Introduction
Poultry diets are mostly composed of plant feed ingredients, and the phosphorus (P) contained in plant-based feeds is in the form of phytate. Because there is no phytase enzyme that dissolves phytate P in digestive systems, broilers can only use it to a limited extent (Cowieson and Bedford, Reference Cowieson and Bedford2009; Selle et al., Reference Selle, Ravindran, Cowieson and Bedford2011; Ghasemi et al., Reference Ghasemi, Toghyani and Landy2019). Undigested phytate P is excreted in faeces as an environmental pollutant (Landy and Toghyani, Reference Landy and Toghyani2018; Fallah et al., Reference Fallah, Karimi, Sadeghi and Behroozi-Khazaei2020). To supply the P requirement of poultry, inorganic phosphate sources are added to the diets (Akter et al., Reference Akter, Graham and Iji2016) and phosphate is a component that increases feed cost more than other minerals (Rodehutscord, Reference Rodehutscord2013; Anjum et al., Reference Anjum, Javaid and Nadeem2018). The phytase enzyme releases P and other nutrients bound to phytic acid. Exogenous phytase decreases the need for phosphate supplementation in the diet, reducing total P excretion (Hamdi et al., Reference Hamdi, Perez, Létourneau-Montminy, Franco-Rosselló, Aligue and Solà-Oriol2018). For these reasons, including phytase enzyme in broiler diets, either alone or in combination with other enzymes, has become a standard practice.
High dietary calcium (Ca) reduces P absorption by forming insoluble complexes in the intestinal lumen and raising the pH of the gizzard and digestive tract (Walk et al., Reference Walk, Bedford and McElroy2012), whereas a low Ca diet forces bone mineralization, resulting in increased P excretion (Ajith et al., Reference Ajith, Shet, Ghosh, Awachat, Bhat, Pal and Elangovan2018). As a result, it is critical that the Ca:AvP ratio in the diet be 5:1 to maximize P availability while decreasing excretion (Ajith et al., Reference Ajith, Shet, Ghosh, Awachat, Bhat, Pal and Elangovan2018). Calcium and P contents can be decreased without having an adverse effect on birds if this ratio is maintained (Akter et al., Reference Akter, Graham and Iji2016). Cardoso et al. (Reference Cardoso, Rodrigues, Bertechini, Freitas, Lima and Lima2010) stated that reducing Ca from 8.50 to 6.50 g/kg and AvP from 4.25 to 3.25 g/kg in the diet resulted in a similar performance. Akter et al. (Reference Akter, Graham and Iji2016) noted that decreasing Ca and AvP contents in the diet to 6.00 and 3.00 g/kg, respectively, had no negative effect on bone development in broilers.
Bone development is critical for the growth of muscular tissue, which is the primary source of income in broiler breeding. Bone mineralization and bone strength are two indications of bone development. Insufficiency in bone development would cause bone fractures during the catching, transport and slaughter stages, marketable carcass losses and subsequent economic losses (Cardoso et al., Reference Cardoso, Rodrigues, Bertechini, Freitas, Lima and Lima2010). Calcium and P are the most important minerals present in the bone structure and require for bone formation; and for bone development taking them in the diet not only in sufficient amounts, but also in available forms is a requisite (especially P) and also at optimal rates (Olgun and Aygun, Reference Olgun and Aygun2016).
The jejunum is the digestion and absorption zone for majority of nutrients (Svihus, Reference Svihus2014), so jejunal development is an important indicator of digestion and absorption ability. Dietary low P (Martínez-Vallespín et al., Reference Martínez-Vallespín, Männer, Ader and Zentek2022) and high Ca negatively affect villus development. However, there are limited studies on the effect of Ca and P contents on villus development, and no changes were found (Abdulla et al., Reference Abdulla, Loh, Akit, Sazili and Foo2016; Oikeh et al., Reference Oikeh, Sakkas, Blake and Kyriazakis2019; Noruzi et al., Reference Noruzi, Hassanabadi, Golian and Aziz-Aliabadi2022).
In poultry nutrition, meeting the need for AvP has been given more importance than Ca, and considering the rate and form of P, it has been emphasized that the Ca need is dependent on the dietary AvP content (Rodehutscord, Reference Rodehutscord2013). However, excess Ca can have negative effects on digestion and performance, including the production of insoluble Ca–protein–phytate complexes and insoluble salts with dietary fatty acids, which reduce nutrient digestibility (Tamim et al., Reference Tamim, Angel and Christman2004). Therefore, the central hypothesis of this study posits that reducing the contents of calcium (Ca) and available phosphorus (AvP) in corn–soybean meal-based diets, while maintaining a Ca:AvP ratio of 2:1 and incorporating phytase enzyme, will not adversely impact the performance and bone characteristics of male broilers. Furthermore, this study aimed to explore the potential alterations in the morphology of the jejunum resulting from these dietary modifications.
Materials and methods
The experiment was designed according to the completely randomized design and completed in a local farm in Selçuklu (Konya, Türkiye) at 38°1′36″, 32°30′45″ coordinates.
Birds and experimental design
In the experiment, 480 one-day-old Ross 308 male broiler chicks were used. The research was conducted in four treatment groups with eight subgroups with 15 birds each. Chicks were distributed randomly, 120 in each experimental group. Except for Ca and AvP, all nutritional requirements of broiler chickens (Aviagen, Reference Aviagen2019) were met or exceeded by the corn and soybean meal-based diets formulated (Table 1).
Ingredient and nutrient composition of experimental basal diets (as fed)
LCP1, LCP2 and LCP3: compared to control diet; 0.3 g/kg lower AvP in the starter period; it contains 0.2, 0.4 and 0.6 g/kg lower AvP in the grower period, respectively, and 0.45, 0.9 and 0.9 g/kg lower AvP in the finisher period, respectively. Dietary Ca contents were determined to be at a Ca/AvP ratio of 2/1.
Bold values indicate the calcium and available phosphorus contents in the experimental diets.
a Supplied per kg diet: 13 000 IU vitamin A, 5000 IU vitamin D3, 80 mg vitamin E, 5 mg vitamin B1, 9 mg vitamin B2, 5 mg vitamin B6, 0.020 mg vitamin B12, 4 mg vitamin K3, 25 mg pantothenic acid, 70 mg niacin, 0.35 mg biotin, 2.5 mg folic acid, 110 mg zinc, 120 mg manganese, 16 mg copper, 20 mg iron, 0.3 mg selenium, 1.25 mg iodine.
b Values in parentheses show analysis results for calcium.
c Analysed values.
For each period of the study, four different diets were used, including a control diet containing Ca and AvP at recommended rates and three trial diets containing reduced Ca and AvP. The Ca and P analysis was carried out according to AOAC (2005). During the starter period (0–10 days of age), birds received the control diet (9.60 g/kg Ca and 4.80 g/kg AvP) and LCP1, LCP2 and LCP3 diets (9.00 g/kg Ca and 4.50 g/kg AvP). During the grower period (11–24 days of age), Ca and AvP contents were 8.70, 8.30, 7.90 or 7.00, and 4.40, 4.20, 4.00 or 3.80 g/kg in the control, LCP1, LCP2 and LCP3 diets, respectively. During the finisher period (25–42 days of age), Ca and AvP contents were 7.80, 7.10, 6.00 or 5.00, and 3.90, 3.50, 3.00 or 3.00 g/kg in the control, LCP1, LCP2 and LCP3 diets, respectively. Calcium and AvP contents of the diets were reduced and the recommended Ca/AvP ratio (2/1) was applied. All the dietary treatments contain equal contents of phytase enzyme (1000 FTU/kg). The experiment lasted for 42 days, and water and feed were provided ad-libitum.
Determination of performance and carcass yield
Body weights (BWs) were determined by group weighing on the 0th, 11th, 25th and 42nd days of the experiment, and the feed intake (FI) of each period was determined on the same days. Body weight gain (BWG) was calculated by subtracting the average body weight of the previous period from the average body weight obtained at the end of each period. The feed conversion ratio (FCR) was obtained by dividing the FI by the BWG in each period. To determine the carcass yield on the 42nd day of the experiment, two birds were randomly selected from each group and slaughtered, and visceral was cleaned, then the hot carcass weight was determined, and carcass yield was calculated as: carcass weight/BW × 100.
Determination of tibia characteristics
At the end of experiment two birds/replicate were chosen and slaughtered. The left tibia of two broilers was removed to assess their mechanical characteristics. They were cleaned and maintained in a freezer at −20°C until further examination. Bone mechanical properties were determined by the load–deformation curve with a three-point bending test using an Instron Universal Testing Instrument (Model 1122; Instron, Canton, MA) and the TestWorks 4 software package (version 402; MTS System Corporation, Eden Prairie, MN), as described by Wilson and Ruszler (Reference Wilson and Ruszler1996) and Gül et al. (Reference Gül, Olgun, Yıldız, Tüzün and Sarmiento-García2022).
To examine the ash weight, right tibia of two broilers randomly selected from each replicate was taken; tibia separated, adhering tissue removed and dried at 100°C for 24 h. The fat was removed from the tibias by Soxhlet extraction using 1000 g/kg ethyl ether. Then fat-extracted tibias were dried for 24 h at 100°C and subsequently ashed for 24 h at 600°C. Ash weight (g/100 g tibia) was determined with the following formula: Ash weight = (ash weight/sample weight) × 100.
Determination of jejunum histomorphological traits
For histological analysis, segments of 5 cm long were taken from the midpoint of jejunum (towards the proximal duodenum) and flushed with a saline solution. These segments were fixed in 100 g/kg neutral buffered formalin at room temperature for 72 h. After the routine histological procedures, samples were embedded in paraffin wax. Sections having thickness of 4 μm were mounted on a glass slide by using microtome. Histological sections were examined under a light microscope (Leica DM2500, Leica Microsystems GmbH, Wetzlar, Germany) and photographed with a digital microscope camera (Leica DFC450, Leica Microsystems GmbH, Wetzlar, Germany). The images were evaluated using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, MD). The variables measured were villus height (VH), villus width (VW), crypt depth (CD), villus height to crypt depth ratio (VH:CD) and villus surface area. VH was measured from the tip of the villus to the junction of villus and crypts, and the CD was measured from its base up to the region of transition between the crypt and villus. Villus surface area (VSA) was determined using the following formula: (2π) × (VW/2) × (VH)/106 (Santos et al., Reference Santos, Tellez, Farnell, Balog, Anthony, Pavlidis and Donoghue2005; Golzar Adabi et al., Reference Golzar Adabi, Hajibabaei, Casey and Bayraktaroglu2016; Gül et al., Reference Gül, Olgun, Yıldız, Tüzün and Sarmiento-García2022).
Statistical analysis
In the research, the means of four different trial groups consisting of diets containing different contents of Ca and AvP were analysed according to one-way analysis of variance. Differences among the means were determined by the Duncan multiple comparison test.
Results
Performance and carcass yield
The effects of diets based on corn–soybean meals with reduced Ca and AvP contents on the performance and carcass yield of broilers are demonstrated in Table 2. BW, BWG, FI, FCR, mortality and carcass yield were not affected by reducing Ca and AvP contents in the diet (P > 0.05). In the study, initial BW was found to be 42.48–42.56 g, on 42nd day 2621–2733 g, BWG 2579–2690 g, FI 4167–4295 g, FCR 1.582–1.618, mortality 1.67–6.67 per/100 chicks and carcass yield 72.87–73.51 g/100 g BW. Compared to the control group, it was observed that reducing Ca and AvP contents by approximately 100 g/kg (LCP2) tends to improve the feed efficiency of male broilers but reducing it further (LCP3 group) tends to negatively affect.
Effect of low dietary Ca and AvP on broiler performance and carcass yield
BW, body weight; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio; s.e.m., standard error mean.
LCP1, LCP2 and LCP3: compared to control diet; 0.3 g/kg lower AvP in the starter period; it contains 0.2, 0.4 and 0.6 g/kg lower AvP in the grower period, respectively, and 0.45, 0.9 and 0.9 g/kg lower AvP in the finisher period, respectively. Dietary Ca contents were determined to be at a Ca/AvP ratio of 2/1.
Bold values indicate the cumulative averages of the trial groups for each variable at the final of growing period (42nd day).
Tibia characteristics
Table 3 shows that the effect of reducing Ca and AvP contents in diets based on corn/soybean meals on tibia characteristics of broilers. Compared to the tibia ash of the control group (35.86 g/100 g tibia), the LCP2 and LCP3 (32.24 and 33.12 g/100 g tibia, respectively) groups were found to be significantly lower and similar to the LCP1 (35.96 g/100 g tibia) group (P < 0.05). The effect of treatments on tibia diameter (9.13–9.55 mm), cortical bone thickness (1.32–1.53 mm), cross-sectional area (32.37–36.15 mm2) and shear force (1015–1273 N) was found to be statistically insignificant (P > 0.05). Although the treatment groups (LCP2 and LCP3) caused a decrease in the tibia ash, it was determined that there was a tendency to improve in tibia diameter, cortical bone thickness and cross-sectional area by reducing the Ca and AvP contents in the diet. This tendency was obtained in the LCP1 and LCP2 groups for shear force.
Effect of low dietary Ca and AvP on tibia characteristics
s.e.m., standard error mean.
LCP1, LCP2 and LCP3: compared to control diet; 0.3 g/kg lower AvP in the starter period; it contains 0.2, 0.4 and 0.6 g/kg lower AvP in the grower period, respectively, and 0.45, 0.9 and 0.9 g/kg lower AvP in the finisher period, respectively. Dietary Ca contents were determined to be at a Ca/AvP ratio of 2/1.
Jejunum histomorphological traits
The effect of reducing Ca and AvP contents in broiler diets based on corn/soybean meals on jejunum histomorphology is shown in Table 4. No significant effects were found for VH (1331–1459 μm), CD (113.35–129.05 μm) and goblet counts (192.3–219.2 units) (P > 0.05). Jejunum VW (P < 0.01), VH/CD (P < 0.05) and VSA (P < 0.01) were statistically affected by reducing Ca and AvP in the diet. The jejunum VW of the LCP2 (198.41 μm) and LCP3 (202.55 μm) groups was significantly higher than the control (148.80 μm) and LCP1 (164.44 μm) groups. Compared with the control (10.37) group, VH/CD considerably increased in the LCP2 (12.96), but it was similar in the LCP1 (11.25) and LCP3 (11.29) groups. On the other hand, VSA was significantly higher in the LCP2 (0.909 mm2) and LCP3 (0.882 mm2) groups than in the control (0.621 mm2) and LCP1 (0.689 mm2) groups. The highest VW was obtained in the LCP3, and VH/CD and VSA were obtained in the LCP2. Additionally, reducing Ca and AvP contents in the diet improved VH numerically and the highest value was obtained in the LCP2 group.
Effect of low dietary Ca and AvP on jejunum histomorphological traits
VH, villus height; VW, villus width; CD, crypt depth; VH/CD, villus height to crypt depth; VSA, villus surface area; s.e.m.: standard error mean.
LCP1, LCP2 and LCP3: compared to control diet; 0.3 g/kg lower AvP in the starter period; it contains 0.2, 0.4 and 0.6 g/kg lower AvP in the grower period, respectively, and 0.45, 0.9 and 0.9 g/kg lower AvP in the finisher period, respectively. Dietary Ca contents were determined to be at a Ca/AvP ratio of 2/1.
Discussion
Reducing dietary Ca and AvP contents did not affect the performance variables of broilers in the current study. This finding is consistent with the previous studies which reported that reducing Ca (from 8.50 to 6.50 g/kg) and AvP (from 4.25 to 3.25 g/kg) contents by maintaining a 2:1 ratio (Cardoso et al., Reference Cardoso, Rodrigues, Bertechini, Freitas, Lima and Lima2010) or decreasing dietary Ca and AvP contents by 300 g/kg (Imari et al., Reference Imari, Hassanabadi and Moghaddam2020) did not affect performance during the grower and finisher periods. Additionally, the results obtained in the current study agree with those documented by Wilkinson et al. (Reference Wilkinson, Bradbury, Bedford and Cowieson2014), Akter et al. (Reference Akter, Graham and Iji2016), Valable et al. (Reference Valable, Narcy, Duclos, Pomar, Page, Nasir, Magnin and Létourneau-Montminy2018) and Noruzi et al. (Reference Noruzi, Hassanabadi, Golian and Aziz-Aliabadi2022). Balancing the Ca:AvP ratio (2:1) is important in diet formulation (Gautier et al., Reference Gautier, Walk and Dilger2017). Birds fed with diets of reduced Ca and AvP and maintaining a 2:1 ratio can adapt to low dietary P content as they grow (Yan et al., Reference Yan, Angel, Ashwell, Mitchell and Christman2005). Also, low Ca and AvP contents in the diet promote the retention of these minerals (Mitchell and Edwards, Reference Mitchell and Edwards1996). Actually, the performance did not affect by reducing dietary Ca and AvP in the current study may be due to the fact that sufficient Ca and P gets absorb into the circulatory system from the intestines for sustainability.
Reducing dietary Ca and AvP contents decreased tibia ash. The current study agree with findings of Wilkinson et al. (Reference Wilkinson, Bradbury, Bedford and Cowieson2014) who clarified that the interactions created by 5.00 or 10.0 g/kg Ca and 2.50, 3.50, 4.50 or 5.50 g/kg AvP contents did not affect the tibia ash rate of broilers on 7–35th days. Similarly, Cardoso et al. (Reference Cardoso, Rodrigues, Bertechini, Freitas, Lima and Lima2010), Akter et al. (Reference Akter, Graham and Iji2016), Valable et al. (Reference Valable, Narcy, Duclos, Pomar, Page, Nasir, Magnin and Létourneau-Montminy2018), Imari et al. (Reference Imari, Hassanabadi and Moghaddam2020), Kop-Bozbay et al. (Reference Kop-Bozbay, Akdag, Atan and Ocak2021) and Noruzi et al. (Reference Noruzi, Hassanabadi, Golian and Aziz-Aliabadi2022) reported that the tibia ash decreased with a reduction in dietary Ca and AvP contents. In this study, reducing Ca and AvP contents by maintaining a 2:1 ratio in diets did not affect performance, but caused a decrease in the amount of tibia ash. This reveals that performance and tibia ash are affected by different dietary manipulations, because bones contain 800 g/kg of the P and 990 g/kg of the Ca present in the body (Proszkowiec-Weglarz and Angel, Reference Proszkowiec-Weglarz and Angel2013). Therefore, although performance is unaffected, bone mineralization is expected to be affected. It is possible that bone mineralization is negatively affected as the existing amounts may be used for growth and development as a result of reducing the Ca and AvP contents in the diet by 100 g/kg or further. Accordingly, more detailed information can be obtained through bone, blood and faecal mineral analysis. Although reducing the contents of Ca:AvP in the diet decreased tibia ash, it had no effect on biomechanical characteristics. These findings were similar to results published by Valable et al. (Reference Valable, Narcy, Duclos, Pomar, Page, Nasir, Magnin and Létourneau-Montminy2018), who found that reducing dietary Ca and AvP contents had no effect on the tibia shear force of 37-day-old broilers. However, Noruzi et al. (Reference Noruzi, Hassanabadi, Golian and Aziz-Aliabadi2022) stated that reducing the Ca and AvP contents in the diet caused a linear decrease in the shear force of broilers at the age of 42 days. Similarly, reports of Imari et al. (Reference Imari, Hassanabadi and Moghaddam2020) demonstrated that reducing the Ca and AvP requirement by 100, 200 and 300 g/kg decreases tibia strength. Gautier et al. (Reference Gautier, Walk and Dilger2017) documented that the tibia showed more strength with a Ca (10.00 g/kg) and AvP (2.00 g/kg) ratio of 2:1 compared to higher Ca (10.00 g/kg) and AvP (5.00 g/kg) contents. However, in that study, Ca (4.00 v. 6.00 g/kg) and AvP (2.00 v. 3.00 g/kg) contents were lower compared to the current study. In the research, although the tibia mineralization decreased, the biomechanical properties were not affected and even tended to improve. Bone biomechanical properties can be affected not only by Ca and P but also by trace elements (Olgun and Aygun, Reference Olgun and Aygun2016). Examining the accumulation of macro and micro minerals in the tibia in the current study may provide more details.
Although the intestinal morphology of broilers shows an alteration depending on various factors, optimum values for VH, CD, VH/CD, VW and VSA were reported as 954.26 and 1376.83 μm, 133.8 and 209.67 μm, 6.38–7.33, 58.37–178.56 μm and 0.174–0.765 mm2, respectively (Banaszak et al., Reference Banaszak, Biesek, Bogucka, Dankowiakowska, Olszewski, Bigorowski, Grabovicz and Adamski2020; Ceylan et al., Reference Ceylan, Koca and Golzar Adabi2023; Olyayee et al., Reference Olyayee, Javanmard, Janmohammadi, Kianfar, Alijani and Mir Ghelenj2023). In the current study, the jejunum histomorphological traits, VW, VH/CD and VSA were positively affected by reducing the dietary Ca and AvP contents (LCP2 and LCP3). These results disagree with Imari et al. (Reference Imari, Hassanabadi and Moghaddam2020) and Noruzi et al. (Reference Noruzi, Hassanabadi, Golian and Aziz-Aliabadi2022), who stated that reducing the content of AvP in the diet by 300 g/kg did not change the intestinal morphology in terms of VH, CD and VH/CD of broilers. Abdulla et al. (Reference Abdulla, Loh, Akit, Sazili and Foo2016) found that increasing the total Ca and P (Ca: 10.00–15.00 g/kg, P: 4.50–6.70 g/kg) contents in the diet did not change the intestinal morphology. Villus, which are the absorption zones of nutrients in the digestive tract, play a key role in the development of animals. Butyric acid ensures the continuity of the health of the small intestine epithelial tissue (Zou et al., Reference Zou, Ji, Qu, Wang, Shu, Wang, Liu, Li and Luo2019). As the intestinal pH decreases, the presence of beneficial bacteria such as Faecalibacterium boosts the production of butyric acid, which has a positive effect on villus development (Wu et al., Reference Wu, Yin, Wang, Mahmood, Shahid, Yin and Yuan2020) and the decrease in Ca content in the diet causes the pH in the jejunum to increase (Zanu et al., Reference Zanu, Kheravii, Morgan, Bedford and Swick2020). Low Ca (8.00 g/kg) and AvP (2.50 g/kg) contents in the diet positively affect the ileal microbiota of broilers (Kumar et al., Reference Kumar, Shang and Kim2019). The pH in the small intestine may have been lower in the current study as the Ca content declined, and the production of the beneficial microorganism population may have been positively affected. Therefore, the production of butyric acid, which is important for villus development, may have increased. However, to better understand this issue, further studies are needed to examine intestinal pH, microbiota and volatile fatty acid production, especially butyric acid, by reducing dietary Ca and AvP contents while maintaining the Ca:AvP (2:1) ratio. In the current study, it was shown that VH/CD achieved the highest value in the LCP2 treatment group. Higher VH/CD indicates more effective absorption of nutrients (Paiva et al., Reference Paiva, Walk and McElroy2014). However, considering the FCR, the improvement in the VH/CD of the LCP2 group did not affect the performance and was only reflected as a numerical improvement.
Conclusions
The performance of male broilers was not affected by reducing Ca and AvP contents in a diet. The tibia ash was adversely affected by reducing Ca and AvP by 100 g/kg (LCP2) and 200 g/kg (LCP3), but this negative effect was not reflected in the biomechanical properties. The jejunum histomorphological traits improved by reducing dietary Ca and AvP, and this improvement was observed numerically in performance. As a result, it can be concluded that by reducing Ca and AvP in male broiler diets by 100 g/kg of the recommended content, jejunum histomorphology is improved without affecting performance and tibia biomechanical characteristics. Since lowering Ca and P contents without compromising the Ca:AvP ratio did not negatively affect performance or bone biomechanical properties in the current study, it is possible to decrease these macrominerals to reduce diet costs and possible environmental pollution, but more research is needed to identify the critical contents.
Author contributions
Conceptualization: Y. C. and O. O.; methodology: Y. C. and O. O.; validation: Y. C., O. O., E. T. G. and S. A. G.; investigation, resources, supervision and project administration: Y. C. and O. O.; data curation: E. T. G., S. A. G. and B. S.; writing – original draft preparation: O. O. and E. T. G.; writing – review and editing: Y. C. and O. O. All authors have read and agreed to the published version of the manuscript.
Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
Competing interests
None.
Ethical standards
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, The European National Research Council's guidelines for the Care and Use of Laboratory Animals have been followed.
