Nutritional composition, microbial safety and sensory attributes of eggs produced from Bovans Brown hens fed different levels of Azolla (Azolla pinnata) meal in their diet

Seid A, Beruk B¹ and Berhe H¹

School of Animal and Range Sciences, Hawassa University, Hawassa, Ethiopia P O Box 05, Hawassa, Ethiopia
seid@hu.edu.et
¹ School of Nutrition, Food Science and Technology, Hawassa University, Hawassa, Ethiopia

Abstract

The effect of inclusion of various levels of Azolla (Azolla pinnata) in diets of Bovans Brown hens on nutritional composition, microbial safety and sensory attributes of eggs of eggs was evaluated. Experimental eggs were obtained from 72 hens which were fed diets with 0% (Az0), 2.5% (Az2.5), 5% (Az5) and 7.5% (Az7.5) levels of Azolla from 26 to 36 weeks of age. Sampled eggs were analyzed for their proximate, mineral and vitamin A compositions. Eggs also assayed for S aurous, aerobic mesophilic bacterial count, E coli, Salmonella and yeast and mold counts. Further a total of 25 untrained panelists were allowed to test the sensory attributes of hard-boiled fresh eggs with respect to organoleptic characteristics using five-point hedonic scale for each attribute. Results showed that diets have insignificant effect on the proportion of egg parts, except the shell which was the highest for maximum level of Azolla inclusion p<0.05). More important than proximate composition, diet effect was pronounced significantly in micronutrient value, especially at the maximum level of Azolla inclusion. Inclusion of Azolla at 7.5% in the diet led to an increment of 41.53, 74.4, 70.90, 125.53, 77.16, 107.84, 111.83, 200.31, 71.45 and 95.76% in Ca, Mg, K, Na, Fe, P, S, Cu, Zn and B in eggs from the control group, respectively. In parallel, egg yolks from hens fed on 2.5%, 5% and 7.5% levels of Azolla containing diets composed by189.20, 218.52 and 271.66%, more vitamin A in egg yolk compared with the control group, respectively. Eggs obtained from the hens fed Azolla have better acceptability in terms of texture, color, and taste than the control. The overall acceptability and best egg quality was observed in group containing 7.5% Azolla. Salmonella did not detect in any of egg samples, while AMBC and S aurous counts were not significantly varied. Compared with the Azolla fed group, eggs from control group contain the highest count for E. coli and YMC. In conclusion, the mineral and vitamin A compositions, and sensory attributes of eggs produced from hens had positively influenced by incorporating different levels of Azolla in the diet, but without compromising their microbial loads.

Key words: azolla, egg quality, micronutrients, salmonella, sensory attributes, vitamin A

Introduction

Animal-source foods are nutritionally dense sources of energy, protein, and various essential micronutrients. They match particularly well with the nutrients needed by people to support normal development, physiological functioning, and overall good health (Smith et al 2013). Egg in particular remains a food product of high nutritional quality for adults including elderly people and children and is extensively consumed all over the world. The perfect balance and diversity in its nutrients along with its high digestibility and its affordable price has put the egg in the forefront as a basic food for humans (Réhault-Godbert et al 2019). Eggs are of particular interest from a nutritional point of view, containing essential lipids, proteins, vitamins, minerals, and trace elements (Nys and Sauveur 2004). Egg production and quality are greatly influenced by the nutrition of laying hens in terms of composition in nutrients, energy content, feedstuff texture and presentation, presence of anti-nutritional factors, and the mode of daily feed delivery (Bouvarel et al 2011). Of particular interest the concentration of such minerals and micronutrients in egg (vitamin A, E, zinc, selenium, and iodine) may be significantly increased depending on hen’s diet (Bouvarel et al 2011; Schiavone and Barroeta 2011).

Due to the current inflation rate in staple feed ingredient prices, recent years have witnessed an increasing interest in exploitation of unconventional feeds such as Azolla fern in practical poultry diets. Azolla, a prolific aquatic fern, possess high levels of both macro- and micro-nutrients which are suitable for poultry nutrition. Azolla contained 7.9–16% lipids (Brouwer et al 2018) and produces a high-protein (20–40%) biomass with an amino acid profile that mirrors the complete amino acid content of soybean meal (Kumar and Chander 2017; Brouwer et al 2018). Azolla fern was also a rich source of essential minerals, including Ca, P, K, Mn, Cu, Mg, Fe, and Zn (Kumar et al 2018). Beyond the amino acid and mineral contents, Azolla contains biologically active compounds that increase healthy gut bacteria, reduce intestinal inflammation and boost the metabolism. It was reported to gather appreciable quantities of β-carotene, vitamin B-12 and biopolymers (Pillai et al 2004), which are missing in many poultry feed ingredients. On fresh material, the carotene content of Azolla ranged from 206 to 619 mg/kg DM (Lejeune et al 2000). Overall, Azolla gather a superior nutritional profile and a lower ecological footprint which can help build a more sustainable food system.

Several authors have reported feeding trials using Azolla in laying hen diets at inclusion levels of 2.5 to 20% (Khatun et al 2008; Boitai et al 2018; Wasihun et al 2020; Seid 2023). The inclusion rates of Azolla in diets is limited due to the presence of tannins (Brouwer et al 2018), which can reduce protein digestion in birds. Notably, numerous studies have demonstrated that egg-laying performance and physical characteristics of eggs were improved by the addition of varying levels of Azolla in layers diets with an economical advantage in concentrate feed cost (Khatun et al 2008; Seid 2023). An example is that the pigment of egg yolk increased with the increased level of Azolla in layers diets and a longer period of feeding (Khatun et al 2008; Boitai et al 2018; Seid 2023). The observed improvement in yolk color score is presumably because of the natural richness of Azolla in β-carotene pigments and bioactive substances (Pillai et al 2004). Another important trait in laying hens that is improved by Azolla feeding was eggs shell in terms of its strength, weight, and thickness (Boitai et al 2018; Seid 2023). In nature, the shell and the shell membranes serve to protect and extend the life of the egg, but also to prevent penetration of microorganisms.

Microbial safety and sensory attributes of eggs are of additional concerns when considering the effect of replacing certain feedstuffs with others in poultry diets. Due to their rich nutritive value of their contents, eggs are potential hosts and carriers for pathogenic microbes like Salmonella enteritidis (Sumashree et al 2019). The widely utilized cultivation protocol of Azolla is the provision of a mixture of cow dung and bed soil in pond water of 10–12 cm depth, in some cases, with periodic supplementation of phosphorous fertilizer (Kumar and Chander 2017; Seid 2023). The cultivation pond seemingly contains a wide range macro-and microorganisms, which may translate in to the egg content if birds reared on Azolla containing diets. Eggs are considered to be a perishable food partly due to the low efficiency of their natural protection barriers (Sogunle et al 2017). This observation is particularly interesting, knowing that eggs absorb off-odors and off-flavours if stored near pungent foods. Scientific evidence existed that probiotics or vitamins supplementation can improve the egg defense by altering the caecal microbiota (Chousalkar et al 2021), suggesting that Azolla may plays a yet unknown role in egg safety. Besides microbial safety, the sensory or organoleptic properties of eggs, such as flavor, aroma, color, and palatability, are essential for customers (Hayat et al 2020; Dilawar et al 2021).

Although it has become clear that Azolla-derived diets improved layers performance, their influence on nutritional, safety concerns and overall acceptability of eggs produced from Azolla-fed birds remain unknown. With the above background, there is therefore the need for further documentation of the nutritional composition, microbial safety and sensory attributes of eggs produced from Azolla-fed birds. The objective of this study was to investigate whether the inclusion of varying levels of Azolla (Azolla pinnata) in diets would influence the nutritional, microbial and sensory attributes of eggs produced from Bovans Brown hens reared under tropical environment. The findings will expand our understanding of the nutritional and microbial composition of eggs and provide additional insights into Azolla utilization in sustainable chicken production systems.

Materials and methods

Source of eggs and Azolla production

The eggs used in the present investigation were obtained from a project designed to assess the effect of inclusion of Azolla meal on egg quality and production performance in layers, in which a total of 72 Bovan Brown layers of aged 26 weeks were provided with diets containing Azolla meal at 0%, 2.5%, 5% and 7.5% levels. Azolla has been cultivated on polyethylene sheet covered pits as described by Kumar and Chander (2017). It was harvested weekly and dried under shade by using framed bed nets to produce meal (Photo 1). Particular reference on Azolla cultivation protocols, layers management, experimental diets composition, and results with regard to hen production performance, and physical characteristics of eggs were reported earlier (Seid 2023).The proportion of eggs as to albumen, yolk, and shell percentages was determined from 6 randomly collected eggs per pen on weekly basis. On 8^th week of feeding the experimental diets, eggs (n = 24/treatment) were collected for the investigation of their nutritional composition, microbial safety and sensory attributes.

Photo 1. Azolla cultivation, harvesting, dying and meal production protocols

Sample processing and nutritional analysis of eggs

The nutritional composition of the whole eggs, yolk and albumen were analyzed by collecting 48 eggs (12 eggs per treatment) in the 8^th week of the experiment. Six eggs were used for the whole egg sample, while six were broken and separated into yolk and albumen parts from each treatment. The eggs were broken manually and then homogenized with a homogenizer at Animal Nutrition Laboratory, Hawassa University, Ethiopia. Eggs were broken into a stomacher bag and each sample of whole egg, yolk and albumen were stomached for one minute as described by Anderson et al (2011). The moisture, crude ash, crude protein, and crude fat or ether extract contents of the whole edible egg (yolk and albumen combined), yolk and albumen were determined according to the AOAC (1995). Minerals composition of whole egg samples were determined with the method of flameless atomic absorption spectrometry. Dry ashed samples were prepared for minerals analysis by acid digestion, and concentrations ofCa, Mg, K, Na, Mn, Fe, P, S, Cu, Zn, and B were determined at Horticoop Laboratory, Bishoftu, Ethiopia. In parallel, an aliquot of yolk sample was taken for determination of vitamin A composition in the hen's egg at Jije laboratory, Addis Ababa, Ethiopia. The spectrophotometric method of assay has been used in the quantification of vitamin A (Gillam and Heilbron, 1935). Samples were transported on dry ice to the Horticoop and Jije Laboratories.

Microbial analysis of eggs

The egg samples (n = 6/treatment) were analyzed for Staphylococcus aurous, Aerobic Mesophilic Bacterial Count (AMBC), E.coli, Salmonella, and Yeast and Mold Count (YMC) at the Nutrition, Food Science and Technology Laboratory, Hawassa University, Ethiopia. During sample preparation, a sterile knife was used to crack the egg, and a sterile spatula was used to evenly combine the egg's contents in a beaker. A sterile stomacher bag containing 225 ml of 0.1% sterile buffered peptone water was filled with 25 ml of the egg content. For a 10^-2 dilution, the sample was homogenized in the stomacher for one minute. After that, the homogenate was allowed to stand at room temperature for 15 minutes. One milliliter of the original suspension was aseptically transferred with a sterile pipette into a test tube containing nine milliliters of sterile peptone water, and then well mixed to create a dilution of 10^-2, from which additional decimal serial dilutions at seven levels were also performed. Enumeration tests were carried out for Aerobic Mesophilic Bacterial Count (AMBC), Staphylococcus aurous, Escherichia coli, Salmonella,and yeast and mold.

For aerobic mesophilic bacterial count, serial dilutions of the egg content were poured on plate count agar, and the plates were incubated at 30 °C for 72 hrs. For the presumptive enumeration of Staphylococcus aurous, 0.1 ml of the primary sample and its serial dilutions were spread on Mannitol salt agar in duplicates. Plates were incubated at 37 °C for 24 hours. Enumeration of E. coli was carried out by the spread-plating of the primary sample and its serial dilutions on Eosin Methylene Blue (EMB) agar, which were then incubated at 30 °C for 24 hours. For detection and enumeration of Salmonella species, the sample was pre-enriched in sterile buffered peptone water, followed by selective enrichment in Rappaport-Vassiliadis broth (RVS), streaking on XLD agar, and incubating at 37 °C for 24 hrs. Unconfirmed positive colonies were stabbed on both Lysine Iron agar and Triple Sugar Iron agar (TSI) and incubated for 24 hours at 37 °C. Positive black tubes with the colonies were verified with enzyme tests after incubation. Enumeration of yeast and molds was performed on the yeast extract glucose chloramphenicol bromophenol blue agar (YGCA) incubated at 25 °C for 5 days. At the end of the incubation, for all microbial counts, plates with countable colonies (30 to 300) were calculated based on the following formula, and converted to log CFU/g.

Sensory evaluation of eggs

A total of 25 untrained panelists were used to test hard-boiled fresh eggs with respect to organoleptic characteristics at the Nutrition, Food Science and Technology Laboratory, Hawassa University, Ethiopia. Six fresh eggs laid within 48 hours from each treatment were added to a stainless steel pot with a lid, which contained approximately 1350 ml of tap water. Electrical stove was turned on and kept at high heat level for 10 minutes until the eggs were brought to a low-rolling boil. After turning off the heat, the eggs were kept in the pot with a lid for 20 min. The water was drained, and the eggs were kept under running tap water until the temperature of the egg was cooled to room temperature. The eggs were peeled and cut into quarters (lengthwise) for the sensory evaluation. Sliced eggs were 3-digit blind-coded and each was placed in duplicate sample plates. Panelist were provided with cold water in between the samples to rinse their mouth. The sensory attributes tested were as follows: (a) texture (softness/ stickiness) of the whole boiled egg; (b) color of the egg albumen (whiteness); (c) color of the egg yolk (yellowness); (d) taste of the whole egg; (e) odor (aroma/ smell) of the egg; and (f) the overall acceptability of the whole egg based on the above parameters. The panelists were asked to rate the eggs on a five-point hedonic scale for each attribute, where 1 = dislike very much, 2 = dislike moderately, 3 = neither like nor dislike, 4 = moderately like, and 5 = like very much.

Stastical analysis

The data were analyzed by one-way analysis of variance (ANOVA) using the GLM procedure of the SAS Statistical Package Program (SAS 2012). The following statistical model was used to determine the effects of Azolla meal: Y_ij = µ + t_ij + e_ij, where Y_ij = response variable, µ = general mean, t_ij = effect of Azolla meal, and e_ij = random error. The means were calculated and presented with the standard error of the mean (SEM). The differences among treatments were compared by Duncan’s multiple range test, and p values equal to or less than 0.05 were used to declare statistical significance.

Results and discussion

Proportion of egg parts

The effect of Azolla meal on the proportion of egg components is shown in Figure 1. On average, the eggs from the current experiment were composed of 64.82% albumen (egg white), 26.21% yolk, and 8.97% shell. These findings were slightly different from USDA/AMS (2000) which states eggs on average consists of 58% albumen, 32% yolk, and 10% shell. The egg white represents about 64.7%, 64.75%, 65.07%, and 64.75% of the weight of the egg in Az0, Az2.5, Az5, and Az7.5, respectively. On the other hand, the egg yolk represents about 26.37%, 26.25%, 26.17% and 26.06% of the weight of the whole egg in Az0, Az2.5, Az5, and Az7.5 groups, respectively.

Figure 1. Effects of Azolla meal on the proportion of eggs (Az0 = 0% inclusion of Azolla; Az2.5 = 2.5% inclusion of Azolla; Az5 = 5% inclusion of Azolla; Az7.5= 7.5% inclusion of Azolla)

Proportionate results showed that experimental diets have insignificant effect on the proportion of egg parts. It was reported that the nutrition of laying hens affect egg-yolk and egg-white proportion to a lesser extent (Réhault-Godbert et al 2019). On the other hand, the shell with its membrane was significantly highest for maximum level of Azolla inclusion in hens diet (P = 0.0036; SEM = 0.041). The shell in eggs from group Az7.5 represents about 9.18% of the weight of the egg and consists mainly of tiny crystal calcium carbonate particles. Egg shell weight and thickness are among the most important traits of laying hens. Besides serving a packaging role, the shell and the shell membranes have a biological function; namely to regulate evaporation and air circulation, but also to prevent penetration of microorganisms. Generally, the observed improvement in percentage of egg shell from group Az7.5 can be explained by the increased levels of calcium in Azolla fern. It had been reported that the shell thickness and thus the strength depends on egg size, breed, the age of the hen, and feed composition (Bouvarel et al 2011). In fact, one of the major problems in the production of high quality eggs is the production of eggs having good shells, suggesting that Azolla may plays important role in this regard.

Proximate composition of eggs

Table 1 presents the proximate composition of edible parts of eggs produced from hens fed diets with or without Azolla inclusion. On average, eggs from the current experiment contain 75.06% moisture (24.94% DM), 15.54% crude protein, 26.96% crude fat and 1.12% ash (Table 1). This findings are partially consistent with the reports of 76.1% water, 12.6% protein, 9.5% fat, 0.7% carbohydrates, and 1.1% ash for whole freshly laid egg by Techer et al (2014). Breed, diet composition and age all affect the egg composition. It was observed that the dry matter, moisture and crude protein of egg albumen was unaffected by diets provided. Compared with other parts, egg yolk contains the highest DM. The lowest dry matter was exhibited from eggs from 5% inclusion of Azolla in hens’ diet. Eggs from the control group and were found to contain the lowest ash value. Significantly higher crude fat in yolk was obtained from the eggs collected from hens fed the maximum level of Azolla inclusion in diets.

Crude protein of yolk and albumen were unaffected, but the whole egg from control group contains the highest value compared with others. The nutritional analysis revealed that crude protein values of whole raw eggs, yolk and albumen were, on average, 15.54%, 20.09% and 13.43%, respectively. Divergent to the present finding, Réhault-Godbert et al (2019) reported that the concentration of proteins is, on average, 12.5%, 15.90% and 10.90% of whole raw fresh egg, egg yolks and egg white, respectively. It seems that these values are slightly modified by hens’ diet, age and genetics. Egg white and egg yolk are highly concentrated in proteins. Crude fat of whole eggs was significantly higher in Az2.5 and Az5 groups than Az0 and Az7.5 groups. To the best of our knowledge, there is no data available regarding the effects of Azolla fern on the proximate composition of eggs in commercial laying hens. It is noteworthy that diet effect is generally exhibited minor changes on the proximate composition of edible egg parts. These findings are in agreement with earlier studies that the proximate composition of eggs was unaffected regardless of the dietary treatments which contain Mentha arvensis and Geranium thunbergii (Dilawar et al 2021).

Micronutrient composition of eggs

The mineral composition of eggs produced from Bovans Brown hens fed diets containing graded levels of Azolla meal are presented in Table 2. The effect of Azolla meal on vitamin A value of egg yolk is shown in Figure 2. The vitamin A value of egg yolk was the most affected nutrient by hen diets. The precise values of vitamin A were 364.09, 688.87, 795.60 and 989.08 µg/100g for Az0, Az2.5, Az5 and Az7.5, respectively.

It is noteworthy that inclusion of Azolla meal in diets resulted in a linear increase in mineral concentration in the whole eggs and vitamin A content of the egg yolk as well (p < 0.05). The levels of nutritional enrichment are most pronounced at the maximum level of Azolla inclusion. The inclusion of Azolla meal at 7.5% in the diet led to an increment of 41.53%, 74.41%, 70.90%, 125.53%, 77.16%, 107.84%, 111.83%, 200.31%, 71.45% and 95.76% in Ca, Mg, K, Na, Fe, P, S, Cu, Zn and B in eggs from the control groups, respectively. Detailed explanation of benefits of each of the minerals is beyond the scope of this paper, however, these are important minerals needed for good health. Iron and zinc in eggs are bound in compounds that are more bioavailable and may be more easily digested than those in which they are bound in plant-based foods (Smith et al 2013). Iron is required for oxygen to travel to the tissues and organs, while zink prevents illness by supporting the immune system. Calcium in conjunction with magnesium, chlorine and proteins are involved in the formation of bones (Abullude et al 2007). Phosphorus is required for normal function of the body since it is a major structural component of bone and teeth and helps to maintain normal pH (Knochel 2006). It is crucial for production of ATP and also plays important role in the growth, maintenance and repair of cells and tissues. Magnesium is an antioxidant micronutrient and its presence may boost the immune system and aid in removing magnesium deficiencies which could lead to severe metabolic disorders and compromise the health of the organism (Hassan et al 2007). The presence of such minerals in eggs of Azolla-fed hens is quite interesting as a deficiency in some of these (Zn, Mg, and Cu) has been associated with depression and fatigue, and development of pathological diseases (Wang et al 2018).

Figure 2. Effect of Azolla meal on vitamin A value of egg yolk (p<.0001; SEM = 85.64)

Besides mineral enrichment, the present study also showed that the vitamin A component of egg yolks increased with increasing levels of Azolla meal in the diet. The value of vitamin A in egg yolk had been enriched by189.20%, 218.52% and 271.66%, respectively when Bovan brown hens fed 2.5%, 5% and 7.5% levels of Azolla meal in their total diet for a period of 8 weeks. It has been reported that the content of liposoluble vitamins such as vitamins A in egg yolk is highly dependent on the hen’s diet. According to Réhault-Godbert et al (2019) egg content in vitamin A can be enhanced 10-fold from its initial value when hens are supplied with 30,000 IU retinol. Vitamin A contributes to the maintenance of normal skin and the normal function of the immune system. It also contributes to normal vision. In light of the present findings, it may be concluded that eggs enriched with micronutrients such as vitamin A, Ca, Fe, Cu and Zn can be produced by minor dietary modifications without affecting overall production and egg quality parameters in commercial laying hens. The trends in child under nutrition including stunting and wasting, deficiencies in essential micronutrients in children continue to be of great concern in many areas of the world. Eggs are easily digested and absorbed, provide several essential nutrients and are therefore valuable in diets for infants, convalescent or elderly patients who may have greater difficulty digesting and absorbing nutrients (Nys and Sauveur 2004). Thus, Azolla-derived eggs could be of interest in complementary food formulation for infants, convalescent patients, and pregnant women as well.

Microbial load of eggs

Table 3 shows that microbial analysis of eggs produced from Bovans Brown hens fed Azolla was better or comparable regardless of the diets provided. Nevertheless, Azolla had been either improved or maintained the microbial quality of eggs.

The highest count for E. coli, yeast and mold was observed in eggs from Azolla free diet group, but not much significant variation exist. Eggs were not significantly different in aerobic mesophilic bacterial count and Staphylococcus aurous counts. Salmonella is not detected in any of the egg groups at all, suggesting the absence of the risk of transmission of food-borne microbial diseases and spoilage due to Azolla. Microbial analysis of eggs from laying hens fed Azolla meal has not been reported before, which limits the possibility of comparing data. However, the observed microbial quality of eggs collected from Azolla fed hens may be associated with the improved egg shell thickness or shell percent. Egg shell (weight and thickness) is the most important trait of laying hens. It is required to form the structure and handling of eggs. In particular, it is very important in upholding the hygiene of eggs, as any damages or cracks will make the eggs more susceptible to bacterial contamination. It is well established that eggs having thin shell are more prone to microbial contamination (Sumashree et al 2019), implying that Azolla fern has something to do towards protection of eggs against microorganisms.

Sensory attributes of eggs

The inclusion of Azolla meal in commercial layer diets improved the sensory parameters of eggs, except odor which is insignificant among the treatments (Table 4). A significant increase was observed in sensory attributes of eggs as a result of Azolla inclusion in the diets of laying hens.

In this study, the panelists did not identify any negative impact that is caused by inclusion of Azolla in commercial layer diets at graded levels. The reverse is true in this respect. The sensory evaluation of eggs from laying hens fed Azolla meal has not been reported before, which limits the possibility of comparing data. The eggs collected from the birds fed Azolla meal have better acceptability in terms of texture, color, and taste than those eggs from the control group. These data suggest that the characteristic smell of Azolla biomass are not transferred to the eggs. In terms of the flavor, the eggs obtained from the hens fed Azolla meal were better than the control. This may be due to the antioxidant potential of Azolla pinnata (Mithraja et al 2011; Noor et al 2014), which prevents the oxidation of proteins and lipids in egg yolk, resulting in better flavor (Dilawar et al 2021).

It was also found that the most liked yolk color (yellowness) by the panelist is eggs obtained from Azolla-fed hens. The diet of the hen determines the colour of the egg yolk. If the hen is mainly fed by yellow and orange-pigmented food, this is the colour that is more prominent in the egg yolk. A relatively colorless feed, gives almost colorless yolks. Yolk color (yellow/orange shading) is apparently determined by the content in carotenoids in the diet (Bouvarel et al 2011). To this end, several sources of carotenoids (lutein, xanthophylls, and zeaxanthin) for laying hens such as yellow maize and green feeds are incorporated in a hen’s diet to meet consumer demand for a more orange-yellow yolk. The improvement of egg quality traits related to appearance, such as color, is quite critical for product marketing and to the economic value of fresh eggs. Unacceptable color is of course most vital for its impact on consumer attitude and can cause a huge economic damage in the processing industry. Beside its interest in increasing the visual aspect of the yolk, the high content of carotenoids in the yolk may also have a positive incidence for human health in increasing visual performance and reducing the risk of age-related macular degeneration (Ranard et al 2017).

Conclusions

• The feeding of Azolla-derived diets to laying hens had a positive effect in terms of nutritional, microbial and sensory attributes of eggs.
• The albumen and yolk parts of the egg were unaffected, the shell with its membrane was the highest in maximum Azolla group.
• The diet effect is more pronounced in mineral and vitamin A composition of eggs, especially at the maximum spectrum of Azolla inclusion in layers diets.
• Vitamin A value of egg yolks had been increased from 364.09 µg/100g to 688.87, 795.60 and 989.08 when hens fed diets with 2.5%, 5%, and 7.5% of Azolla containing diets, respectively.
• Microbial count of eggs produced from hens fed diets with Azolla were better or comparable regardless of the diets provided.
• Compared with other egg groups, eggs from maximum Azolla group were excellent in their taste, color and overall acceptability as well.
• Utilizing Azolla in hens’ diet have multiple advantages both for the poultry producers and egg consumers/processors.
• The development of alternative feed in poultry industry may represent a great opportunity for human nutrition, health and economy.

Conflict of interest

The authors declare no conflict of interest to reveal.

Acknowledgements

This investigation was funded by the Office of Research and Technology Transfer of Hawassa University for which the authors are highly grateful. The unlimited assistance from the Laboratory of Animal Nutrition (esp. Mr Feleke T.), Nutrition, Food Science and Technology, and Horticoop Ethiopia (esp. Ms Zufan G.) are acknowledged for the nutritional analysis undertaken.

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