology by day 42, particularly in the context of the phosphorus-deficient diet. Notably, birds fed the control diet with acidifier alone exhibited significantly increased villus height and villus width compared to those receiving the reduced-phosphorus diet supplemented with both phytase and acidifier. This suggests that acidifier plays a primary role in promoting jejunal villus development, likely by modulating gut pH or enhancing digestive enzyme activity. Organic acids are known to improve villus architecture and functionality, enhancing nutrient absorption efficiency by optimizing villus height and surface area (Ebeid and Al-Homidan, 2022). In contrast, crypt depth was significantly greater in birds fed the reduced-phosphorus diet supplemented with both phytase and acidifier compared to the control. This increased crypt depth in the combined treatment group likely indicates a compensatory mechanism by the gut to maintain epithelial turnover and overall gut health under conditions of reduced phosphorus availability, supporting an enhanced rate of cell proliferation to optimize nutrient absorption. Birds fed the LPHAC diet exhibited greater crypt depth compared with the control group (Table 6), which may indicate enhanced epithelial turnover under reduced dietary phosphorus. This adaptive response, in combination with high-level phytase supplementation, may have supported nutrient utilization despite the increased maintenance demand (Moita et al., 2021). The unchanged VH: CD ratio further suggests that overall intestinal architecture remained proportionally balanced, indicating maintained mucosal integrity rather than pathological alteration (Wickramasuriya et al., 2022). The overall improvements in intestinal morphology underscore the importance of a healthy gut for efficient nutrient utilization, as proposed by Zanu et al. (2020). Furthermore, while not explicitly measured as changes in total bacterial populations in the cecum (as discussed previously), the efficacy of organic acids in potentially suppressing pathogenic bacteria (Nhara et al., 2024) can reduce inflammatory responses within the gut mucosa, leading to improvements in intestinal morphology. This, in synergy with phytase's ability to release bound phosphorus, supports optimal secretory, digestive, and absorptive functions of the intestinal lining (Khan and Iqbal, 2016), enabling the birds to better cope with phosphorus-deficient diets.

The absence of significant differences in hepatocyte nucleus and cell diameters among treatment groups suggests that dietary supplementation with phytase and acidifier did not adversely affect hepatic cellular morphology. These findings indicate that the combined use of these additives, even under reduced phosphorus conditions, was not associated with detectable histomorphological alterations in the liver. The stability of hepatocyte structure observed in the present study supports the physiological compatibility of this nutritional strategy (Akaichi et al., 2022; Derakhshan et al., 2023). Collectively, these findings imply that while phytase and acidifier supplementation can modulate intestinal morphology, their influence on hepatic immune markers, as reflected by hepatocyte morphology, appears limited under the conditions tested. Future studies should investigate the underlying mechanisms by which these additives affect gut morphology and immune function, potentially by evaluating varying supplementation levels or earlier developmental stages.

Conclusions

This study demonstrated that combined supplementation of phytase and acidifier (PHAC and LPHAC; 2000 FTU/kg phytase + 3 g/kg acidifier) improved growth performance primarily during the early growth phases. Although overall growth performance, carcass characteristics, and cecal microbiota were largely unaffected, dietary inclusion of 3 g/kg acidifier significantly enhanced intestinal morphology by increasing villus height and width. Collectively, these findings suggest that phytase and acidifier supplementation may contribute to reducing reliance on inorganic phosphorus sources without adversely affecting growth performance or physiological health in broiler chickens.

Ethics statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes.

Availability of data and materials

The datasets produced and/or analyzed during the current study are available from the corresponding author on reasonable request.

CRediT authorship contribution statement

Arman Farzanegan: Writing – original draft, Project administration, Methodology, Data curation. Seyed Davood Sharifi: Writing – review & editing, Supervision, Conceptualization. Hasan Rouhanipour: Writing – original draft, Investigation, Formal analysis, Data curation. Melika Jourablou: Writing – original draft, Visualization, Methodology.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can in appropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.