Introduction
Poultry products are widely recognized for their efficient land usage and relatively low carbon footprint compared to other major livestock industries, making them a critical component in sustainable food systems (Gaillac and Marbach, Reference Gaillac and Marbach2021). As the global population is expected to surpass 9.7 billion by 2050 (Benito-Diaz et al., Reference Benito-Díaz, Sarmiento-García, García-García, Vieira, Domínguez, Bodas-Rodríguez, Gómez-Gordo and Vicente-Galindo2024), the demand for eggs will continue to rise, underscoring the importance of sustainable egg production practices (Costantini et al., Reference Costantini, Ferrante, Guarino and Bacenetti2021).
Traditionally, the poultry industry relies heavily on soybean meal as a primary protein source. However, the increasing cost of soybean meal and environmental and ethical concerns associated with its production pose significant challenges (Sarmiento-García et al., Reference Sarmiento-García, Palacios, González-Martín and Revilla2021; Pirgozliev et al., Reference Pirgozliev, Whiting, Mansbridge and Rose2023). Soybean cultivation is often associated with deforestation, particularly in vulnerable ecosystems such as the Amazon rainforest, which contributes to biodiversity loss and increased carbon emissions (Richards et al., Reference Richards, Myers, Swinton and Walker2012). Furthermore, the expansion of soybean agriculture frequently disrupts local communities and traditional land uses, raising ethical concerns in global supply chains (Grossi et al., Reference Grossi, Massa, Giorgino, Rossi, Dell’Anno, Pinotti, Avidano and Rossi2022). Given the steady increase in soybean prices over recent decades, finding alternative protein sources has become essential for economically viable and environmentally responsible poultry farming (IDH, 2021). The combination of ethical and economic challenges underscores the urgent need for alternative, locally sourced protein options to enhance the sustainability of modern poultry production systems (Abdulla et al., Reference Abdulla, Rose, Smackenzie and Pirgozliev2017; Whiting et al., Reference Whiting, Pirgozliev, Rose, Wilson, Amerah, Ivanova, Staykova, O. Oluwatosin and Oso2017). Addressing these challenges requires innovative approaches to feed formulation and collaborative efforts among researchers, industry stakeholders and policymakers to foster the adoption of sustainable practices in poultry farming. By prioritizing integrating alternative protein sources, the poultry sector can contribute to improved feed security while minimizing its ecological footprint (Sarmiento-García et al., Reference Sarmiento-García, Palacios, González-Martín and Revilla2021).
Sunflower (Helianthus annuus L.) is a globally cultivated oilseed plant known for its adaptability to diverse growing conditions (Laudadio et al., Reference Laudadio, Ceci, Nahashon, Introna, Lastella and Tufarelli2014). A notable byproduct of sunflower cultivation is sunflower meal (SFM), which serves as a valuable source of vegetable protein and fibre for livestock (Mbukwane et al., Reference Mbukwane, Nkukwana, Plumstead and Snyman2022). SFM offers several advantages, including high levels of polyunsaturated fatty acids, such as linoleic and oleic acids, which are beneficial for poultry health (Saleh et al., Reference Saleh, El-Awady, Amber, Eid, Alzawqari, Selim, Soliman and Shukry2021; Rakita et al., Reference Rakita, Koki´c, Manoni, Mazzoleni, Lin, Luciano, Ottoboni, Cheli and Pinotti2023). Its adaptability to diverse agricultural environments and cost-effectiveness further enhances its appeal in the feed industry, especially in regions where sunflowers are locally cultivated (Laudadio et al., Reference Laudadio, Ceci, Nahashon, Introna, Lastella and Tufarelli2014). However, SFM has certain limitations: its lower digestible protein content, reduced lysine and methionine levels and high-fibre content may impair nutrient absorption and utilization, particularly in monogastric animals (Nolte et al., Reference Nolte, Jansen, Weigend, Moerlein, Halle, Simianer and Sharifi2021; Saleh et al., Reference Saleh, El-Awady, Amber, Eid, Alzawqari, Selim, Soliman and Shukry2021). Indigestible components, such as phytate, may form insoluble complexes with minerals, further limiting nutrient availability. Additionally, chlorogenic acid, present in SFM, can inhibit digestive enzymes, including trypsin, potentially reducing nutrient absorption (Mbukwane et al., Reference Mbukwane, Nkukwana, Plumstead and Snyman2022).
Several strategies, such as enzyme and organic acid supplementation, have been explored to mitigate these limitations (Tüzün et al., Reference Tüzün, Olgun, Yıldız and Şentürk2020; Yaqoob et al., Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022; Zhang et al., Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022; Gül et al., Reference Gül, Golzar Adabi, Cufadar and Mızrak2025). Enzyme supplementation, which includes xylanase, protease and cellulase, has shown promise in breaking down complex fibres and enhancing nutrient digestibility (Baghban-Kanani et al., Reference Baghban-Kanani, Hosseintabar-Ghasemabad, Azimi-Youvalari, Seidavi, Ayaşan, Laudadio and Tufarelli2018; Khan et al., Reference Khan, Aziz, Khan, Khan and Ayasan2021). Several studies have highlighted the ability of enzymes to maintain performance levels, increase egg quality and improve feed efficiency when adequately supplemented (Berwanger et al., Reference Berwanger, Nunes, Oliveira, Bayerle and Bruno2017; Kargopoulos et al., Reference Kargopoulos, Bonos, Basdagianni and Nikolakakis2017; Baghban-Kanani et al., Reference Baghban-Kanani, Hosseintabar-Ghasemabad, Azimi-Youvalari, Seidavi, Ayaşan, Laudadio and Tufarelli2018; Souza et al., Reference Souza, Freitas, Alencar, Costa, Santos, Freire, Rocha, Coelho and Nepomuceno2020; Koçer et al., Reference Koçer, Bozkurt, Ege and Tüzün2021). Moreover, butyric acid has gained attention for its role in improving intestinal health and nutrient absorption by supporting villus development and intestinal epithelial integrity (Elnesr et al., Reference Elnesr, Ropy and Abdel-Razik2019; Pires et al., Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020). Sodium butyrate, specifically, has been demonstrated to improve performance as it reduces pathogenic bacteria, enhances feed efficiency and stimulates immune function in poultry (Sobczak and Koz1owski, Reference Sobczak and Kozłowski2016; Kazempour and Jahanian, Reference Kazempour and Jahanian2017), making it a valuable additive in high-fibre diets (Abdelqader and Al-Fataftah, Reference Abdelqader and Al-Fataftah2016; Ahsan et al., Reference Ahsan, Cengiz, Raza, Kuter, Chacher, Iqbal, Umar and Çakır2016; Kazempour and Jahanian, Reference Kazempour and Jahanian2017; Nari and Ghasemi, Reference Nari and Ghasemi2020; Zhang et al., Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022). The supplementation of sodium butyrate in poultry diets has been consistently linked with enhanced intestinal health and physiological function (Elnesr et al., Reference Elnesr, Ropy and Abdel-Razik2019). Additionally, as a primary energy source for intestinal epithelial cells, butyrate plays a crucial role in promoting gut development and preserving the integrity of the intestinal barrier (Liao et al., Reference Liao, Shao, Sun, Yang, Zhang, Guo, Luo and Lu2020).
Building on these findings, this study hypothesises that incorporating SFM in the diet of laying quails may negatively impact jejunal morphology and pancreatic enzyme activity due to its high fibre and anti-nutritional factors. It also posits that the inclusion of exogenous enzymes and/or sodium butyrate could alleviate these adverse effects. Therefore, this study aims to investigate the effects of SFM-based diets supplemented with multi-enzymes and sodium butyrate on productive performance, eggshell quality, pancreatic enzyme activity and jejunal histomorphology in laying quails.
Material and method
Ethical approval
The study was conducted in collaboration with a local farm and adhered to the ethical guidelines outlined in the European Union Directive 2010/63/EU (EPCEU, 2010) on protecting animals used for scientific purposes.
Experimental design
The experiment followed a completely randomized design and was conducted on a local farm (38°1′36″N, 32°30′45″E) in Selçuk (Konya, Türkiye). A total of 140 female 24-week-old Japanese quails (Coturnix coturnix Japonica) with similar body weight (274.73 ± 11.63) were randomly assigned to five treatment groups, each with 14 subgroups and 28 females per group. The pens (30 × 45 cm) were uniform, well-ventilated, clean and sanitized. The temperature was set at 22°C (± 2.0°C), with a lighting schedule of 16 hours of light and 8 hours of darkness. All pens were equipped with individual feeders and drinkers to allow ad libitum access to feed and water.
The diets were prepared in a mash form and formulated to contain 20% crude protein and 2898 kcal/kg of metabolizable energy suitable according to the National Research Council (1994). The negative control diet (NC) was based on corn and soybean meal, while the positive control diet (PC) included 25% SFM (Table 1). The treatment diets were prepared by adding either 500 g/tonne of enzyme to the positive control diet (PC+E), 1000 g/tonne of sodium butyrate (PC+B), or a combination of both 500 g/tonne of enzyme and 1000 g/tonne of sodium butyrate (PC+EB). An enzyme preparation containing xylanase, amylase and protease enzymes was used as a multi-enzyme (Avizyme® 1505X; Nutriline Yem ve Besin Tic. Ltd. Sti. Atasehir, Istanbul, Türkiye). All inclusion rates expressed as percentages (%) throughout the manuscript refer to the proportion of the diet on an as-fed basis, unless otherwise stated. The experiment spanned 84 days. The basal diet was formulated to meet the NRC (1994) guidelines, and Table 1 presents the chemical composition of the basal diet, as determined following the AOAC (Reference Hortwitz and Latimer2006) procedures.
Negative and positive control diets and their chemical content (as fed)
1 Premix supplied per kg diet: 13000 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.
2 Analysed values.
Determination of productive performance parameters
To assess performance parameters, group body weights were measured at the start and end of the experiment with an accuracy of ± 0.01 g. Body weight gain (BWG) was calculated from the difference between the initial and final group weights. Feed provided to each subgroup, as well as the leftover feed at the end of the trial, was recorded, and then feed intake (g/quail/day) was measured according to the method described by Olgun et al. (Reference Olgun, Gül, Kılınç, Yıldız, Çolak and Sarmiento-García2022). Eggs were collected daily at 10:00 a.m. and recorded, with egg weights determined over the last three days of the experiment. Egg production was determined by dividing the number of eggs collected by the total number of birds and multiplying by 100. Additionally, egg weight, egg mass (g/quail/day), and feed conversion ratio were assessed in line with those described by Sarmiento-García et al. (Reference Sarmiento-García, Olgun, Kılınç, Sevim and Gökmen2023).
Determination of eggshell quality parameters
To determine eggshell quality, 300 eggs collected during the final three days of the study were analysed at the Egg Quality Laboratory, Faculty of Agriculture, Selcuk University, Konya, Türkiye. The proportion of cracked, broken, and damaged eggs was recorded and expressed as a percentage of the total eggs. Shell strength was measured by applying pressure to the blunt end of the egg using an Egg Force Reader (Orka Food Technology Ltd., Israel). Eggshell thickness (μm) was measured at three separate points using a micrometre (Mitutoyo, Japan), and the relative eggshell weight (g/100 g egg) was calculated by weighing the cleaned, dried shells and dividing by the egg weight.
Determination of digestive enzyme activities
Pancreas samples were excised from one quail per subgroup (n = 14) within five minutes post-mortem and immediately weighed using a precision scale. Relative pancreas weight was calculated as a percentage of the bird’s body weight. The samples were then wrapped in aluminium foil, snap-frozen in liquid nitrogen, and stored at –80°C until analysis. Subsequent procedures followed the method described by Chen et al. (Reference Chen, Nakthong, Chen and Buddington2005). The activities of key digestive enzymes, including amylase, protease, and lipase, were assessed following the protocols reported by Tüzün et al. (Reference Tüzün, Olgun, Yıldız and Şentürk2020).
Determination of histomorphometry of jejunum
A 3 cm segment of the jejunum was carefully excised and immediately fixed in 10% buffered formalin for 72 hours. Each sample (n = 70) was divided into triplicate cross-sections to ensure the inclusion of intact, well-oriented crypt-villus units. The preparation, fixation, and subsequent histological measurements of villus and crypt dimensions adhered to the procedure outlined by Gül et al. (Reference Gül, Olgun, Yıldız, Tüzün and Sarmiento-García2022). Villus height (VH) was measured from the crypt-villus junction to the brush border at the villus tip, while villus width (VW) was measured at the midpoint between the opposing brush borders of epithelial cells. Crypt depth (CD) was recorded at the level of the basement membranes of opposing crypt epithelial cells. The VH/CD ratio was calculated to assess the functional morphology of the intestine. Villus surface area (VSA) was determined using the next formula (1) as described by Sakamoto et al. (Reference Sakamoto, Hirose, Onizuka, Hayashi, Futamura, Kawamura and Ezaki2000)
$$VSA\; = \;\left( {2\pi } \right)\; \times \;\left( {VW/2} \right)\; \times \;\left( {VH} \right),\;$$
Statistical analysis
The effects of dietary treatments on the measured parameters were analysed using one-way ANOVA (SPSS, version XX, IBM, Armonk, New York). The treatment groups served as the fixed factor within the statistical model. The normality of residuals was assessed using the Shapiro–Wilk test. No data transformation was required, as the residuals were normally distributed and satisfied the assumptions of the ANOVA model. Post hoc comparisons among the treatments were conducted using Duncan’s multiple comparison tests to identify specific differences between the means. Average values were calculated, including the standard error of the mean (SEM), and the significance level for all statistical evaluations was set at P < 0.05. For the statistical evaluation of the jejunum histomorphometry, the statistical unit was the individual sample, with comparisons made on a treatment basis. All data analyses were performed using the Statistical Package for the Social Sciences (SPSS, IBM, 2012), ensuring comprehensive data processing and evaluation throughout the research.
Results
Productive performance
The impact of SFM with enzyme and/or sodium butyrate supplementation on laying quail performance is summarized in Table 2. The treatments did not significantly affect body weight, body weight change, egg weight, egg mass or feed conversion ratio (P > 0.05). However, egg production was significantly influenced by the dietary treatments (P < 0.01), with the PC, PC+E and PC+EB groups achieving higher production rates (92.0, 91.6, 92.4 eggs/100 quails/day, respectively), than the NC group (90.2 eggs/100 quails/day). Feed intake also showed significant differences (P < 0.001) with the PC group (25% SFM) recording the highest intake (34.4 g/quail/day) among all groups. Although feed intake remained higher than NC (29.9 g/quail/day) in the enzyme and/or butyrate-supplemented groups (PC+E, PC+B, PC+EB), it was lower (32.7, 32.8 and 32.6 g/quail/day, respectively), than in the PC group.
Effects of enzyme and/or sodium butyrate addition to diets containing sunflower meal (SFM) on quail performance (n per treatment group = 28)
NC: Negative control without SFM, PC: Positive control containing 25% SFM, PC+E: Positive control + 500 g/tonne enzyme mix, PC+B: Positive control + 1000 g/tonne sodium butyrate, PC+EB: Positive control + 500 g/tonne enzyme mix + 1000 g/tonne sodium butyrate; IBW: Initial body weight, FBW: Final body weight, BWC: Body weight change, EP: Egg production, EW: Egg weight, EM: Egg mass, FI: Feed intake, FCR: Feed conversion ratio; S.E.M.: Standard error of means.
A,B,C : Means with different superscripts in the same row were significantly different (P < 0.05).
Egg external quality
The effect of dietary SFM, enzyme and sodium butyrate on eggshell quality is summarized in Table 3. The rate of damaged eggs was the only eggshell quality parameter that was not significantly affected by the treatments (P > 0.05). In contrast, eggshell-breaking strength showed a significant improvement (P < 0.01) in the PC+B group (13.1 N) compared to the rest of the experimental groups except for the PC+E group (12.6 N). Additionally, both eggshell ratio and eggshell thickness were significantly higher in all treatment groups compared to NC (P < 0.05)
Enzyme and/or sodium butyrate addition to diets containing sunflower meal (SFM) on quail egg external quality (n per treatment group = 60)
NC: Negative control without SFM, PC: Positive control containing 25% SFM, PC+E: Positive control + 500 g/tonne enzyme mix, PC+B: Positive control + 1000 g/tonne sodium butyrate, PC+EB: Positive control + 500 g/tonne enzyme mix + 1000 g/tonne sodium butyrate; S.E.M.: Standard error of means.
A,B : Means with different superscripts in the same row were significantly different (P < 0.05).
Pancreatic enzyme activities
As shown in Table 4, the relative pancreas weight was not significantly affected by any of the dietary treatments (P > 0.05). Specifically, lipase activity did not differ significantly between the PC and NC groups (0.685 U/mg, 0.448 U/mg, respectively), but both groups exhibited significantly lower values (P < 0.001) compared to the PC+E group (1.36 U/mg) and PC+E showed similar values to the PC+B and PC+EB groups (1.18 U/mg, 0.94 U/mg, respectively). In contrast, amylase activity was significantly lower (P < 0.001) across all treatment groups (0.154, 0.200 0.15, 0.174 U/mg, respectively), compared to NC (0.358 U/mg). Similarly, protease activity followed the same trend, with all treatment groups showing reduced levels compared to NC. The lowest protease activities were observed in the PC+B and PC+EB groups, registering 0.70 U/mg and 0.57 U/mg, respectively, indicating a pronounced reduction.
Enzyme and/or sodium butyrate addition to diets containing sunflower meal (SFM) on pancreas weight and enzyme activity in layer quails (n per treatment group = 14)
NC: Negative control without SFM, PC: Positive control containing 25% SFM, PC+E: Positive control + 500 g/tonne enzyme mix, PC+B: Positive control + 1000 g/tonne sodium butyrate, PC+EB: Positive control + 500 g/tonne enzyme mix + 1000 g/tonne sodium butyrate; S.E.M.: Standard error of means.
A.B.C : Means with different superscripts in the same row were significantly different (P < 0.05).
Jejunum histomorphology
The data presented in Table 5 demonstrate the substantial effects of dietary treatments on jejunal histomorphology (P < 0.001). The VH was significantly lower in the PC group (249 µm) compared to the NC group (291 µm), but significantly higher in PC+E (305 µm) and PC+EB (350 µm). The PC+EB group recorded the highest VH, followed by PC+E, with NC and PC+B exhibiting intermediate values, and PC showing the lowest VH. VW varied significantly (P < 0.001), with the PC+E (50.5 µm) and PC+EB (51.0 µm) groups having the widest villi, significantly different from NC (46.1 µm), PC (47.0 µm) and PC+B (46.7 µm). Crypt depth (CD) was significantly greater in all treatment groups compared to NC (34.3 µm), with the deepest crypts in PC+EB (42.6 µm), followed by PC+B (38.3 µm), PC+E (38.3 µm) and PC (36.2 µm). The VH/CD ratio was lowest in PC (7.04) and highest in NC (8.60), with PC+EB (8.37) showing similar values to NC. VSA followed a similar trend, with the lowest value in PC (0.369 mm2), intermediate values in NC (0.421 mm²) and PC+B (0.439 mm²) and the highest in PC+E (0.484 mm²) and PC+EB (0.561 mm²). These findings suggest that enzyme and butyrate supplementation counteracted the negative effects of SFM on gut structure.
Enzyme and/or sodium butyrate addition to diets containing sunflower meal (SFM) on jejunum histomorphology in layer quails (n per treatment group = 14)
NC: Negative control without SFM, PC: Positive control containing 25% SFM, PC+E: Positive control + 500 g/tonne enzyme mix, PC+B: Positive control + 1000 g/tonne sodium butyrate, PC+EB: Positive control + 500 g/tonne enzyme mix + 1000 g/tonne sodium butyrate; S.E.M.: Standard error of means; VH: Villus height, VW: Villus width, CD: Crypt depth, VH/CD: Villus height/crypt depth ratio, VSA: Villus surface area.
A,B,C,D Means with different superscripts in the same row were significantly different (P < 0.05).
Discussion
In the current research, the treatments did not affect quail development. Given SFM’s high crude fibre content, a negative impact on poultry digestive health might be expected, particularly regarding performance outcomes (de Morais Oliveira et al., Reference de Morais Oliveira, de Arruda, Silva, de Souza, de Queiroz, da Silva Melo and Holanda2016). However, the lack of a marked effect on performance observed here and in similar studies may be due to the advanced digestive capacity of laying hens compared to younger birds like chicks, allowing them greater tolerance to SFM’s fibre. This assertion aligns with the findings of Mtei et al. (Reference Mtei, Abdollahi, Schreurs, Girish and Ravindran2019) who suggested that mature birds can better tolerate higher fibre levels in their diets. Regarding feed intake, the PC group recorded the highest values, while egg production improved significantly in the PC and supplemented groups (PC+E and PC+EB) compared to the NC group. It is important to point out, that no feed aversion was observed when sodium butyrate was introduced. The quails adapted normally, maintaining stable feed intake across the supplementation period. The lack of negative effects on intake suggests that sodium butyrate did not alter diet palatability. Furthermore, the improvement in egg production may be attributed to the increase in feed intake associated with SFM, suggesting that SFM could positively contribute to overall productive performance. Similarly, studies by Koçer et al. (Reference Koçer, Bozkurt, Ege and Tüzün2021) and Saleh et al. (Reference Saleh, El-Awady, Amber, Eid, Alzawqari, Selim, Soliman and Shukry2021) found that SFM at levels up to 9.7% and 10.0% (as fed), respectively, improved egg production, reinforcing the positive role of SFM in layer diets when balanced with other dietary components. Nevertheless, the effects of SFM on performance in layer diets remain a topic of debate. For instance, de Oliveira Costa et al. (Reference de Oliveira Costa, Nepomuceno, Souza, de Melo, de Souza, Silva, Gomes, Watanabe and Freitas2023) observed that SFM at 7–14% (as fed) in laying hen diets negatively impacted egg mass and feed conversion ratio but had no significant effect on other performance indicators. By contrast, studies by Koçer et al. (Reference Koçer, Bozkurt, Ege and Tüzün2021) and Saleh et al. (Reference Saleh, El-Awady, Amber, Eid, Alzawqari, Selim, Soliman and Shukry2021) found that SFM at levels up to 9.7% and 10.0% (as fed), respectively, improved egg production. Additionally, de Morais Oliveira et al., (Reference de Morais Oliveira, de Arruda, Silva, de Souza, de Queiroz, da Silva Melo and Holanda2016), reported that replacing 10–30% of soybean meal with SFM in laying hen diets did not affect performance, except for egg mass. Similarly, other studies have demonstrated that SFM inclusion levels as high as 20–24% (as fed) can be used without detrimental effects on performance parameters (Shi et al., Reference Shi, Lu, Tong, Zou and Wang2012; Pinheiro et al., Reference Pinheiro, Fonseca, Silva, Oba, Balarin and Brunelli2013).
Moreover, in this study, although not statistically significant, all groups supplemented with enzymes and/or sodium butyrate showed numerically higher feed intake than the NC group. These results align partially with those of Baghban-Kanani et al. (Reference Baghban-Kanani, Hosseintabar-Ghasemabad, Azimi-Youvalari, Seidavi, Ayaşan, Laudadio and Tufarelli2018), who reported that multi-enzyme supplementation at 0.02% in diets containing 10–20% (as fed) SFM increased feed intake in laying hens. In addition, the combination of enzymes and sodium butyrate (PC+EB) led to numerically higher egg production than all other groups, indicating a potential synergistic effect. Although studies examining the combined effects of SFM and sodium butyrate are limited, evidence supports the beneficial impact of butyric acid in layer diets. For instance, Wang et al. (Reference Wang, Wang, Lin, Gou, Fan and Jiang2021) observed that butyric acid supplementation improved both egg production and egg weight, while Miao et al. (Reference Miao, Zhou, Li, Zhu, Dong and Zou2021) found that coated sodium butyrate at 500 mg/kg increased egg production without influencing feed intake. However, Dahiya et al. (Reference Dahiya, Berwal, Sihag and Patil2016) reported a reduction in egg production when sodium butyrate was included at levels between 0.5% and 1.5% (as fed). In this sense, other studies have reported that sodium butyrate either does not significantly affect general performance parameters (Sobczak and Kozłowski, Reference Sobczak and Kozłowski2016; Baghban-Kanani et al., Reference Baghban-Kanani, Hosseintabar-Ghasemabad, Azimi-Youvalari, Seidavi, Ayaşan, Laudadio and Tufarelli2018; Pires et al., Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020) or has a positive effect (Jahanian and Golshadi, Reference Jahanian and Golshadi2015; Zhang et al., Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022). These findings suggest that while SFM and its supplements may have variable effects on performance, the combined use of enzymes and sodium butyrate appears promising. Future studies are needed to better understand the optimal levels and interactions of these supplements to maximize the benefits of SFM in layer diets.
In this study, supplementing diets with sodium butyrate alone (PC+B) resulted in significantly higher eggshell-breaking strength compared to the NC, PC and PC+EB groups. This finding may be attributed to sodium butyrate’s known ability to improve mineral absorption and utilization (Miao et al., Reference Miao, Zhou, Li, Zhu, Dong and Zou2021). Sodium butyrate, as shown in prior studies, enhances intestinal health, which can increase the availability of essential minerals like calcium for eggshell formation (Rattanawut et al., Reference Rattanawut, Pimpa and Yamauchi2018). This mechanism likely supports shell strength more effectively when sodium butyrate is used alone, as observed here, than when combined with enzymes, possibly due to interactions that influence mineral uptake efficiency (Miao et al., Reference Miao, Zhou, Li, Zhu, Dong and Zou2021). Comparing these findings with previous studies, there is some variability in outcomes. Research by Pires et al. (Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020) found that sodium butyrate at varying levels improved eggshell thickness and breaking strength, although it reduced the eggshell ratio. In contrast, Sobczak and Kozłowski (Reference Sobczak and Kozłowski2016) and Arabshahi et al. (Reference Arabshahi, Ghasemi, Hajkhodadadi and Farahani2021) observed that butyrate supplementation enhanced eggshell thickness without affecting breaking strength, indicating that the specific effects of butyrate may vary based on dosage and dietary composition. Here, the higher breaking strength in the PC+B group suggests that sodium butyrate alone may optimize the structural integrity of the shell when used with SFM, likely by enhancing calcium and phosphorus absorption essential for shell formation (Miao et al., Reference Miao, Zhou, Li, Zhu, Dong and Zou2021).
Regarding eggshell ratio and thickness, including SFM in all treatment groups appears to have a beneficial effect. Both eggshell ratio and eggshell thickness were significantly increased across all treatment groups (PC, PC+E, PC+B and PC+EB) compared to the NC group, indicating that the inclusion of SFM, whether alone or with added enzymes or sodium butyrate, contributes positively to these parameters. This outcome aligns with studies by de Morais Oliveira et al., (Reference de Morais Oliveira, de Arruda, Silva, de Souza, de Queiroz, da Silva Melo and Holanda2016), who reported that SFM (12 and 20% (as fed), respectively), inclusion could enhance eggshell thickness. These effects may be attributed to SFM’s fibre content, which, although high, can positively impact gut motility and mineral absorption in laying hens due to their more developed digestive systems (Mtei et al., Reference Mtei, Abdollahi, Schreurs, Girish and Ravindran2019).
While studies on the combined effects of SFM and enzyme or butyrate supplementation remain limited, the present findings suggest that SFM can enhance eggshell quality when balanced with enzyme or butyrate additions. However, the variations in outcomes between sodium butyrate and enzyme combinations in this study indicate that each supplement may act through different mechanisms, with sodium butyrate possibly exerting a more pronounced effect on shell strength. Future research should explore these supplements’ individual and combined impacts to optimize SFM-based diets for eggshell quality in poultry.
The economic feasibility of using SFM with enzyme and sodium butyrate supplements depends on the local cost of SFM compared to soybean meal and the price of the additives (de Morais Oliveira et al., Reference de Morais Oliveira, de Arruda, Silva, de Souza, de Queiroz, da Silva Melo and Holanda2016). In many regions, SFM is more cost-effective and our study shows that its inclusion at 25% with enzyme and sodium butyrate supplementation does not negatively impact egg production or quality while improving eggshell thickness and strength. Although the initial cost of additives may increase feed expenses, the improved feed efficiency and bird health could lead to long-term economic benefits. Additionally, using SFM aligns with sustainable farming practices by reducing reliance on soybean meal, which is often linked to environmental concerns (Sarmiento-García et al., Reference Sarmiento-García, Palacios, González-Martín and Revilla2021; Pirgozliev et al., Reference Pirgozliev, Whiting, Mansbridge and Rose2023). Future studies should include a detailed cost-benefit analysis to provide clearer insights into the economic feasibility for farmers.
In this study, relative pancreas weight remained unaffected across all treatments, consistent with findings from Koçer et al. (Reference Koçer, Bozkurt, Ege and Tüzün2021), Tüzün et al. (Reference Tüzün, Koçer, Ege and Bozkurt2021) and Ciurescu et al. (Reference Ciurescu, Vasilachi, Grosu and Grigore2019), who reported that SFM at various inclusion levels did not alter pancreatic weight in birds. This stability in pancreatic weight suggests that the dietary inclusion of SFM, even with enzyme and butyrate supplementation, does not elicit significant physiological adaptations in pancreatic size in response to fibre content or other nutritional components.
In this study, the highest pancreatic lipase activity was observed in the group receiving SFM with added enzymes (PC+E), which was significantly higher than in the non-SFM control group (NC) and the group with SFM only (PC). Interestingly, lipase activity in the PC+E group was comparable to that in the groups supplemented with sodium butyrate and the combined enzyme and butyrate treatment (PC+B and PC+EB, respectively). This finding suggests that enzyme supplementation alone may stimulate pancreatic lipase activity more effectively than SFM or butyrate alone, potentially by compensating for the high fibre and anti-nutritional factors in SFM, as lipase facilitates lipid hydrolysis and assimilation (Du et al. Reference Du, Sarwar, Ahmad, Suheryani, Anjum, Andlib, Kakar and Arain2024).
The differences in starch, crude cellulose, and fat ratios between the PC and NC diets are primarily due to the inclusion of SFM in the PC diet. SFM is known to have higher crude fibre and lower starch content compared to soybean meal, which can influence feed consumption and digestive enzyme activity, particularly amylase and protease (Saleh et al., Reference Saleh, El-Awady, Amber, Eid, Alzawqari, Selim, Soliman and Shukry2021; Mbukwane et al., Reference Mbukwane, Nkukwana, Plumstead and Snyman2022). Increasing the amount of SFM beyond 25% (as fed) could further exacerbate these differences, potentially leading to reduced nutrient digestibility and altered enzyme activity. However, the inclusion of exogenous enzymes and sodium butyrate, as demonstrated in this study, can mitigate some of these negative effects by improving nutrient utilization and gut health. Future studies should explore the effects of higher SFM inclusion levels (e.g. 30% or more (as fed)) in combination with enzyme and sodium butyrate supplementation to determine the optimal inclusion rates for maintaining performance and nutrient digestibility in laying quails.
Conversely, amylase and protease activities were significantly reduced across all SFM-fed groups compared to NC. The reduction in amylase may be linked to the higher fibre and non-starch polysaccharide (NSP) content in SFM, which can alter carbohydrate digestion and reduce the need for amylase (Saksrithai and King, Reference Saksrithai and King2019). Additionally, the lower protease activity could be due to chlorogenic acid in SFM, a known protease inhibitor (Yaqoob et al., Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022). These results suggest that the composition of SFM, especially its NSPs and anti-nutritional factors, influences the activity of certain digestive enzymes by potentially reducing the accessibility or need for these enzymes. These findings align partially with Fisinin et al. (Reference Fisinin, Vertiprakhov and Grozina2018), who reported elevated lipase activity in hens-fed diets containing SFM, suggesting an adaptive pancreatic response to high-fibre diets. De Oliveira Costa et al. (Reference de Oliveira Costa, Nepomuceno, Souza, de Melo, de Souza, Silva, Gomes, Watanabe and Freitas2023) similarly noted that increased lipase activity with SFM inclusion reflects an effort to maintain nutrient digestibility. However, other studies have found inconsistent results regarding enzyme addition with SFM. For instance, Yaqoob et al. (Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022) found that adding 100 mg/kg multi-enzymes in SFM-based broiler diets (3, 6 and 9% SFM (as fed)) did not alter lipase but increased amylase and protease activity, indicating that enzyme effects may vary depending on the formulation and fibre content. Alagawany et al. (Reference Alagawany, Attia, Ibrahim, Mahmoud and El-Sayed2017) observed that 1 g/kg enzyme supplementation and 25, 50 and 75% SFM replacing soybean meal reduced amylase and protease activities, potentially due to fibre-enzyme interactions, which might alter enzyme efficacy or nutrient accessibility and are in line with the current research.
In this study, amylase activity showed no significant differences across groups receiving butyrate (alone or combined with enzymes, PC+B and PC+EB, respectively), compared to PC and PC+E. This stability suggests that butyrate’s role may not directly influence amylase, as amylase secretion responds more to carbohydrate content than to fibre modification or mineral absorption, which are the primary benefits of butyrate. For protease, both the PC+B and PC+EB groups had significantly lower activity than the PC+E group, with values similar to the PC group. This decrease could result from butyrate’s enhancement of nutrient absorption (Du et al. Reference Du, Sarwar, Ahmad, Suheryani, Anjum, Andlib, Kakar and Arain2024), reducing the need for protease secretion. Additionally, anti-nutritional factors in SFM, like chlorogenic acid, may inhibit protease activity, an effect that butyrate’s absorption benefits may partially offset (Yaqoob et al., Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022). Thus, butyrate appears to improve digestive efficiency, reducing the demand for high protease levels without altering amylase production. Nevertheless, the current results contrast with findings from Miao et al. (Reference Miao, Zhou, Li, Zhu, Dong and Zou2021), who observed increased amylase and trypsin activity at doses of 500 mg/kg and higher. Butyric acid enhances nutrient utilization by lowering digestive pH and stimulating the release of hormones such as secretin and cholecystokinin, which promote pancreatic enzyme secretion. However, variations in its effects on enzyme activity may result from differences in the coated forms used, which influence its release rate and bioavailability in the gastrointestinal tract. This would suggest that while butyrate can stimulate enzyme secretion under specific conditions, its impact may vary with diet composition and formulation.
In the current research, SFM inclusion reduced VH, VH/CD and VSA compared to all experimental diets, while CD was lowest in the NC group. Shortening of the villi and a deep crypt can lead to poor nutrient absorption and decreased overall performance, as observed in recent studies (Yaqoob et al., Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022; Zhang et al., Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022). The negative impact of SFM on intestinal morphometry may be linked to the high-fibre content, which can increase cell turnover rates, leading to an accelerated loss of epithelial cells and reducing the rate of cellular renewal (Miao et al., Reference Miao, Zhou, Li, Zhu, Dong and Zou2021). High-fibre diets, such as those containing SFM, exert an erosive effect on the intestinal lining, increasing endogenous nutrient losses and thinning the protective mucin layer (Pires et al., Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020). In this context, a well-developed intestinal structure with long villi and shallow crypts enhances nutrient absorption, as it maximizes the mucosal surface area available for nutrient transfer from the intestine to the bloodstream (Zhang et al., Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022). Previous studies have shown varied impacts of SFM on intestinal structure. For example, Koçer et al. (Reference Koçer, Bozkurt, Ege and Tüzün2021) reported that including 4.73–9.74% SFM in laying hen diets did not affect ileum VH and CD in laying hens, VW and VSA decreased quadratically decreased while the VH/CD ratio increased which are in disaccord with the current results. This suggests that intestinal response to SFM inclusion may depend on factors such as fibre levels and bird species or age (Singh and Kim, Reference Singh and Kim2021). Moreover, it is important to note that VH, CD reached its highest value in the PC+EB group, indicating a positive effect of the combined enzyme and butyrate supplementation on villus structure. VW was also significantly higher in both PC+E and PC+EB groups compared to other groups, suggesting enhanced nutrient absorption capacity. The increase may indicate an adaptive response to the high-fibre diet, where deeper crypts contribute to a more robust epithelial cell turnover necessary to maintain intestinal integrity. Hence, the observed improvements in VH and VSA in the PC+E and PC+EB groups may be attributed to the beneficial effects of enzymes and butyrate on nutrient digestibility and gut health. Previous studies support the positive impact of enzymes on intestinal integrity, noting improvements in villus structure and nutrient digestibility with enzyme supplementation in poultry diets (Khan et al., Reference Khan, Sardar and Siddique2006; Tavernari et al., Reference Tavernari, Albino, Morata, Dutra Júnior, Rostagno and Viana2008; Oliveira et al., Reference Oliveira, Nunes, Eyng, Berwanger and Bayerle2016). In this sense, Yaqoob et al. (Reference Yaqoob, Yousaf, Imran, Hassan, Iqbal, Zahid, Ahmad and Wang2022) reported that multi-enzyme supplementation in broiler diets with SFM reduced VH at higher inclusion rates (9% as fed) and increased CD at 3%, suggesting that enzymes help maintain villus structure, though outcomes may vary by dosage. Moreover, the positive effect of sodium butyrate has also been shown to enhance intestinal morphology by serving as a direct energy source for epithelial cells, promoting cell proliferation and improving VH and overall gut barrier function (Pires et al., Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020). The ability of butyrate to stimulate blood flow and the secretion of gastrointestinal hormones, such as peptides that enhance enterocyte proliferation, supports VH and mucosal repair (Elnesr et al., Reference Elnesr, Ropy and Abdel-Razik2019). Butyrate additionally promotes the production of short-chain fatty acids, which fuel epithelial cells, thus contributing to taller villi (Tomaszewska et al., Reference Tomaszewska, Dobrowolski, Muszyński, Kwiecień, Kasperek, Knaga, Tomczyk-Warunek, Kowalik, Jeżewska-Witkowska and Grela2018). Zhang et al. (Reference Zhang, Zhang, Wang, Bai, Zeng, Peng, Zhang, Xuan and Ding2022) reported that sodium butyrate at 800 mg/kg increased jejunal VH, reduced CD and expanded the absorptive surface area in laying hens, aligning with the structural improvements observed here in the PC+EB group. Similarly, Miao et al. (Reference Miao, Zhou, Li, Zhu, Dong and Zou2021) found that sodium butyrate at 500 mg/kg and higher improved VH and the VH/CD ratio without affecting CD in laying hens. Similarly, Pires et al. (Reference Pires, Leandro, Jacob, Carvalho, Oliveira, Stringhini, Pires, Mello and Carvalho2020) demonstrated that 105–300 mg/kg coated sodium butyrate linearly increased VH and VH/CD ratio while quadratically reducing CD. The enhanced intestinal morphology observed in the PC+EB groups suggests that the combination of these additives help counteract the negative effects of SFM on gut structure, supporting nutrient absorption and overall intestinal health in laying quails. Nevertheless, future research should investigate optimal dosages and combinations of enzymes and butyrate to maximize their benefits in high-fibre poultry diets.
Conclusion
This study demonstrates that SFM can be effectively included in laying quail diets without detrimentally affecting performance or egg quality, particularly when enzyme and sodium butyrate supplements are used. Enzyme supplementation alone, or in combination with butyrate, supported higher egg production, improved eggshell quality and positively influenced intestinal morphology by enhancing VH and surface area. These findings suggest that SFM, when paired with these additives, can serve as a viable alternative to traditional protein sources, offering potential economic and nutritional benefits in poultry diets. Future studies should explore optimal dosages and combinations of these supplements in SFM-based diets to maximize nutrient utilization and production outcomes. Long-term evaluations across different production stages and poultry species are also recommended to better understand the sustained effects of SFM and its supplements on health and productivity.
Acknowledgements
We would like to thank Selçuk University Faculty of Agriculture Dean’s Office.
Author contributions
AET, ETG, OO, AY and AS-G contributed to the conceptualization, methodology, investigation, writing – original draft and writing – review and editing. AET contributed jejunum histomorphology and pancreatic enzyme analyses. AET, ETG, OO, AY and AS-G contributed to supervision, review and editing. All authors have read and approved the final 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
The authors declare no conflicts of interest exist.
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 were followed.
Open access
