ABSTRACT
The rising cost and environmental impact of conventional protein sources in poultry feed necessitate exploration of sustainable alternatives. Azolla pinnata (AP), a nutrient-rich aquatic fern, has emerged as a promising candidate. Our objective was to evaluate the potential of AP as a sustainable feed ingredient for broilers, focusing on its impact on growth, feed efficiency, gut health, and profitability. To obtain AP, we harvested green Azolla from river banks using plastic sieves, sun-dried it, and pulverized it. Three-hundred-day-old Cobb chicks were individually weighed and randomly assigned to four dietary groups, namely: Group A (the control, 0g APM/kg feed), Group B (1g APM/kg feed), Group C (2g APM/kg feed), and Group D (3g APM/kg feed). The study used a completely randomized design with four treatments, each having three replicates of 15 broilers. Broiler weight gain, feed intake, feed conversion ratio (FCR), villus height, villus-crypt ratio, and economic returns were assessed. The results showed that broilers fed with 3g APM/kg feed had significantly improved weight gain, optimal feed intake, and the lowest FCR compared to other groups. This group also exhibited increased villus height and villus-crypt ratio, suggesting enhanced nutrient absorption. Notably, the 3g APM/kg feed group showed a 22% increase in net returns compared to the control. This study provides compelling evidence for the potential of APM as a sustainable and cost-effective protein source in broiler diets. It offers economic benefits to farmers while promoting gut health and improved broiler performance. Further research can explore the long-term effects and optimal APM inclusion levels for large-scale adoption in the poultry industry.
Keywords: Azolla Pinnata meal; feed intake; FCR; gut histomorphology; broiler
INTRODUCTION
The global population is projected to reach approximately 9.6 billion inhabitants by 2050. This substantial increase necessitates a parallel augmentation in food supplies, particularly dietary proteins (Khan et al., 2023). Regarding dietary proteins, the insufficiency of animal-derived proteins is a recurrent issue. Addressing this deficiency and enhancing the availability of animal proteins in both human and animal food supplies are a paramount concern for specialists in human and animal nutrition (Gibson & Jung, 2006). The poultry sector is a vital player in this scenario, having a critical role in ensuring an adequate supply of animal protein. The 2020 report from the Food and Agriculture Organization (FAO), anticipated an expansion in poultry meat production, projecting a 2.4% increase in poultry meat production compared to the figures recorded in 2019 (FAO, 2019), reaching 137 million tons.
However, the poultry industry is faced with the costs of production, of which feed costs constitute 70% to 80%. Corn and soybean are the primary energy and protein sources, respectively (Mwesigwa, 2021). Because of the competition between the human and livestock industry for this limited grain, animal nutritionists actively seek ways to curtail feed ingredient expenses by exploring the utilization of by-products from diverse origins and unconventional food items. Azolla pinnata, a nutrient-rich aquatic fern, has been shown as a promising feed ingredient.
Azolla pinnata is not only high in proteins, fatty acids, amino acids and vitamins (Nasir et al., 2022), but also demonstrates cost-effectiveness (affordability) and adaptability in both natural (non-cultivated environments) and controlled farming settings (Saikia et al., 2014). It can be a valuable nutritional resource for various species, including poultry, livestock, and aquatic organisms. Previous research has demonstrated that it can improve meat quality, promoting enhanced colour, flavour, tenderness, and juiciness (Al-Rekabi et al., 2020; Joysowal et al., 2018).
Basak et al. (2002) established Azolla pinnata’s nutritional profile, indicating its suitability as a component in broiler feed rations. It provides essential amino acids such as leucine and alanine, as well as essential minerals such as iron, calcium, and magnesium. Rich in chlorophyll a, chlorophyll b, and carotenoid compounds (Lejeune et al., 2000; Mudgal et al., 2022), Azolla pinnata contains linolenic acid (9.8% to 37.95%) and linoleic acid (5.11% to 15.38%), surpassing some edible oils in linolenic acid content and showcasing its nutritional superiority (Lejeune et al., 2000).
Studies conducted by Rana et al. (2017), Rengma et al. (2019) and Shambhvi et al. (2021) confirm Azolla spp.’s minimal aflatoxin content (0.01 microgram/kg). This makes it suitable for animal consumption without adverse health effects.
Previous research has shown that supplementing broiler diets with Azolla up to a level of 2.5g APM/kg feed enhanced blood biochemical parameters and positively improved broiler gizzard weight and breast muscle yield (Al-Rekabi et al., 2020; Shambhvi et al., 2021).
Given the potential benefits of Azolla supple-mentation in broiler diets, it is important to understand its impact on gut histomorphology. This will help to assess its potential health benefits or risks. Therefore, evaluating the effects of Azolla pinnata meal (APM) supplementation on key indicators such as Villus Height (VH), Crypt Depth (CD), Villus Width (VD), and the Villus-Crypt Ratio (VCR) is crucial. Positive changes in these parameters may suggest improved nutrient absorption and overall gut health. Conversely, negative changes could indicate potential adverse effects (Refaey et al., 2023a).
Thus, this study aims to examines the impact of supplementing Azolla pinnata meal on gut histomorphology. The core objective is to enhance nutrient absorption, improve overall gut health and advocate for sustainable, locally sourced alternatives in the poultry production industry. This will contribute to a resilient future in poultry production.
MATERIALS AND METHODS
Ethical Statement and IRB Approval
This study, involving Cobb broiler chickens, was conducted in accordance with the ethical guidelines of the Faculty of Animal Husbandry and Veterinary Sciences (FAHVS) at The University of Agriculture, Peshawar, Pakistan. The study received approval from the FAHVS ethical committee on 11/12/2022 (approval number 1145/ASRB/UAP). Additionally, the study adhered to the principles outlined in the Basel Declaration, the Guidelines for the Care and Use of Poultry published by ALAW, and the Animal Research Tomorrow (ART). During the study, great care was taken to ensure that the birds had a comfortable environment, access to enrichment opportunities, and appropriate feed and water. Every effort was made to minimize the pain and discomfort experienced by the birds throughout the experiment.
Study Area, Collection, and Processing of APM Supplementation
The experiment took place at the Department of Poultry Science, and sample analysis was conducted at the pathology laboratory of the College of Veterinary Science, Faculty of Animal Husbandry and Veterinary Science, The University of Agriculture, Peshawar.
Wild Azolla pinnata (mosquito fern) was collected in accordance with all relevant local and national guidelines at the University of Agriculture Peshawar, Pakistan. Permission for collection was obtained from the Botanic Department, responsible for managing such resources. Green Azolla was harvested from river banks and shallow water areas using plastic sieves to minimize any disturbance to the environment and impact on other organisms. The collected material was sun-dried until completely dehydrated and then pulverized. Importantly, Azolla pinnata is not listed as an endangered or protected species in Pakistan. This research adheres to responsible and ethical plant collection practices, ensuring minimal environmental impact and compliance with local regulations.
The dried Azolla sample was chemically analyzed before it was incorporated into the broiler diet. The nutritional constituents of Azolla spp., including dry matter (DM), moisture, crude protein (CP), crude fiber (CF), and ether extract (EE) were evaluated following the methods specified in AOAC (2007) - specifically, 934.01, 976.05, 942.05, and 920.39, respectively. Additionally, acid detergent fiber (ADF), neutral detergent fiber (NDF), and acid detergent lignin (ADL) were analyzed following the methodology outlined by (Goering & Van Soest, 1970).
Experimental design and diets
Three-hundred-day-old Cobb chicks were weighed individually and randomly assigned to four treatments (A, B, C, and D). Group A, the control, was fed with a basal diet (0g APM/kg feed of APM) and Groups B, C, and D were respectively fed with a basal diet containing 1g APM/kg feed, 2g APM/kg feed, and 3g APM/kg feed replacing the soybeans of the basic feed. The chicks were weighed using a “Camry” brand scale with a range of 4800g and accuracy of 0.1g before being transferred and divided into five replicates, with fifteen birds of similar body weight (43g) each. The chicks were provided with starter feed from days 0 to 19, and finisher feed from days 20 to 35.
The nutrient compositions of the starter and finisher feeds comprising the experimental basal diet are respectively presented in Tables 1 and 2 respectively. Feed and water were provided ad libitum from days 1 to 35. The experiment lasted five weeks, maintaining biosecurity to prevent disease spread, with adjustment of temperature and duration of light according to the standard rearing practices of broiler production used in commercial farms. The temperature was set at 36°C during the starting phase, transitioned to 23°C in the growing phase, and gradually reduced to 20°C in the finishing phase, while ensuring proper ventilation (with the help of exhaust fans) and adjusting feed types.
Data collection
Weekly feed intake, body weight gain (BWG), and feed conversion ratio (FCR) were calculated as described by Ullah et al. (2023). The dressing percentage was calculated using the formula:
For the calculation of the giblet ratio, liver, heart and gizzard weights were measured and represented as a percentage of the total body weight. Later the giblet percentage were calculated using the formula below:
Weight of Giblets is the combined weight of the heart, liver, and gizzard, and Live Body Weight is the total weight of the live bird before slaughter.
Furthermore, digestibility testing was conducted to assess the nutrient digestibility of the diets containing Feathered Mosquito fern (Azolla pinnata, APM). Two Cobb broilers aged 35 days were selected from each treatment group at five weeks. These birds were not placed in digestibility cages; instead, fresh morning feces were collected directly from the pen. After collection, the feces were weighed, dried for three days and then analyzed to determine nutrient digestibility using the following formula:
For histomorphological analysis, a total of 15 sam-ples (three per replication) of each group/treatment were collected from the ileum portion of the small intestine, which, considering four dietary groups (0g APM/kg feed, 1g APM/kg feed, 2g APM/kg feed, and 3g APM/kg feed), amounted to a total of 60 samples. Each sample was placed in sterile containers with 10% buffered formalin to prevent autolytic changes during fixation. After proper labeling, the formalin was drained through washing and treated with alcohol to dehydrate. L-molds were used for the casting blocks, followed by placing the tissue sample in benzene to maintain firmness, facilitating section cutting using a rotatory microtome (model RX-11B, Volts 220; AMPS 2; Hz 50; manufactured by Sakura Seiki Co.Ltd.Nagano Japan). Subsequently, xylene was used to deparaffinize the tissue before staining with haematoxylin and eosin to observe pathomorphological changes with an OLYMPUS microscope (Model CX41RF; Made in Philippines), following the method described by (Lillie, 1965). All histometric measurements were made using ImageJ software (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation. LOCI, University of Wisconsin, USA).
Economic parameters were determined by calculating input and output costs. A cost-benefit analysis was conducted to evaluate the economic viability of using Azolla pinnata meal (APM) compared to conventional feed methods. The total cost of production included expenses for chicks, feed, vaccines, electricity, and casual labor.
The nutritional composition of APM
The proximate analysis of Azolla pinnata revealed a well-balanced nutritional composition, with a moisture content of 94.84%. It also had a notable concentration of crude protein (23.25%), and a harmonious distribution of crude fat, crude fiber, ash, and nitrogen-free extract (Table 3). By comparing Azolla pinnata with recognized nutritional standards, it becomes evident that it aligns well with recommended values for essential nutrients, demonstrating its potential as a valuable dietary ingredient.
Metabolizable Energy = 37% x Protein + 81.8 x % Fat + 35.5 x % NFE (Pauzenga, 1985) (AOAC, 1990).
Nitrogen-corrected apparent Metabolizable Energy (AMEn) proposed by (Mariano et al., 2013).
Statistical Analysis
The data collected, including weekly feed intake, body weight gain (BWG), feed conversion ratio (FCR), dressing percentage, nutrient digestibility, and giblet ratios, were analyzed using SAS® version 9.1.3. The general linear model (GLM) was employed to evaluate the effects of the feeding systems on feed intake, BWG, FCR, and dressing percentage, allowing for a detailed assessment of how these variables were influenced by the different treatments. Additionally, analysis of variance (ANOVA) was applied to compare nutrient digestibility and giblet ratios across the treatment groups, identifying any significant differences attributable to the dietary interventions. The least significant difference (LSD) test was used to distinguish means, with the significance of means investigated at a 0.05 level of probability.
RESULTS
Effect of APM supplementation on feed intake (g), weight gain and feed conversion ratio in broilers.
Table 4 provides a comprehensive overview of the impact of Azolla pinnata meal (APM) supplementation on feed intake, weight gain, and feed conversion ratio (FCR) in broiler chickens. The treatments include the control (Group A) with no APM supplementation and Groups B, C, and D with increasing APM levels (respectively 1g APM/kg feed, 2g APM/kg feed, and 3g APM/kg feed). A discernible trend in feed intake was observed, with Group B consuming the most feed at 3083g, followed closely by Group A (3080g). Group D exhibited the lowest intake at 3072g, showing no statistically significant difference from Group C (3078g).
As depicted by the correlation matrix (Figure 1), a positive correlation emerged between the FCR and both weight gain and feed intake. This indicates an overall trend of increasing FCR values as both these factors progressed over the experiment period. Specifically, we observed an effect of increased APM supplementation in weight gain, with Group D leading at 1816.0g. Despite APM supplementation, Group B showed the lowest weight gain, at 1765.0g. However, there was a decrease in FCR with higher APM levels, with Group D achieving the lowest FCR at 1.69, denoting improved feed utilization.
The correlation matrix depicted in the heatmap generated by the corrplot (Figure 1) serves as a comprehensive visualization of the relationships between key variables. The colour-coded representation highlights the strength and direction of correlations, unveiling distinct patterns within the dataset.
Effect of APM supplementation on ileum Histomorphology and Giblet Weight.
This study evaluates the impact of varying APM levels on gut histomorphology, examining parameters such as Villus Height (VH), Crypt Depth (CD), Villus Width (VD), and the Villus-Crypt Ratio (VCR) (Table 5 and Figure 2).
The study revealed a substantial increase (p<0.05) in VH with increasing levels of APM. This trend is evident when comparing Group D, which had the highest VH at 0.91, to Groups C (0.70), B (0.66), and the control group (0.48). Intriguingly, there was no significant variation (p<0.05) in CD across the groups.
Regarding VCR, Group D also had the highest value at 3.93, indicative of a healthier gut environment. Group B closely followed, while Group A (control group) had the lowest VCR in this study.
Clear micrograph (10X) of ileum segments of broilers from different treatments (A, B, C and D).
Effect of Azolla pinnata meal supple-mentation on Dressing Percentage and Giblet Weight.
The effect of APM meal supplementation on dressing percentage and giblet weight is shown in Figure 3. There was a significant enhancement (p<0.05) in dressed body weight percentage with increasing APM levels. Group D achieved the highest dressing percentage at 71%, followed by Group C (2g APM/kg feed) at 68.00%, Group B at 67.00%, and finally Group A (1g APM/kg feed) with the lowest dressing percentage at 66.00%. This highlights a positive correlation between APM supplementation and dressed body weight percentage.
In contrast, figure 4 shows no significant difference (p>0.05) in giblet percentage across treatments, suggesting that APM supplementation does not substantially alter giblet percentage in broilers.
Effect of Azolla pinnata meal supplemen-tation on economic parameters
Table 6 indicates the economic implications of different APM levels in broiler feed, focusing on feed expenses, gross return, and net return per chicken.
Higher APM supplementation was associated with increased total feed costs, leading to a substantial rise from 445.49 Pakistani rupees (PKR) in the control group (Group A) to PKR 497.47 in Group D (3g APM/kg feed). However, this cost increase is offset by positive trends in both gross and net returns, which are particularly evident in Groups C and D. Group D stands out with an impressive mean net return of PKR 54.42, emphasizing the potential financial gains from increased APM supplementation.
DISCUSSION
The present study emphasizes the promising potential of Azolla pinnata as a feed additive for improving broiler performance, particularly feed intake, weight gain and feed conversion ratio. The study consistently showed that Group D, which received the highest APM supplementation (3g APM/kg feed), had the most favorable results, including low feed intake and feed conversion ratio (FCR), and the highest weight gain. Importantly, lower levels of APM (groups B and C) did not have a significant impact on feed efficiency.
A dose-dependent response was observed, with the highest level (3g APM/kg feed) yielding the most significant improvements in weight gain and FCR, which suggests a specific optimal dosage within this range. Improved digestion and absorption of nutrients from feed, particularly protein, could explain the enhanced growth performance observed in the current study. In fact, the substitution of soybean meal by APM increased the fiber in the diet, and thus we noted a slowing in the transit of the food bolus, which increases the absorption of nutrients. The high crude protein content (23.25%) of Azolla is also promising, as it provides readily available essential amino acids for muscle building. These findings align with similar benefits observed with optimized APM inclusion at 2.5g APM/kg feed in studies conducted by Al-Rekabi et al. (2020) and Shambhvi et al. (2021).
Furthermore, the observed decrease in feed intake despite improved weight gain in Group D hints at enhanced nutrient utilization. This could be attributed to Azolla supplementation’s impact on gut microbiota, as suggested by Chatterjee et al. (2013) and (Abdelatty et al., 2020). Chatterjee et al. (2013) demonstrated heightened growth performance and enhanced feed conversion efficiency in crossbred cattle receiving Azolla microphylla supplementation. In a similar context, (Abdelatty et al., 2020) observed improved growth performance, intestinal morphology and cecal microbiota in broiler chickens with inclusion of Azolla leaf meal in their diet. Taken together, these findings strongly suggest that Azolla supplementation holds the potential to exert a beneficial influence on the microbiota of animal gut. The study’s strengths are its clear dose-dependent response and statistically significant results. However, a limitation lies in the lack of investigation into the specific mechanisms behind APM’s action. Future research on its impact on gut microbiota, enzyme activity, or potential metabolic effects could strengthen the argument. The lack of significant effect at lower APM levels could be due to insufficient dosage or failure to reach the threshold for activation of relevant mechanisms. Alternatively, saturation of potential effect sites might also explain the plateau effect observed (Refaey et al., 2023b). Exploring a wider range of dosages could provide further insights. While not observed in this study, high APM intake could potentially affect liver or kidney function due to increased protein intake. Further studies examining long-term effects and optimal dietary protein balance are recommended.
The final weight gain of broilers in Group D, fed with 3g APM/kg feed of Azolla, had the highest weight gain, attributed to Azolla’s enriched nutrient profile. Mineral elements highlighted in the study by Dhumal et al. (2009) and the antioxidant properties of carotenoids and biopolymers, as noted by Acharya et al. (2015), likely contributed to the observed benefits.
While these findings align with previous studies such as Samad et al. (2020) and Ara et al. (2015), which showed an increase in body weight gain with Azolla supplementation, there are discrepancies with Sujatha et al. (2013), Paudel et al. (2015) and Ibrahim et al. (2024). These discrepancies suggest that the effects of Azolla supplementation may vary depending on the levels of supplementation, age of the broilers, and the type of feed used.
The current study provides convincing evidence of a dose-dependent relationship between Azolla pinnata (APM) levels and improvements in gut health. Group D, which had the highest APM inclusion, showed the most significant increases in villus height and VCR. These findings support the idea that APM can promote gut health, as previous studies by Chichilichi et al. (2015), Bhattacharya et al. (2016) and Balaji et al. (2022) have demonstrated. Bhattacharya et al. (2016) reported that Azolla pinnata supplementation did not have negative effects on growth and enhanced broilers’ immune response. This observation is further supported by Chichilichi et al. (2015), who found that incorporating APM into the diet improved the immune response when combined with enzyme supplementation. Mishra et al. (2016) also found that Azolla pinnata replacement groups had similar blood biochemical parameters and a higher cell-mediated immune response, indicating an immune-regulating effect.
The increase in villus height in Group D directly correlates with an increased surface area for nutrient absorption in the small intestine. This explains the higher APM levels and improved weight gain. Taller villi allow broilers to extract more nutrients from their feed, resulting in better growth performance, as Disetlhe et al. (2017) have also observed. These findings are consistent with Abdelatty et al. (2020), who observed a significant effect of Azolla pinnata meal on broiler intestinal morphology. However, there are differences with ADIL et al. (2022), which suggests some variation across studies, emphasizing the need for further research to investigate the impact of APM on specific bacterial populations and their potential prebiotic activity.
The current study provides evidence of a clear dose-dependent relationship between Azolla pinnata meal (APM) levels and improved gut health and broiler performance. While the preservation of gut barrier function suggests overall support for APM, it is essential to conduct further studies, as the lack of specific bacterial analysis and comprehensive exploration of gut health markers raises concerns. Future studies should analyze bacterial populations and other markers to gain a deeper understanding of the effects of APM supplementation on broiler health.
Concerning giblet percentage, no significant effects were observed in broilers fed with Azolla-supplemented diets compared to the control group. This consistency with established theories underlines the importance of amino acids in poultry diets and suggests that Azolla can be integrated without adverse effects on giblet development, highlighting its potential as a sustainable feed ingredient. These results align with previous research by Yadav et al. (2014), Naghshi et al. (2014) and Abdelatty et al. (2020), which emphasize the lack of a significant impact of Azolla pinnata meal on giblet percentage. Additionally, the variations in dressing percentage among treatments, as reported by (Shambhvi et al., 2021), underscore the complexity of dietary effects on broiler physiology.
The current study unveils a fascinating trade-off: the enhanced broiler performance it triggers outweighs the cost spike. In particular, Groups C and D showed significantly higher gross and net returns due to their improved weight gain and feed conversion ratio (FCR). This trend culminates in Group D, boasting an impressive 22% increase in net return compared to the control group, with a remarkable PKR 54.42 per chick - suggesting an optimal dosage range for maximizing economic benefit.
These findings align with the cost-benefit analyses of other feed additives conducted by Ara et al. (2015), Rawat et al. (2015), Acharya et al. (2015), Paudel et al. (2015) and Attia et al. (2023) . Their studies revealed that supplementing broiler feed with probiotics at specific levels enhances performance and profitability, mirroring our study’s positive economic impact of APM. Furthermore, Ullah et al. (2023), Soisuwan et al. (2023) and Morais et al. (2023) demonstrated that optimizing dietary enzyme blends could lead to improved FCR and net returns, similar to the results achieved with Group D’s APM level.
The profitability potential of APM extends beyond simply offsetting the increased feed cost. Analyzing these findings through a cost-benefit lens highlights the clear advantage for poultry farmers seeking to optimize financial gains and broiler performance. This is particularly relevant in today’s competitive market, where maximizing profit margins is crucial for sustainable business operations.
This study opens doors for further research to solidify the economic viability of APM. Investigating the long-term economic impact of sustained APM supplementation over multiple production cycles would provide valuable insights for farmers planning their production strategies. Additionally, conducting a comprehensive cost-effectiveness analysis comparing APM with other performance-enhancing additives would offer more nuanced comparative data for informed decision-making. Finally, considering re-gional and farm-specific factors like feed prices, market conditions, and farm size could enable the development of tailored economic recommendations for optimal APM utilization, further expanding the practical application of these findings.
CONCLUSION
The current study has revealed significant improvements in broiler performance and gut health attributed to the inclusion of Azolla meal in birds’ diets. Notably, the addition of 3g APM/kg feed had a profound impact on several key parameters. This study demonstrated that supplementation with 3g APM/kg feed of Azolla meal significantly enhanced essential aspects of broiler growth performance, including weight gain and dressing percentage. The most compelling discovery was the remarkable improvement in feed conversion ratio (FCR) achieved by the group receiving the highest Azolla supplementation level, underscoring the critical importance of fine-tuning dietary formulations for optimal feed efficiency in broiler production.
Furthermore, our research shed light on the profound effects of APM on gut histomorphology, particularly the substantial increase in ileal villus height. This positive change in gut structure causes enhanced nutrient absorption capacity, potentially contributing to the improved growth observed in the study.
Therefore, further research should address the long-term effects of Azolla supplementation on broiler performance and gut health, investigate optimal inclusion levels for diverse broiler breeds and production systems, and elucidate the underlying mechanisms responsible for the favorable effects of Azolla pinnata meal on broiler growth and gut histomorphology.
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Funding
This research was funded by the Researchers Supporting Project, number (RSPD2024R637), King Saud University, Riyadh, Saudi Arabia.
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Data availability statement
All data are included in the manuscript; however, if further information regarding data 481 availability is needed, the corresponding author will provide it upon special request.
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Disclaimer/Publisher’s Note
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
Data availability
All data are included in the manuscript; however, if further information regarding data 481 availability is needed, the corresponding author will provide it upon special request.
Publication Dates
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Publication in this collection
25 Nov 2024 -
Date of issue
2024
History
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Received
21 May 2024 -
Accepted
16 Sept 2024