Effect of inclusion of graded levels of Azolla (Azolla pinnata) meal in layers’ diets on productive performance and egg quality parameters

Seid A

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

Abstract

An experiment was conducted to investigate the effect of inclusion of dried Azolla (Azolla pinnata) meal on the feed intake, production performance and egg quality parameters in laying hens. Four experimental diets were formulated by incorporating Azolla meal at 0 (Az0), 2.5% (Az2.5), 5% (Az5) and 7.5% (Az7.5) levels and in which an attempt was made to maintain a constant crude protein. Seventy-two 28-week-old Bovans Brown layers were randomly divided into four dietary treatments with three replicate pens having six hens per pen. The actual experiment lasts for 8 weeks. Daily feed intake, egg production and egg weight were monitored. Initial and final body weights of hens were taken. On weekly basis, 6 randomly collected eggs per pen were used to determine internal and external egg quality parameters. Azolla meal contained 25.56% crude protein; 3.15% ether extract; 14.23% crude fibre; 28.42% total ash, 23.55% nitrogen free extract, 1.88% Ca and 0.97% P. The performance data indicate that dietary inclusion of Azolla meal did not affect the daily feed intake and body weight gains of layers (p>0.05). Both hen-day egg production and egg mass were significantly higher for hens offered the Azolla containing diets (p<0.05). The eggs were found to have optimum weight (55.32^_56.82 g). The most satisfactory egg weight was obtained on a diet containing 5% added Azolla meal. The data further indicate that at the higher levels of Azolla meal inclusion, the albumen height, albumen index, Haugh unit, and shell thickness were increased (p<0.05). Yolk color score increased with increasing levels of dietary Azolla inclusion (p<0.05). The efficiency of feed utilisation (g feed/g egg mass) also improved with increasing Azolla inclusion rate. Above all, feed cost/kg and total feed cost/hen statistically reduced with increasing levels of Azolla in the diets. In conclusion, the inclusion of Azolla meal up to 7.5% in the diet of laying hens can enhance laying performance and egg quality traits while lowering feed costs.

Key words: Azolla meal, bovans brown, egg mass, feed cost, shell thickness, yolk color score

Introduction

Poultry production may be the solution to sustainably feeding a projected human population of 10 billion by the year 2050. Poultry production have the potential to enhance and sustain food and nutrition security with considerable impact on livelihoods of the most at risk through production of safe, high-quality food within a comparably short period. However, higher price and scarcity of conventional feed ingredients are ruining its prospects. Feed consumption accounts for the major cost of production which is responsible for more than 70% of the total cost. Prices for staple feed ingredients are up to 100% more expensive than they were before some years in Ethiopia (Seyoum et al 2018). Although conventional feed ingredients are becoming increasingly expensive, evidence shows alternative resources can maintain productivity and profitability in poultry. Azolla is one of such feeds which have rich nutrients and mineral profile. Azolla is a small free-floating aquatic fern native to Asia, Africa, and America (Pillai et al 2002). Azolla pinnata in particular is widely adapted in the tropical climates as in Ethiopia. It grows naturally in stagnant water bodies (Kumar and Chander 2017).

There are multiple reasons for considering Azolla as a potential feed resource for sustainable, viable and profitable small-scale poultry production. In terms of production benefits, Azolla is rapidly growing, self-perpetuating, high N₂ fixer, high biomass per unit area, takes up much less space and water for propagation than the traditional feed resources (Kumar and Chander 2017). Evidence existed that Azolla is rich source of nutrients containing protein, lipids, minerals and vitamins (Alalade and Iyayi 2006; Brouwer et al 2018). Azolla is of interest as a protein feed due to the high protein content of its biomass, which was reported to be between 20–40% of the dry weight (Kumar and Chander 2017). Azolla contained a higher proportion of all essential amino acids viz. lysine, arginine, methionine, isoleucine, leucine, phenylalanine, glycine and valine (Brouwer et al 2018). Azolla was further reported to contain 7.9–16% lipids, which may contribute to the gross energy of whole Azolla feed (Brouwer et al 2018). It is also found to contain essential minerals including iron, calcium, magnesium, phosphorus, manganese and potassium (Kumar et al 2018), along with appreciable quantities of Vitamins A and Vitamin B-12 (Pillai et al 2002). Lejeune et al (2000) reported that on fresh material, the carotene content ranged from 206 to 619 mg/kg on a dry matter basis.

As far as the bird is concerned, Azolla contain tannins (Brouwer et al 2018), which can reduce protein digestion, so it is imperative to evaluate low dietary inclusion rates with a view to determine the optimal amount that can be added safely. On feed trials, dietary inclusion of dried Azolla meal has been reported to replace about 10% of soybean meal in the diets of broilers without any adverse effects on performance indices (Wasihun et al 2019). Kumar et al (2018) also recorded improvement in body weight gain and feed conversion ratio with inclusion of Azolla up to 7.5% in the diets of broiler, without any ill-health signs. Research on layers performance and egg quality parameters has been very limited when compared with broilers performance. Khatun et al (2008) indicated that Azolla meal could be considered a sources of protein and pigment for laying hens without any harmful effects on performance up to 10%. Nevertheless, the effect of feeding Azolla pinnata on layers diet on feed intake, egg laying performance and quality characteristics has not been fully investigated and pertinent data for poultry is lacking. The main objective of this study was to investigate the effect of inclusion of Azolla meal at different levels on the feed intake, laying performance and egg quality parameters of layer hens at tropical environment.

Materials and methods

Description of the study area

The feeding trial was conducted at poultry research station, Hawassa University, Ethiopia. The farm is located at 7^o11’ N latitude and 38^o31’ E longitude. The altitude of the area is 1750 masl, annual rainfall is 955 mm and the mean temperature is 20 ^oC (12-27 ºC).

Experimental design and birds management

A total of 72 Bovans Brown laying hens aged 26 weeks were assigned to four dietary treatments in a Completely Randomized Design (CRD). Each treatment had three replicated pens. Each replicate had six hens. The hens were housed in experimental pens of wire-mesh partitioned. The pens were installed with feeding troughs, drinking troughs, laying nests, and lighting facilities. The concrete floor was covered with 6-10 cm depth of sawdust as litter. Feed was offered twice a day. The hens were given two weeks of adaptation period to the diets followed by the actual feeding trial. Fresh clean water was provided at all times. Communal laying nests, covered with sawdust, were placed in each pen. A lighting schedule of 16 h/day was applied during laying period. Sanitation was observed at all times in the birds’ house. The birds received identical care and management. Any ill health signs are monitored throughout the study period.

Azolla cultivation and meal production

The culture of Azolla (Azolla pinnata) was obtained from Sebeta Fishery and Aquatic Life Research Center, Ethiopia. Azolla has been cultivated on large scale on polyethylene sheets covered pits (two pits of uniform dimensions of 3 x 1.5 x 0.2 m) as described by Kumar and Chander (2017). A mixture of soil and cow dung was added as a nutrient source. The water level was 10-15 cm. Azolla biomass was manually harvested weekly. After harvest, the biomass was washed with clean water, and air dried under shade. Briefly, Azolla biomass was subjected to shade drying by placing the fresh biomass on framed nets in a well ventilated shade with constant turning. Clean bed nets were used for drying and sieving purpose (Photo 1). Azolla meal was then prepared for feeding chicken in concentrate mix.

Photo 1. Azolla cultivation and meal production process

Preparation of experimental diets

Four experimental diets were formulated by incorporating Azolla meal at 0 (Az0), 2.5% (Az2.5), 5% (Az5) and 7.5% (Az7.5) levels. The levels of limestone, layer premix, methionine and salt were kept constant in all the diets. Attempts were made to make the diets isonitrogenous. Composition of the experimental layer diets are shown in Table 1.

Data collection, measurements and calculations

Production performance

Each day, a measured quantity of feed was offered to hens. Feed refused was recorded daily and feed intake was calculated on group basis. Individually, initial and final body weights of hens were taken; and body weight gain was calculated. Eggs were collected twice a day. The number and weight of eggs laid were recorded on the same day of collection. Egg production was calculated on a hen-day basis. Hen-day egg production (HDEP) was calculated as the number of eggs laid divided by the number of hens (Ahmed et al 2013). The egg mass was calculated by multiplying the average egg weight with HDEP. Then, feed conversion ratio (FCR) was calculated by dividing the feed intake by the egg mass.

Egg quality measurements

The egg quality parameters were determined every week by randomly collecting six eggs from each replicate. The individual eggs were weighed on a digital balance to the nearest of 0.01 g accuracy. The egg length (upper end to the lower end) and width (centre of the egg or equator) were measured using Vernier caliper with the least count of 0.01 mm. The shape index (SI) was determined from the egg length and width that were described by Duman et al (2016).

The eggs were broken out on a flat transparent glass surface using a spatula to obtain various internal parameter measurements. Yolk diameter was measured end to end by using Vernier caliper. Albumen width was measured at the narrowest and widest point of thick albumen from the yolk, and their average value was used for analysis. Tripod micrometer was used for determining the height of the yolk and tick albumen of eggs on a table glass. The egg yolk was gently separated from the albumen and weighed. The yolk colour was determined by the Roche Yolk Colour fan ranging from values 1 (pale yellow) to 15 (dark orange) (Beardsworth and Hernandez 2004).

The egg shell was allowed to dry at room temperature and weighed after 48 hours. The weight of albumen was determined indirectly by deducting the yolk weight and shell weight from egg weight. The eggshell thickness was measured using a Vernier caliper after removing the shell membrane and it is represented as the average thickness of the upper, middle, and lower end of the shell. Egg shell ratio in percentage was determined as a result of egg shell weight divided by egg weight. The shell surface area was determined as 4.67 x SW^0.667; Where: SW= Shell weight (Carter 1975).

Albumen and yolk ratios were calculated taking their individual weights as the percentage of total egg weight. Yolk albumen ratio was calculated as weight of yolk/weight of albumen. Yolk index (YI) was estimated in percentage, taking the ratio of the height (YH) to the average diameter (YD) by using the formula: YI = (YH/YD)*100 (Kul and Seker 2004). Haugh Unit (HU) was calculated from the height of the albumen and the weight of the egg. Haugh unit = 100 x log (Albumen height + 7.57 ­ 1.7 x Egg weight ^0.35), where 7.57, 1.7 and 0.35 are constant (Haugh 1937).

Chemical analysis

The content of dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF) and crude ash in the feed samples was determined according to standard procedures (AACC, 1995). Atomic absorption spectrophotometer was used to determine the concentration of Calcium (Ca) and Phosphorous (P) after dry ashing of samples in the furnace for 3 hrs. Metabolisable energy was then estimated as ME (kcal/kg DM) = 3951 + 54.4 EE - 88.7CF - 40.8 Ash (Wiseman 1987). Samples were analyzed in triplicates at Animal Nutrition and Soil Laboratory of Hawassa University.

Partial budget analysis

Economic efficiency analysis took into considerations the cost of feed and selling price of eggs at the time of experiment. The variable costs were kept constant among the dietary groups except the feed cost. Feed cost was calculated based on the market price of each ingredient and percent of inclusion. The cost of Azolla meal, on the other hand, consider the farming and harvesting cost per kg biomass produced. The partial budget was calculated as the difference between the feed cost incurred during the experimental periods and sale of the eggs. The net return (NR) was calculated by subtracting total feed cost (TFC) from total return (TR).

Statistical analysis

The data were analyzed by using the ANOVA procedure of the SAS Statistical Package Program (SAS 2009). The following statistical model was used to determine the effects of the treatment: Y_ij = µ + t_ij + e_ij, where Y_ij = response variable, µ = general mean, t_ij = effect of dietary treatments 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 the results were considered to be significant if the p-values were equal to or less than 0.05.

Results and discussion

Nutritional composition of feeds

The chemical composition of the major feed ingredients and experimental layer diets are shown in Table 2. Chemical analysis revealed that dried Azolla meal contained 25.56% crude protein (CP); 3.15% ether extract (EE); 14.23 % crude fibre (CF) and 28.42% total ash; 23.55% nitrogen free extract (NFE) and 1614.07 kcal/kg DM metabolizable energy (ME). Similar CP content was reported by earlier researchers (28.54%, Khatun et al 2008; 24.89%, Wasihun et al 2020). Slightly higher values of CP were also obtained by various researchers: 28.24% (Indira et al 2009) and 28.59% (Ahirwar and Leela 2012). However, lower values of CP were reported by other workers (21.40%, Alalade and Iyayi 2006 and 21.89 %, Boitai et al 2018). The differences in CP might be associated with the soil type and the drying process. Shade drying retained significantly higher crude protein levels than both sun and oven drying (Ncube et al 2015).

Similar to the present finding, Boitai et al (2018) found 3.52% EE for Azolla. However, the EE (3.15%) was higher than the 2.7% reported by Alalade and Iyayi (2006). Comparatively higher values for EE were reported by Wasihun et al (2020) and Khatun et al (2008). Their findings are 3.74% and 4.54 %, respectively. Crude fiber (CF) findings of 12.7% (Alalade and Iyayi 2006), 12.38% (Khatun et al 2008), 14.21% (Wasihun et al 2020) and 13.92% (Boitai et al 2018) were reported in earlier studies. The results were consistent with the present finding of 14.23%. The total ash content of Azolla meal was 28.47%. This is higher than reported values of 16.20% (Alalade and Iyayi 2006), 16.92% (Khatun et al 2008), 18.01% (Boitai et al 2018) and 18.79% (Wasihun et al 2020). The growth morphology and composition of Azolla could be affected by environmental conditions, worth mentioning temperature, light intensity and soil nutrient (Alalade and Iyayi 2006). Also, contamination with fallen tree leaves and other epiphytic algae may affect the nutritional composition of Azolla meal. The calculated value of metabolizable energy is 1614.07 kcal/kg DM, and this is in accordance with the previous result of 1759 kcal/kg DM by Parashuramulu et al (2013). In contrast, the obtained ME value is lower than earlier report of 2431 kcal/kg (Khatun et al 2008). In fact, fiber-rich ingredients are often reduce metabolizable energy value. The results in general implies that Azolla could be a good source of protein and minerals for poultry, with moderate source of energy.

Productive Performance of Layers

The production performance of Bovans Brown hens fed different levels of Azolla meal is shown in Table 3 and Figure 1-3. The dietary inclusion of Azolla meal did not affect the daily feed intake and body weight gains of layers. The average egg weight was significantly higher in Az5 relative to the other treatment groups. Hens offered Az2.5 and Az7.5 diets had similar egg weights. Hens on Az5 diet were more efficient compared to those on Az0 and Az2.5 and but similar to hens on Az7.5. The poorest egg weight, egg mass and feed conversion ratio (FCR) were obtained from the control group (p < 0.05). Neither ill health signs nor mortality occurred during 8 weeks of the study period.

Figure 1. Daily feed intake of laying hens fed graded levels of Azolla meal	Figure 2. Feed conversion ratio of laying hens fed graded levels of Azolla meal

Figure 3. Effect of dietary incorporation of Azolla meal on egg mass

The dietary inclusion of dried Azolla meal did not affect the daily feed intake (Figure 1) and body weight gains of layers (Table 3). Azolla meal had no effect on palatability of the diet and livability of the birds (Khatun et al 2008). Similar reports of unaffected feed intake was reported when Koekoek chicken offered diets with Azolla meal up to 15 % substitution of soybean meal (Wasihun et al 2020). In contrary, Boitai et al (2018) found that dietary incorporation of Azolla meal at either 5 or 10% level reduced the body weight of laying hens.

In the present study, Azolla meal improve egg output by hens in terms of daily egg production, egg weight and egg mass. Scientific evidence existed that addition of Azolla in layers diets resulted in increasing egg production and egg weight at the expense of feed cost. Kumar and Chander (2017) observed an appreciable increase in egg laying capacity (10-15%) in chickens after consumption of Azolla pinnata. The experiments carried out by Lakshmanan et al (2017) showed that feeding Azolla meal raised the poultry egg-laying performance by 6.64% while egg weight increased by 6.62%. A similar better egg production performance was detected by Kannaiyan and Kumar (2005) in terms of higher egg yield after Azolla inclusion at 100 g/bird/day. However, Khatun et al (2008) found non-significant difference in egg production and egg mass in laying hens up to 10% level of Azolla meal. During 8 weeks of trial period, it was found that egg weight increases with increasing levels of Azolla meal inclusion. These results indicate inclusion of Azolla meal up to 7.5% in diets of laying hens have beneficial effect on egg size. A similar confirmation of Azolla meal also reported by Khatun et al (1999) who found that feeding Azolla up to 15% on a total or digestible nutrient basis had improved egg size.

The results of feed conversion ratios are consistent with the observations of Wasihun et al (2020). Lower FCR values indicate higher efficiency. Several studies indicate Azolla meal have beneficial effects on feed conversion rates. Against the present finding, Khatun et al (2008) observed no variation in feeding efficiency of laying hens received diets of varying levels of Azolla meal up to 10%. There was no mortality of birds across the dietary treatments during the whole period of study. Similar to the finding of the present study, previous studies indicate Azolla meal have no effect on bird livability (Khatun et al 2008; Boitai et al 2018).

Egg Quality Parameters

The effect of Azolla meal on the egg quality parameters is presented in Table 4 and 5. The Albumen height, Albumen index, Haugh unit and shell thickness were significantly better in Az7.5 when compared with the control and other treatments (p < 0.05). Yolk diameter was unaffected by the different levels of Azolla meal inclusion. Eggs from the control group contain the lowest yolk weight. Yolk color score increases with increasing levels of Azolla meal in the diet and the highest yolk colour score (10.05) obtained at 7.5% Azolla inclusion.

As evident from Table 4 and 5, most of the studied egg quality parameters were improved by the inclusion of Azolla meal in layers diets, especially at 7.5%. Egg length and width are increased by Azolla meal feeding. These findings are inconsistent with Wasihun et al (2020) who found non-significant measurements. Several factors influence the size of an egg, including breed and age of the birds. The current results clearly indicates that eggshell weight and thickness were significantly improved at 7.5% inclusion of Azolla meal. These findings are inconsistent with Alalade et al (2007) and Khatun et al (2008) who reported non-significant effect on eggshell thickness due to dietary incorporation of Azolla meal up to 15% and 20%, respectively. In another study, Boitai et al (2018) reported that Azolla meal up to 10% in the diet had no influence on shell thickness. Eggshell 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.

In this trial, yolk diameter was unaffected by Azolla meal inclusion in layer diets. These findings are in line with observations of Khatun et al (2008). Fresh yolk weight (14.42-14.80 g) and yolk index (44.74-45.90) were improved by the inclusion of Azolla meal in layers diets in particular at 2.5%, 5% and 7.5%. These findings are partially inconsistent with Wasihun et al (2020) who obtained yolk weight (15.24-16.47 g) and yolk index (41.56-52.43) values. Concomitant to Haugh unit, the yolk index provides indication on the freshness of the egg. It was reported elsewhere that eggs with yolk index above 38% are considered as extra fresh. The highest Haugh unit (HU) value was obtained from eggs laid by birds fed the maximum level of Azolla inclusion in the diets (Az7.5). Divergent findings with non-significant HU due to Azolla feeding up to 10% were reported by Khatun et al (2008) and Boutai et al (2018). Similar to the present finding, Wasihun et al (2020) found an improved HU value from eggs laid by Koekoek chicken received diets with varying levels of Azolla meal. This measurement determines the protein content and freshness of the egg. Higher value for HU observed in the experiment is attributable to the freshness of eggs and proper age of hens. It was generally agreeable that the higher the score of HU, the better the quality of the egg (fresher, higher quality eggs have thicker whites). Breed, egg weight and age all affect these quality parameters. Yolk ratio estimated in the experiment (26.21±0.16) is lower than the earlier estimate (Wasihun et al 2020) that means yolk contribute less to the total egg weight.

Shape index (76.96 – 78.66) observed during 8 weeks of the trial indicates that the eggs are round in shape. According to Sarica and Erensayin (2009) eggs are characterized by the shape index values as sharp, normal (standard) and round if they have an shape index value of <72, between 72 and 76, and >76, respectively. Shape index values of 69 - 75 were reported by Khatun et al (2008) when commercial hens (43-59 weeks) offered diets of varying levels of Azolla meal up to 10%. Values of 74.21-75.19 also reported by Wasihun et al (2020). Results of experiments may differ according to the stage and breeds of chicken.

Photo 2. Effect of Azolla meal on yolk color

As expected, Azolla meal evidently contributes to better yolk pigments (Photo 2). Concomitant to the findings of the present study, Khatun et al (1999) found that yolk colour was significantly improved with increasing levels of Azolla meal and a longer period of feeding. Several of previous studies affirmed Azolla meal have beneficial effects on yolk colour (Khatun et al 2008; Boitai et al 2018; Wasihun et al 2020). Yellow yolk colour is determined by animal genetic or xanthophyll (plant pigment with beta-carotene) content in the diet. The deep orange color of egg yolk in Azolla fed hens might be due to the presence of β-carotene pigments in Azolla. Pillai et al (2004) reported that Azolla contains appreciable quantity of β-carotene, vitmin B₁₂ and biopolymers.

Economic efficiency of Azolla feeding

Economic analysis for egg production is presented in Figure 4. All production costs except the feed cost as well as the selling price of eggs were kept constant across the dietary treatments. The feed cost of the dietary treatments Az2.5, Az5 and Az7.5 reduced than that of the control (Az0).

Figure 4. Economic efficiency of Azolla feeding

Feed cost/kg and total feed cost/hen significantly reduced with increasing levels of Azolla meal in the diets. Several studies showed that the utilization of Azolla in layers diets saved the concentrate feed cost (Khatun et al 2008; Lakshmanan et al 2017; Wasihun et al 2020). Azolla makes the difference and have to do with lower feed costs. Azolla cultivation is non-competitive with human needs. These findings suggest that Azolla meal production is both technically and economically feasible strategy without requiring fertile land or high volumes of water for cultivation. The farmer needs no more than stagnant water, cow dung and bed soil of which are mostly free of cost. Although there were variations with regards to total revenue and net return on hens fed diets with varying levels of Azolla meal, the data did not differ significantly during 8 week of the trial. The observed non-significance net return may be associated with the small level of Azolla meal inclusion in the diets.

Conclusions

Despite of lower ME value, Azolla contains valuable protein, micronutrients and pigment which are suitable for feeding laying hens. Azolla meal evidently play an important role in improving laying performance and feed efficiency without any adverse effect on feed intake and weight changes. The economically important egg quality attributes like Haugh unit, shell thickness and yolk color score are clearly improved when hens offered diets containing graded levels of Azolla meal. In addition, economic analysis results show that feed cost significantly reduced with increasing levels of Azolla meal in the diets up to 7.5%. The combined effects observed in the experiment implies that Azolla can make small scale poultry production more profitable and more sustainable. A much greater emphasis is recommended for improving plant protein supply through cultivation of Azolla in marginal growing environments.

Acknowledgement

The author is grateful to the Office of Research and Technology Transfer of Hawassa University for financial and logistic provision to undertake this research. The School of Animal and Range Sciences is acknowledged for providing premises for Azolla cultivation and poultry experiment. Sebeta Fishery and Aquatic Life Research Center (especially Dr Marshet A.) also deserves gratitude for providing Azolla culture.

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