Effect of Aquatic Fern, Azolla cristata in Diet on Growth, Serum Biochemistry and Laying Performance of Chicken

Shoukat Ara1, Sheikh Adil2,* and Manzoor Ahmad Khan3

1Division of Environmental Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar-190006, India

2Division of Livestock Production and Management, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar-190006, India

3Division of Veterinary Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar-190006, India

ABSTRACT

To determine the effect of dietary supplementation of aquatic fern Azolla cristata on growth, serum biochemistry and laying performance layer chicken, a study was conducted on 240 key stone golden layer chicks which were randomly assigned into five groups having three replicates of 16 each. Birds in control (T1) group were fed the basal diet, where as other groups the groundnut cake was replaced with 5 (T2), 10 (T3), 15 (T4) and 20% (T5) Azolla. The results revealed that inclusion of Azolla showed a non-significant increase (p>0.05) on the body weight gain of grower birds. Further, the SGPT and SGOT levels of birds did not differ among various treatments at 19 weeks of age. A non-significant (p>0.05) improvement was observed in the egg weight, shell thickness and weight in all the groups fed Azolla in the diet (T2, T3, T4 and T5) when compared with the control group (T1). The differences in the total number of eggs laid, % hen-day-egg production, various external and internal egg parameters under different treatments were statistically non-significant (p>0.05). In conclusion, Azolla is beneficial at lower levels in improving the performance of grower chicken but had no effect on the number of eggs laid. Further, Azolla inclusion in the diet showed an improvement in the shell quality of eggs which could prove beneficial in decreasing the incidence of egg breakage and thereby maximizing profits. The Azolla inclusion in the diet of layer chicken had no effect on SGPT and SGOT levels indicating that Azolla supplementation had no toxic effect in layer chicken even up to 20% levels.

Article Information

Received 13 December 2016

Revised 20 Apri 2017

Accepted 17 July 2017

Available online 16 October 2018

Authors’ Contribution

SA designed the study and provided guidelines during the work. SA carried out the experiments and wrote the manuscript. MAK helped during the trial and in the processing of samples.

Key words

Azolla, Serum biochemistry, Egg quality and layer chicken.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.6.2325.2329

* Corresponding author: aadilsheikh5@gmail.com

0030-9923/2018/0006-2325 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

The poultry industry in India has made remarkable progress during last 3 decades, but the rising cost and unavailability of the feed ingredients are the major hindrances in achieving the target production. The higher price and non-availability of feed ingredients are the major limitations to the growth and profitability of poultry enterprise (Islam et al., 2010). Feed is the most expensive of all inputs and about 70% of production cost is accounted for feed alone (Parthasarathy et al., 2001). This has led to the poultry nutritionists to look for alternative cost effective, non-conventional feed ingredients for economic poultry production (Mishra et al., 2016).

Aquatic weeds are one such alternative and they have been proposed to be used as feed ingredients (Pirie, 1980). They are believed to offer a fairly cheap feedstuff for poultry and can partly substitute the conventional and expensive dietary protein sources of layer chicken (Haustein et al., 1990). Aquatic plant species, because of their growth habit, appear not to accumulate secondary plant compounds and therefore offer a greater potential than tree leaves as a source of protein for monogastric animals (Bacerra et al., 1995). There has been an increased emphasis in the use of aquatic plants in poultry ration because the protein and other nutrient content in them are comparable to certain leguminous plants. Of these, the water fern Azolla is perhaps the most promising, being free crop of high nutritive value (Lumpkin and Plucknett, 1982).

Azolla is a free floating fresh water fern belonging to the family Azollaceae and order Pteridophyta. It is commonly found in tropics and sub-tropics and grows naturally in stagnant water of drains, canals, ponds, rivers, haors-baors and marshy lands. Anabaena-Azollae, living in the cavity of Azolla leaf, can fix high amount of atmospheric dinitrogen due to presence of symbolic algae in the leaves (Becking, 1979). With its sudden appearance in 2002, Azolla has now spread widely in aquatic ecosystems of Kashmir valley. The Azolla species present in aquatic ecosystem of Kashmir has been identified as Azolla cristsa on the basis of presence of bicelled trichomes, hook shaped, multiseptate glochidia, and a 3-float megaspore apparatus with a granular perine surface (Ahad et al., 2012).

Azolla is a potential feed ingredient for poultry and livestock (Singh and Subudhi, 1978; Pannaerker, 1988). Use of Azolla meal up to 5% in the broiler ration has been reported to improve the performance in broiler diets as a safe level (Basak et al., 2002; Ara and Adil, 2012). Since, there is a very scanty literature available regarding the use of Azolla in layer chicken, the present study was undertaken to evaluate the effect of Azolla cristata in grower chicken and its subsequent effect on the layer performance.

 

Table I.- Ingredient composition of experimental diets.

Ingredients	T1	T2	T3	T4	T5
Maize	51.0	51.0	51.0	51.0	51.0
Soya-Full fat	14.0	14.0	14.0	14.0	14.0
Groundnut cake	10.0	9.5	9.0	8.5	8.0
Azolla cristata	-	0.5	1.0	1.5	2.0
De oiled rice bran	8.0	8.0	8.0	8.0	8.0
Wheat bran	7.0	7.0	7.0	7.0	7.0
Fish meal	4.0	4.0	4.0	4.0	4.0
Shell grit	3.0	3.0	3.0	3.0	3.0
Salt	0.5	0.5	0.5	0.5	0.5
Mineral mixture	2.5	2.5	2.5	2.5	2.5
	100.0	100.0	100.0	100.0	100.0
Chemical composition*
Crude protein (%)	17.94	17.84	17.75	17.65	17.56
Metabolizable energy (Kcal/kg)	2659.7	2658.1	2656.7	2655.2	2653.8

Vitamin premix was added @ 10 g/quintal (Each g contained vitamin A, 82500 IU; vitamin D3, 12000 IU; vitamin B2, 50 mg; vitamin K, 10 m), Spectrimix-BE, 10 g/q (Each g contained vitamin B1, 80 mg; vitamin B6,16 mg; niacin, 120 mg; vitamin B12, 80 mg; calcium pantothenate, 80 mg; vitamin E, 160 mg; L-lysine HCl, 10 mg; DL methionine, 10 mg; calcium, 260 mg). *Calculated values.

 

Materials and methods

Bird husbandry and experimental protocol

To achieve the envisaged objectives, 240 key stone golden chicks at two weeks of age were individually weighed and randomly assigned into five groups having three replicates of 16 chicks each. The birds were reared and vaccinated as per the routine recommended procedures. The birds in the control (T1) group were fed the basal diet, where as in other groups groundnut cake was replaced with 5 (T2), 10 (T3), 15 (T4) and 20% (T5) Azolla. The basal diets were formulated to meet the recommendations of Bureau of Indian Standards (1992) and shown in Table I. Up to 8 weeks of age, the birds were fed starter diet having crude protein (CP) 18 % and metabolisable energy (ME) 2750 Kcal/kg of feed. From 8 to 20 weeks, grower diet with 16 % CP and ME 2600 Kcal/kg feed was used. Thereafter, from 20th week onwards, layer diet was used having 18 % CP and ME 2700 Kcal/kg feed. The chemical analysis of Azolla (Table II) was done as per standard procedures of AOAC (1996).

 

Table II.- Analyzed chemical composition of Azolla cristata.

Constituent	% DM basis
Crude protein (CP)	22.06
Ether extract (EE)	3.62
Crude fibre (CF)	14.3
Nitrogen free extract (NFE)	33.4
Total ash	18.1
Calcium	2.04
Phosphorus	0.65

 

Collection, drying and mixing of Azolla

Azolla cristata has engulfed most of the water bodies in Kashmir, from where it was collected and then dried in the sunlight. After sun drying, it was ground and stored in the plastic bags until used for feeding. Azolla was mixed thoroughly in aforesaid quantities to small amount of feed (1 kg) in a premixer. The resultant mixture was then mixed with the rest of the feed in a mechanical blender until a thorough and consistent mixture was obtained.

Parameters recorded

The body weight and feed consumption of pullets was recorded on individual basis at bi-weekly intervals. At the age of 19 weeks, serum samples were collected from 4 birds per replicate for the determination of SGPT (Alanine Aminotransferase) and SGOT (Aspartate Aminotransferase) by means of auto analyzer using commercially available kits purchased from the Accurex biomedical company. The egg production was determined on daily basis while as egg quality parameters like egg weight, yolk weight, albumin height and width, shell weight and thickness were estimated from randomly collected 4 eggs per replicate on weekly basis from 33 to 40 weeks. Egg weight was estimated by means of a digital weighing balance. Each egg was carefully broken at the equator with a spatula and contents emptied on a dry, smooth and flat surface. The albumin height and width were measured by means of a spherometer and vernier caliper respectively. The yolk was carefully separated from albumin and its weight was measured by digital balance. For determination of shell quality, the shells were washed and dried at room temperature for the determination of shell weight and thickness. The shell weight was measured by digital balance and thickness was measured without membranes by taking the mean of 3 pieces (from the 2 ends and the middle) using a micrometer.

Statistical analysis

The data obtained were statistically assessed by the Analysis of Variance (ANOVA) through Statistical Analysis System (SAS, 1996) considering replicates as experimental units. Duncan’s multiple range test (Duncan, 1955) was used to test the significance of difference between means by considering the differences significant at p≤0.05.

 

Results and Discussion

The results of chemical analysis of Azolla (Table II) revealed presence of 22.06 % Crude protein (CP), 3.62% Ether extract (EE), 14.3% Crude fiber (CF), 33.4% Nitrogen free extract (NFE), 18.1% Total ash, 2.04% Calcium and 0.65% Phosphorus on dry matter basis. The values of CP content of Azolla estimated are in close concordance with the findings of Alalade and Iyayi (2006) and Raseena (2006). Further, Basak et al. (2002) reported that EE content of Azolla varied between 3.0 to 3.5%, in present study more or less similar value (3.62%) was obtained. The CF content of 14.3% is in agreement with the findings of Querubin et al. (1986). Parthasarathy et al. (2001) reported that NFE content of the fern varies between 38.85 to 44.06%, however, in our study NFE value of only 33.4% was recorded. The total ash content of 18.1% was recorded which corroborates with the findings of Basak et al. (2002) and Alalade and Iyayi (2006). Azolla contains calcium content of 2.11% (Parthasarathy et al., 2001) which is in close conformity with the value recorded in present study. Ali and Leeson (1995) reported 0.31% phosphorus in Azolla but in our study a value of 0.65% phosphorus was recorded. The variations in the nutrient composition of Azolla could be due to species difference and habitat variation of the taxon.

The performance parameters of grower chicken have been shown in Table III. The body weights of grower birds improved non-significantly (p>0.05) in the group fed Azolla (T2) in the diet when compared with the control group, thus confirming the results of Querubin et al. (1986) and Basak et al. (2002) who reported an increase in the body weight of broiler chicken as a result of Azolla supplementation. However, among the other Azolla fed groups, a proportional decrease in the body weights was observed, thus confirming the results of Parthasarthy et al. (2002). Higher Azolla levels (10, 15 and 20%) result in decreased body weight than low levels which might be due to higher level of (Neutral Detergent Fibre) NDF (Buckingham et al., 1978) and lignin (Tamany et al., 1992) in Azolla meal which are the main limiting factors for its efficient utilization. Alalade and Iyayi (2006) attributed poorer growth rate of birds fed high levels of Azolla to the lower feed intake and consequently a reduced metabolizable energy intake. There was no significant (p>0.05) effect on feed consumption between control and 5% Azolla supplemented group (T2); however a significantly lower feed consumption was observed in higher Azolla supplemented groups (10, 15 and 20%) compared to control and T1. Lowest feed consumption was observed in the group fed 20% Azolla in the diet (T5), confirming the results of Alalade and Iyayi (2006) who reported decrease in feed consumption with increase in the level of Azolla in the diet of poultry birds. The decrease in the feed consumption has been attributed to reduced palatability (Bested and Morento, 1985) and increased bulkiness of Azolla (Bacerra et al., 1995) which reduces its utilization. The feed conversion ratio of chicks fed diets

 

Table III.- Effect of Azolla cristata on the growth performance and liner function enzymes of grower chicken (Mean±SE).

Parameters	Treatments
	T1	T2	T3	T4	T5
Body weight gain (9 to 19 wk in g)	847.3±7.35b	869.5±7.58b	830.7±9.24b	825.6±6.33b	799.3±8.76c
Feed consumption (kg)	4.72±2.51c	4.79±1.84c	4.64±1.16b	4.62±3.01b	4.52±2.37a
Feed conversion ratio	5.6	5.5	5.6	5.6	5.7
SGPT(µ/l)	13.4±0.13	13.7±0.09	12.1±0.25	12.8±0.06	13.1±0.17
SGOT(µ/l)	55.9±0.48	53.4±0.23	55.4±0.37	56.0±0.59	52.6±0.33

Means within the same row with different superscripts are significantly different (p<0.05); each value is the mean of 3 replicates. SGPT, alanine aminotransferase; SGOT, aspartate aminotransferase.

 

Table IV.- Effect of Azolla cristata on laying hen performance (33 to 40 week of age) (Mean±SE).

Parameters	Treatments
	T1	T2	T3	T4	T5
No. of eggs laid	62.37±5.93	60.91±7.68	63.16±4.35	61.51±8.14	62.13±3.47
Egg weight (g)	57.19±1.93	58.16±2.37	58.20±3.14	57.54±2.63	57.82±3.06
Hen day egg production (%) (%)	74.82±4.36	72.61±6.53	75.67±4.76	72.55±7.81	73.14±3.69
Feed/bird/day (g)	148.0	148.0	148.0	147.0	147.0
Yolk weight (g)	13.36±0.46	13.57±0.38	13.14±0.41	13.35±0.27	13.52±0.55
Albumen height (mm)	6.37±0.15	6.36±0.09	6.34±0.22	6.38±0.18	6.35±0.11
Albumen width (mm)	62.49±4.54	61.98±5.39	62.74±7.21	61.57±6.08	62.51±5.36
Eggshell weight (g)	6.32±0.24	6.39±0.21	6.38±0.19	6.36±0.20	6.35±0.87
Eggshell thickness (mm)	0.41±0.07	0.43±0.08	0.43±0.01	0.42±0.04	0.43±0.10

Each value is the mean of 3 replicates.

 

wherein groundnut cake was replaced with Azolla showed no significant (p<0.05) effect among various groups including the control. Best FCR was noticed in the group fed 5% Azolla in the diet. Improvement in the FCR as a result of Azolla feeding was also reported by Ardakani et al. (1996) who found better FCR in birds fed low levels of Azolla compared to control. The mean values of serum constituents in chicken fed Azolla supplemented diets are shown in Table III. Supplementation of Azolla showed no significant (p>0.05) difference in the concentration of SGPT and SGOT levels between the chicks fed diets supplemented with Azolla and the control group reflecting that Azolla could be used up to the level of 20% in the diet of layer chicken without causing any adverse effect on the health of birds.

The results on laying performance (33 to 40 week of age) of chicken fed Azolla based diets are shown in Table IV. The differences in the total number of eggs laid, % hen-day-egg production, various external and internal egg parameters under different treatments were statistically non-significant (p>0.05). However, a non-significant (p>0.05) improvement was observed in the egg weight, shell thickness and weight was observed in the groups fed Azolla in the diet. This improvement in the strength of eggs could be attributed to the presence of good amount of calcium in Azolla, as it has been reported that the eggshell consists primarily of mineral matter most of which is calcium carbonate deposited in organic matrix (Austic and Nesheim, 1990). The thickness of the shell affects the quality of the egg which contributes to eggshell breakage (Rodriguez, 2013). Eggshell quality is one of the most important issues in the poultry industry, influencing the economic profitability as high breaking strength of eggshell and absence of shell defects are essential for protection against the penetration of pathogenic bacteria such as Salmonella spp. into eggs (Swiatkiewicz et al., 2010).

In conclusion, dietary inclusion of Azolla cristata at lower levels was beneficial in improving the performance of grower chicken but had no effect on the number of eggs laid in layer phase. Further, Azolla inclusion in the diet showed an improvement in the shell thickness of eggs which could prove beneficial in decreasing the incidence of egg breakage and thereby maximizing profits. The Azolla inclusion in the diet of layer chicken had no abnormal effect on SGPT and SGOT levels indicating that Azolla supplementation had no hepatotoxic effect in layer chicken even up to 20% levels.

 

Acknowledgement

The authors thankfully acknowledge the financial assistance from Department of Science and Technology, Ministry of Science and Technology, Government of India.

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

References

Ahad, B., Reshi, Z.A. and Ganaie, A.H., 2012. Azolla cristata in the Kashmir. Am. Fern J., 3: 224–227. https://doi.org/10.1640/0002-8444-102.3.224

Alalade, O.A. and Iyayi, E.A., 2006. Chemical composition and feeding value of Azolla (Azolla pinnata) meal for egg type chicks. Int. J. Poult. Sci., 5: 137-141. https://doi.org/10.3923/ijps.2006.137.141

Ali, M.A. and Leeson, S., 1995. Azolla biology and agronomic significance. Bot. Rev., 35: 17-35.

AOAC, 1996. Official methods of analysis, 16th edition. Association of Official Analytical Chemists, Washington, USA.

Ara, S. and Adil, S., 2012. Impact of dietary inclusion of Azolla on production performance of broiler chicken. Geobios, 39: 236-240.

Ardakani, M.H., Shivazad, M., Mehdizadeh, S.S.M. and Novrozian. H., 1996. Utilization of Azolla filliculoides in broiler nutrition. J. agric. Sci., 2: 35-46.

Austic, R.E. and Neshiem. M.C., 1990. Poultry production, 13th edition. Lea and Febiger, Philadelphia.

Bacerra, M., Preston, T.R. and Ogle, B., 1995. Effect of replacing whole boiled soya beans with Azolla in the diets of growing ducks. Livest. Res. Rural Develop., 7: 1-11.

Basak, B., Pramanik, A.H., Rahmnan, M.S., Taradar, S.U. and Roy, B.C., 2002. Azolla (Azolla pinnata) as a feed ingredient in broiler ration. Int. J. Poult. Sci., 1: 29-24. https://doi.org/10.3923/ijps.2002.29.34

Becking J.H., 1979. Environmental requirements of Azolla for the use of tropical rice production. In: Nitrogen and rice. International Rice Research Institute, Los Banos, Leguna, Phillipines, pp. 345-374.

Bested, S.B. and Morento, S.E., 1985. The effect of different percentage of Azolla on fattening pigs. J. Mount. State Agric. Coll., 17: 31-40.

BIS, 1992. Poultry feeds specification, 4th revision. Bureau of Indian Standards, New Delhi, pp. 4.

Buckingham, K.W., Stephen, W.E., James, G.M. and Goldman, C.R., 1978. Nutritive value of the nitrogen fixing aquatic fern Azolla filliculoides. J. Agric. Fd. Chem., 26: 1230-1234. https://doi.org/10.1021/jf60219a051

Duncan, D.B., 1955. Multiple range and multiple F-test. Biometrics, 11: 1-42. https://doi.org/10.2307/3001478

Islam, M.S., Aftabuzzaman, M., Howlider, M.A.R., Kabir, G. and Ali, M.A., 2010. Exogenous phytase for better utilization of parboiled rice polish in broiler diet. Int. J. Biores., 5: 1-6.

Haustein, A.T., Gilman, R.H., Skillicom, P.W., Vergara, V., Guevara, V. and Gastanaduy, A., 1990. Duckweed a useful strategy for feeding chickens: Performance of layers fed with sewage-grown Lemnaceas species. Poult. Sci., 69: 1835-1844. https://doi.org/10.3382/ps.0691835

Lumpkin, T.A. and Plucknett, T.L., 1982. Azolla as a green manure: use and management in crop production. Westview Trop. Agric. Ser., 15: 230.

Mishra, D.B., Roy, D., Kumar, V., Bhattacharyya, A., Kumar, M., Kushwaha, R. and Vaswani, S., 2016. Effect of feeding different levels of Azolla pinnata on blood biochemicals, hematology and immunocompetence traits of Chabro chicken. Vet. World, 9: 192-198. https://doi.org/10.14202/vetworld.2016.192-198

Pannaerker, S., 1988. Azolla as a livestock and poultry feed. Livest. Adv., 13: 22-26.

Parthasarathy, R., Kadirvel, R. and Kathaperumal, V., 2001. Chemical evaluation of Azolla as poultry feed ingredient. Cheiron, 30: 24-26.

Parthasarathy, R., Kadirvel, R. and Kathaperumal, V., 2002. Azolla as a partial replacement for fishmeal in broiler rations. Ind. Vet. J., 79: 144-46.

Pirie, N.W., 1980. Water weed uses. Water Spect., 12: 43-49.

Querubin, L.J., Alcantara, P.F. and Luis, E.S., 1986. Chemical composition and feeding value of Azolla in broiler ration. Philipp. J. Vet. Anim. Sci., 13: 46.

Raseena, 2006. Effect of dietary supplementation of Azolla (A. pinnata) on production performance in Japanese quail (Coturnix coturnix japonica). M.V. Sc. Thesis, Kerala Agricultural University, Thrissur.

Rodriguez, C.M., 2013. Performance of chicken layers as affected by calcium supplement. Int. scient. Res. J., 2: 71-77.

SAS, 1996. SAS users guide: Statistics, ver. 7.0. SAS Inst., Inc., Cary, NC, USA.

Singh, P.K. and Subudhi, B.P.R., 1978. Utilization of Azolla in poultry feed. Ind. Farm., 27: 37-39.

Swiatkiewicz, S., Koreleski, J. and Arczewska, A., 2010. Laying performance and eggshell quality in laying hens fed diets supplemented with prebiotics and organic acids. Czech J. Anim. Sci., 7: 294–306.

Tamany, Y., Samanta, G., Chakraborty, N. and Mondal, L., 1992. Nutritive value of Azolla (Azolla pinnata) and its potentiality of feeding in goats. Environ. Ecol., 10: 755-756.