https://orcid.org/0000-0002-0924-0479
ABSTRACT
Amidst increasing concerns regarding the incorporation of non-traditional protein sources in poultry diets as replacements for protein sources vital for human consumption, such as soybean meal, the utilization of Azolla leaf meal has garnered significant attention and popularity. The experiment commenced with 192 Japanese quail chicks (one day old), originated from a single hatch. Each chick was individually weighed and then randomly assigned to one of four treatment groups, namely control, supplementation of Azolla at the rate of 20 g/kg (Azolla 20), 40 g/kg (Azolla 40), and 60 g/kg (Azolla 60). Weight gain, feed conversion ratio (FCR) and dressing percentage were significantly (P < 0.05) higher in Azolla 40 and Azolla 60 compared to the control. No significant (P > 0.05) difference was found in antibody titre against New Castle disease virus, maleanodialdehyde (MDA) and faecal E. coli excretion. Further, villus height, crypt depth and ratio between them improved significantly (P < 0.05) in Azolla 60 compared to the control. In conclusion, our findings demonstrated a favourable impact of incorporating Azolla meal into the Japanese quail diet, leading to improved growth rates attributed to its positive influence on intestinal morphology. However, no significant effects were observed on immune response, lipid peroxidation, or faecal E. coli levels.
Introduction
The global restrictions imposed on the utilization of dietary antibiotics and the growing awareness among consumers have created an increasing need for natural and secure supplements in animal production. Consequently, there has been a surge in the popularity of herbal-based growth promoters, particularly in the poultry industry (Fatima et al. Citation2022; Khan et al. Citation2023; Saeeda et al. Citation2023; Shah et al. Citation2023). Among the myriad herbal plants, Azolla pinnata emerges as a valuable addition to poultry feed (Riaz et al. Citation2022). Azolla, classified as a aquatic fern, displays a unique floating behaviour, gracefully moving atop the surface of waterlogged rice fields and streams (Kumari et al. Citation2017). Azolla pinnata, a species of aquatic fern, is an unusual yet notable inclusion in feed formulations (Bhattacharyya et al. Citation2016). It has been reported to contain protein content ranging from 21 to 30% Azolla pinnata meal has demonstrated successful application in the feeding of broilers, quail, ducks, and layers (Riaz et al. Citation2022). In terms of nutritional composition, Azolla closely resembles traditional poultry feed, albeit with higher protein content and reduced calcium content. Azolla has long been acknowledged as a cost-effective source of proteins (Joysowal et al. Citation2018). The potential incorporation of Azolla meal into future poultry feed formulations holds promise for reducing feed costs while simultaneously ensuring optimal health and immune function in birds (Mishra et al. Citation2016). Kumar et al. (Citation2018) have demonstrated that incorporating Azolla into broiler diets yielded comparable growth rates and body weights to those achieved with soya bean meal. Azolla pinnata is rich in protein, biopolymers, vitamins and minerals (Bhaskaran and Kannapan Citation2015; Kollah et al. Citation2016).
The inclusion levels of Azolla are much controversial in the published literature. Research findings indicate that incorporating Azolla leaf meal at a 5% inclusion rate leads to improved poultry performance in chickens (Abdelatty et al. Citation2021). Azolla meal (ranging from 0% to 15%) were evaluated and the results suggest that chicken diets can safely incorporate up to 15% Azolla meal (Alalade et al. Citation2007). However, caution should be exercised regarding higher levels of Azolla supplementation. Joysowal et al. (Citation2018) found that supplementation with high levels of Azolla (15% and 20%) negatively impacted bird performance. They suggested that up to 10% supplementation is not only safe but also beneficial for poultry production. In the case of broilers, Saikia et al. (Citation2014) experimented with dietary supplementation of A. pinnata at levels ranging from 0% to 15%. They concluded that a dietary level of 15% Azolla is not ideal from an economic perspective, as it leads to higher feed costs without a proportional increase in weight gain.
Little information are available on the effect of Azolla on the growth performance, faecal E. coli shedding, lipid peroxidation, immune response and gut health in poultry. Therefore, this study was designed to investigate the effect of different levels of Azolla pinnataon growth, immunity, faecal E. coli, antioxidant capacity and gut histomorphology in quails.
Materials and methods
Feeding management and group allotment
The experiment commenced with 192 Japanese quail chicks (one day old), originated from a single hatch (18 ± 0.12 g). Each chick was individually weighed and then randomly assigned to one of four treatment groups, namely control, supplementation of Azolla at the rate of 20 g/kg (Azolla 20), 40 g/kg (Azolla 40), and 60 g/kg (Azolla 60). Each treatment group consisted of four replicates, with each replicate containing 12 quails. The chicks within the four treatment groups were provided with four different starter-cum-grower rations that were nutritionally balanced in terms of protein and energy content. The quails were housed in individual cages organized by replicates, with each cage measuring 75 × 65 × 30 cm. The quails were kept under consistent standard management conditions. Throughout the experimental period, both feed and water were provided to the quails in unlimited quantities (ad-libitum). Light was maintained for 20 h per day. presents the compositions and calculated analysis of all dietary treatments based on the NRC (Citation1994) guidelines. provides the chemical composition of Azolla. The chicks in all treatment groups were subjected to similar hygienic, environmental, and managerial conditions throughout the growth period. The birds were raised for a duration of 6 weeks (42 days), and various parameters related to broiler performance, including body weight gain, feed consumption and feed conversion efficiency were assessed on a weekly basis. On day 42 of the treatment, a random selection was made of two birds from each pen. The selected birds were subjected to weighing and subsequently euthanized in a manner consistent with humane practices using the cervical dislocation method. Following euthanasia, dressing percentage was calculated by removing abdominal organs.
Table 1. Composition and calculated analysis of the experimental diets %.
Table 2. Chemical ingredients of Azolla.
Blood MDA and antibody titre
At the conclusion of the experiment, five birds were randomly chosen from each replicate for the purpose of collecting blood samples. Serum was obtained from the blood samples through centrifugation at 3000 rpm for a duration of 10 min. Subsequently, the serum samples were stored at refrigerated temperatures and reserved for subsequent analyses of Malondialdehyde (MDA) levels and antibody titres. The determination of MDA levels in the serum samples was conducted utilizing the protocol established by Safiullah et al. (Citation2019). Furthermore, the measurement of antibody titres against Newcastle disease (ND) was accomplished using the methodology elucidated by Ahmad et al. (Citation2020) by hemagglutination inhibition assay (HIA).
Intestinal histomorphology
At the conclusion of the study (42 d), three broilers from each pen were euthanized using humane methods. Tissue samples were collected from the ilium and fixed in a 4% formaldehyde solution, dehydrated in xylene, embedded in paraffin, and sectioned into 4 μm slides for subsequent hematoxylin–eosin staining. The resulting sections were examined using a light microscope. To assess villus morphology, villus length, villus width, crypt depth, and their ratio were measured on 10 randomly selected villi per sample using an automatic image analyser.
Faecal Escherichia coli count
At the conclusion of the study, bacterial counts of E. coli were determined by collecting faecal material from each experimental group. Selective Xylose Lysine Deoxycholate (XLD) agar from Oxoid media was employed for this purpose. The samples were initially cultured on nutrient agar at 37°C for 24 h. Subsequently, subcultures were performed on S-S agar, MacConkey agar, and Plate count agar, followed by another 24-hour incubation. After the incubation period, colony characteristics such as shape, size, colour, opacity, and texture were examined using both the colony characteristics approach and the streak plate method, as described by Haq et al. (Citation2020).
Statistical analysis
The data obtained from the trial was recorded in Excel sheets for further analysis. A Complete Randomized Design (CRD) was employed for the experimental setup. To assess the significance of the data, Analysis of Variance (ANOVA) was performed at a significance level of P < 0.05, utilizing the statistical package Statistix 8.1. To group the means, the LSD (Least Significant Difference) test was employed. The performance analysis was conducted using the replicate as the experimental unit, while the analysis of dressing percentage and histo-morphology traits utilized the bird as the experimental unit.
Results
There was no significant difference in weekly and total feed consumption among the treatment groups, as illustrated in . Feeding Azolla affected the weight gain significantly (p < 0.05), as shown in . Birds in Azolla 60 were heaviest (26.00 g) in the 2nd week, than Azolla 40 (22.75 g), Azolla 20 (20.75 g), and Control (19.00 g). During the 3rd week, Azolla 60 (29.00 g) had significantly lower weight gain as compared to Control (34.00 g), groups Azolla 40 (33.00 g) and Azolla 20 (32.75 g). During the 4th week, Azolla 60 (46.00 g) and Azolla 40 (44.00 g) had significantly higher weight gain in comparison to Azolla 20 (43.25 g). Similarly, Azolla 60 had higher weight gain (52.00 g) during the fifth week, as compared with Azolla 20 (47.00 g), Control (47.00 g), and Azolla 40 (46.00 g). For overall period, Azolla 60 (168.60 g) had higher significant weight gain than Control group (155.30 g) and Azolla 20 (159.25 g).
Table 3. Impact of Azolla feeding on feed consumption (g) of quails.
Table 4. Impact of Azolla feeding on gain in body weight (g) of quails.
Feed supplemented with Azolla meal affected overall and weakly FCR significantly (). The FCR of Azolla 60 (2.40) and Azolla 40 (2.64) were significantly lower (P < 0.05) during the second week as compared to Control group (3.29). The FCR was significantly different among the groups in the third week, where the Azolla 60 had the worst FCR (3.28), proceed by Azolla 20 (3.02) and Azolla 40 (2.94), and Control having the lowest FCR (2.84). In the fourth week, the FCR of the Control group (3.25) differs significantly (P < 0.05) from that of Azolla 60 (2.80), Azolla 20 (2.96), and Azolla 40 (2.96). During the fifth week, Azolla 60 had a significantly lower FCR (3.59) than the Control (4.04), Azolla 40 (4.02), and Azolla 20 (4.01). The highest overall FCR (3.26) was achieved by the Control group, followed by Azolla 20 (3.14). For overall period, feed conversion ratio was significantly lower in Azolla 60 (2.97) and Azolla 40 (3.08) as compared to the Control group (3.26).
Table 5. Impact of Azolla feeding on feed conversion ratio of quails.
The effect of Azolla meal on dressing percentage, antibody titre, MDA and faecal excretion of E. coli are given in . The dressing percentage in Azolla 60 was the highest (64.26%), followed by Azolla 40 (62.38%) and Azolla 20 (61.30%), with the Control group having the lowest (60.30%). The rest of the parameters of this table did not change significantly between the treatment groups and the control.
Table 6. Impact of Azolla meal supplementation on the dressing percentage, antibody titre, MDA and faecal E. coli count of quails.
Intestinal histomorphology of quails fed with Azolla pinnata meal in feed is shown in . Intestinal histomorphology improved significantly (P < 0.05), by the supplementation of Azolla meal in the feed. The Azolla 60 displayed the greatest villus height followed by the Azolla 40 and the Azolla 20. The Control group showed a villus height shortest of all. The Control group revealed the deepest crypt, proceeded by groups Azolla 20, Azolla 40, and Azolla 60. illustrates the intestinal villus height and crypt depth of quails.
Figure 1. Effect of Azolla on ilium histology of control (A), Azolla 20 (B), Azolla 40 (C) and Azolla 60 (D) of Japanese quails. The figures show normal villi in the control and treatment groups showing no negative effects on the structural histological features of villi.
Table 7. Impact of Azolla feeding on intestinal histomorphology of quails.
Discussion
The findings of the present study revealed that growth were significantly higher in term of weight gain and FCR in Azolla 60. It is evident that the growth performance was higher at 6% inclusion level followed by 4% and 2%. The inclusion level of Azolla is much controversial and different studies have concluded different levels. The best level has been concluded at 5% in most of the studies on broilers growth (Riaz et al. Citation2022), however, information regarding the inclusion level of Azolla pinnata in Japanese quails is scarce. In the future, Azolla meal shows promise as a potential novel feed component in fowl diets, offering the potential to reduce feed costs while promoting optimal health and production in birds. Shamna et al. (Citation2013) conducted a study with quails and found that incorporating 5% Azolla in the basal ration led to enhanced growth and improved feed efficiency. Additionally, this approach proved to be more economically viable compared to feeding the basal diet alone. Varadharajan et al. (Citation2019) conducted research on quails and concluded that including Azolla meal at up to 6% in their diet did not adversely affect feed consumption or carcase traits.
Furthermore, a study by Rathod et al. (Citation2013) reported that supplementation of Azolla in the diet of Japanese quails did not show any significant effects on feed consumption. Taken together, these findings indicate the potential of Azolla meal as a beneficial and cost-effective component in quails feed, with positive effects on growth and feed efficiency. It seems that addition of Azolla meal up to 6% is safe in quails diet. On the other hand, Azolla meal has been added up to 15% in broilers ration with no health issue (Ara et al. Citation2015). Elevated levels of Azolla have been associated with higher fibre content in Azolla, which has a negative impact on the birds’ appetite, leading to a subsequent reduction in growth rate (Saikia et al. Citation2014). This effect is likely attributed to reduced palatability and increased bulkiness caused by the higher fibre content of the Azolla.
In the current study, the dressing percentage of the birds was found to be significantly higher in the groups that received 4% and 6% Azolla supplementation compared to the control group. The effects of Azolla supplementation on carcase traits have been relatively limited in research. One study reported that broiler chickens fed with 5% Azolla showed higher dressing percentages with 15% giblet percentage, which was attributed to the increased body weight gains (Basak et al. Citation2002). Another investigation found that feeding broiler chicks with 5% Azolla powder led to a significant increase in carcase yield percentages, while the lowest percentage was observed in the group supplemented with 15% Azolla (Naghshi et al. Citation2014). However, Ara et al. (Citation2015) observed no adverse effects on carcase traits when Azolla was fed up to a 20% level. These findings collectively suggest that Azolla supplementation can have varying effects on carcase traits in poultry, with the potential for positive impacts at certain levels of supplementation. Nevertheless, further research is needed to fully understand the optimal level of Azolla inclusion in the diet to achieve the best carcase traits in poultry.
In the current study, no significant difference was found in antibody titre against New Castle disease, MDA and faecal excretion of E. coli, however, ilium villi health was significantly improved in Azolla 60 in term of villus height, crypt depth and the ratio between these two parameters. Recently, Abdelatty et al. (Citation2021) reported that Azolla meal included at 5 and 10% levels significantly decreased the population of Enterobacteriacea and Bacilli but had no effect on Actinobacteria, Lactobacilli and Enterococcus. In addition, MDA concentration was not significantly changed, however, villus histomorphology was improved in the experimental groups. The incorporation of Azolla in the broiler chicken diet has the potential to influence both nutrient absorption and the gut microbial ecosystem. Nutrient absorption is significantly influenced by the morphology and integrity of the intestinal villi, as well as the composition and dynamics of the gut's bacterial community. Scarce information are available on the antioxidant, immune modulating and intestinal histomorphology in animals. Some studies have documented that different doses have improved cell-mediated and antibody titre response in different poultry species (Prabina and Kumar Citation2010; Bhattacharyya et al. Citation2016). A number of factors such as dose and species of Azolla, preparation, mode of inclusion and other experimental conditions may affect the outcome of the experiment. However, further research is recommended.
Conclusion
From the findings of the present study it was concluded that addition of 6% Azolla meal in the diet of Japanese quails improved growth performance and intestinal histomorphology.
Ethical approval
The Committee on Animal Rights and Welfare, The University of Agriculture, Peshawar, Pakistan approved this study (PS/25/2022).
Consent to participate
All authors are agreed to submit the article to this journal.
Consent to publish
All authors are agreed to publish in this journal.
Acknowledgements
The Authors would like to thank the Researchers Supporting Project Number (RSPD2024R591), King Saud University, Riyadh, Saudi Arabia.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
On request.
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