Replacement of vitamin-mineral premix by Spirulina and its effect on the performance of broiler

The main purpose of this study was to explore the effects of Spirulina (Spirulina platensis) as a feed supplement by replacing Vitamin-Mineral Premix on the performance of broiler. The study explored that final body weight was 1039, 1070, 1044, 1065, 1117 and 893 g/bird in group 1, 2, 3, 4, 5 and 6 where significantly (p<0.5) higher weight observed in Spirulina group (5) but lower in negative control group (6). Feed intake observed more or less similar in all groups (1483±24g; p>0.5). Feed conversion ratio (kg FI/kg LWG) observed significantly different like 1.72, 1.65, 1.69, 1.62, 1.61 and 1.99 in group 1, 2, 3, 4, 5 and 6 respectively (p<0.5) where higher in negative group (6) and control group (1), but is lowest and similar in 75% Spirulina group (4) and highest (100%) Spirulina group (5). %Ash of different dietary groups were 46.35, 46.54, 48.93, 49.93, 55.07 and 46.92 of group 1, 2, 3, 4, 5 and 6 respectively. The %ash of bone was gradually increased among dietary groups and the best result was obtained in 100% Spirulina group 5 (55.07). It also revealed that diets containing no vitamin-mineral premix with 100% Spirulina improve the performance of broiler. The findings suggest that poultry farmer can use spirulina as a feed item for broiler for more yield.


Introduction
The blue-green algae (Spirulina platensis) is one of the nutritional algae which has been used as a food source for human and animal.The protein content of spirulina is ranging from 55-65% with essential amino acids [1,2].It provides good energy ranges from 2.5-3.29 kcal/gram with 41% phosphorous [1,3].It also is being used as a feed component for broiler and layer diets since it is a good source of some vitamins like vitamin B12, thiamin, pyridoxine, vitamin C, riboflavin, and carotenoids which enhance yolk color and flesh [2].About 30% of the total production of algae is being used as animal feed production [1].Some studies have already been done by some researcher regarding suitability of algae as an animal and poultry feed [1,[5][6][7][8][9][10].Moreover, the broiler is a very popular poultry item in Bangladesh [11][12][13] and other countries which supplied a major portion of meat demand.The villagers of Bangladesh are rearing broiler by establishing the small firm of the home yard [14,15] and earn money which helps to alleviate poverty [15][16][17][18] and ensuring food security [18][19][20].Many people of the remote village take broiler poultry farming as a profession [2].Not only that broiler meat act as a main meat source in the urban area as well as rural areas [3].But few types of research have been done on an issue of replacement of by Spirulina and its effect on the performance of broiler.The aim of this research was to explore the effects of Spirulina (Spirulina platensis) as a feed supplement by replacing Vitamin-Mineral Premix on the performance of broiler.

Materials and methods
The experiment was conducted in the Shahjalal Animal Nutrition Field Laboratory of the Department of Animal Nutrition, Bangladesh Agricultural University (BAU), Bangladesh.

Collection of Spirulina platensis, stock culture and Kosaric Medium (KM)
Microalgae, Spirulina platensis was collected from the imported stock which was maintained at Animal Nutrition Laboratory, Department of Animal Nutrition, Bangladesh Agricultural University, Mymensingh.The pure stock culture of Spirulina platensis was maintained in the laboratory in Kosaric medium (KM) [4].Kosaric media (KM) was prepared for the culture of S. platensis.Different concentrations of nutrients of the kosaric medium are shown in Table 1.For the preparation of Kosaric Medium, the mentioned amount of ingredients from 1-8 was weight with the help of electric balance and taken in a 5.0 L glass jar [1].The 5ml micronutrient solution was added with the help of pipette in the flask and distilled water was added to make the volume 5.0L, mixing autoclaving (sterilized at 121 0 C for 15 minutes with moist heat) and cooling was carried out and kept it for culture.

Statement of research work and Source of chicks
The experiment was conducted in the poultry rearing unit of Shahjalal Animal Nutrition Field Laboratory, Bangladesh Agricultural University, Mymensingh for a period of 28 days by using 180 DOC (Day old chick) to find out the effect of Spirulina algae as a replacement of vitamin-mineral premix on the performance of broiler.Day-old straight run broiler chicks (COBB 500) were reared for the experiment.The chicks were produced and marketed by Kazi Farm Ltd.

Experimental design and layout
The experiment was conducted in a completely randomized design (CRD).One hundred and eighty chicks (DOC) were randomly divided into six treatment groups having three replicates in each (ten birds in each replication).The six dietary treatment groups were:

Formulation of experimental diets
The ingredients were ground individually using a grinding machine.After weighing, a part of the required amount of ground wheat, soybean meal, rice polish, oyster shell, meat and bone meal and fish meal were mixed thoroughly.Soybean oil was mixed with this mixture step by step.Then the salt, Di-calcium phosphate, Choline Chloride, Lysine, DL-Methionine were added except Vitamin-Mineral Premix and Spirulina [8].Group 1 was controlled and no Spirulina was used.In group 2, 3, 4 and 5 Spirulina was added at replacing the amount of vitamin-mineral premix and mixed homogenously, then sun-dried.In group 6, there was neither vitamin-mineral premix nor Spirulina in the diet considered as the negative control diet [26].It was done by mixing all those premixes with a small amount of mixed feed and then with the gradual increase in quantity by adding the remaining mixed feed [9].The control diet consists of wheat, soybean meal, soybean oil, rice polish, fish meal, common salt, Choline chloride, Dicalcium phosphate, Vitamin-mineral premix, meat and bone meal, Lysine, and DLmethionine [10].Spirulina was calculated on DM basis added minerals in the media.Calculation (in kg) is given below: Where, Control diet vitamin-mineral = 2.5 g/kg; Media mineral = 12.12 g/kg;12.12g mineral in 1 kg media; 0.6g mineral in = (0.6/12.12)=0.05kg=50g (ml) media 1.Control  The ingredients which are used for ration formulation in different dietary treatments are shown in Table 3.

General management of birds and Preparation of house
One hundred and eighty Cobb-500 commercial day-old broiler chicks were reared in this experiment having an average weight of 50.30g/b, divided into six groups, one control group and five treatment groups with 30 chicks per group.The chicks were collected from Kazi Farms Ltd.Mymensingh, Bangladesh by grading, sexing and weighing individually to maintain homogeneity of experimental units.Eighteen cages were considered for this trial each having floor space of 0.91 m 2 (120 cm × 76 cm).
The management house and cages were done as described previously [5].Wall and wire nets were thoroughly disinfected by spraying Vircons-S solution (Antec International Limited, England) in water and kept free to dry up properly.

Chick's management
Freshly dried rice husk was spread on the floor under the cages at a depth of 4 cm.After 3 weeks of age, the upper layer of the litter with droppings was removed and replaced by new litter material.The management was done by following standard methodology [7,12].

Vaccination and Clinical observation
The broilers were vaccinated against Newcastle Disease and Infectious Bursal Disease (Gumboro).Birds from three replicate cages from each treatment were separately vaccinated against ND at the 7 th day of age, Gumboro disease vaccine at 14 th day of age and no vaccine at all respectively.The vaccine which followed during the experimental period is given Table 5.

Record keeping and collection of data
Broilers were weighted in a group at the beginning of the trial and then every week at the age of day 7, 14, 21 and 28.Weighing was done using electric balance before supplying feed at the morning of each week by following standard method [7] and as described by Dadgar et al. [1].

Bone ash% content
Bone ash% was determined from the shank bone of the birds.At day 28, slaughtering the birds and cut the shank of them from randomly selected birds of each replication.After collection of shank bones; those were boiled for 1 hour and then skinning them [13], washed properly and air dried; then weighed the crucibles and shank bones and dried in the oven at 105 0 C for 24 hours.After 24 hours of drying, the oven dry weight of bones was taken; then kept the bones in the muffle furnace at 600 0 C for 5 hours and then calculated the DM% and %ash by the following the formula: W1= Oven dry wt.-Crucible wt.DM% = %Ash =

Blood analysis
Blood analysis was done by following standard methods as explained previously [7].

Economic analysis
The cost of broiler production for each treatment was calculated based on the cost of feed ingredients.Cost for management and other costs are not considered as those are same for all groups.

Chemical analysis
Samples feed ingredients (in duplicate) ware analyzed to determine dry matter (DM), crude protein (CP) and total ash (Ash) following the method described by AOAC [7].

Statistical analysis
All data were analyzed by using statistical SPSS program for one way analysis of variance (ANOVA) and Duncans Multiple Range Test.

Performance of Broilers
The results of growth performance (body weight, feed consumption and feed conversion ratio), mortality, % ash of bone and cholesterol & triglyceride status in blood of broilers are presented in this chapter.

Live weight
The live weight of broilers of different dietary groups at different ages is shown in Table 8.During day 8-14, 15-21 and 22-28 of ages, the live weights of birds are not significantly different (p>0.05) in 1, 2, 3, 4 & 5 dietary treatment groups but have significantly difference (p<0.05) with group 6.At the end of the trial, group 5 attained 1117g live weight, which is higher than negative control (6), and almost similar to group 2 (1070g), group 4 (1065g), group 3 (1044g) and group 1 (1039g).In this experiment, the negative control group was used for observing the effect of no vitaminmineral premix in broiler diet.Neither vitamin-mineral premix nor Spirulina in the diet, birds of the negative control group (6) was lame and unable to move.Their feed intake was irregular; so, their live weight and live weight gain were low at the end of the trial.The result showed that, dietary Spirulina can compensate the performance of broiler by eliminating vitamin-mineral premix level in the diet.The probable reason may be the Spirulina in broiler diets could improve better qualities which give better performance and recover the deficiency of vitamin-mineral premix [29,10].Other studies that replaced groundnut cake [14] or fishmeal [15] with Spirulina in chicken diets found no variation in growth.On the other hand, the negative control group (6) had no vitamin-mineral premix or Spirulina in the diet.As a result, the dietary Spirulina group shows a higher significant difference (p<0.05) from the negative control group in the live weight of broilers [16].The other groups of birds 1, 2, 3 and 4 had no significant differences (p>0.05)among them.But significantly different (p<0.05) from group 5 to group 6.

Feed intake
The weekly feed intake of broilers receiving different diets is presented in Table 10.Feed intake of the bird under different groups during day 8-14, 15-21 and 22-28 of age differed significantly (p<0.05) from each other but feed of group 5 (447g) was highest at day 15-21 of age.At the end of the trial group, 2 (779g) resulted in significantly higher feed intake than other groups.Cumulative feed intake was highest in group 5 (1537g) and lowest in group 4 (1464g) at the day 8-28 of age.Relative to 1 100 97 96 97 100 97 N.B: 1-Control (100% Premix + 0% Spirulina); 2-75% Premix + 25 % Spirulina; 3-50% Premix + 50% Spirulina; 4-25% Premix + 75% Spirulina; 5-0% Premix + 100% Spirulina ; 6-0% Premix + 0% Spirulina *Mean ± SD; abc Means with dissimilar superscripts are significantly different (p<0.05);NS-Not Significant In the present study, feed intake of broilers in different groups was significantly increased.This is might be due to in case of low energy because feed intake was more to recover the energy requirements.

Feed Conversion Ratio (FCR)
The feed conversion ratio (FCR) of broilers receiving different dietary treatments is shown in Table 11.The feed conversion ratio (FCR) of broilers were not significant (p>0.05) at day 8-14.At day 15-21, FCR of group 1, 2, 3, 4 and 5 were not significantly different (p>0.05)among them but had a significant difference (p<0.05) with group 6 [18].Again, there was no significant difference (p>0.05) of FCR in any group at day 22-28.
The highest feed conversion ratio was observed in all groups (p<0.05) at day 22-28.At the end of trial numerically highest FCR was attained in group 6 (1.99).Cumulative FCR was significantly different at day 8-21 and 8-28.Best feed conversion ratio was observed in group 6 (1.99) compared to control (1.72), group 2 (1.65), group 3 (1.69), group 4 (1.61) and group 5 (1.62).As mentioned above, the feed conversion ratio is related to feed intake and live weight gain in all groups of birds [16].In group 1, 2, 3, 4 and 5, birds received the proper nutrients available in their diet.So, the feed intake of these groups is good and the FCR was not significantly different (p>0.05).But in group 6, birds were deficient in their needed vitamins and minerals, so their feed intake was more to recover the nutrient requirements [8].So that, their FCR was higher than another group though there was no significant difference among them (p>0.05).

Bone Ash (%) content
After the experiment, the shank bone from every group of the bird was taken for determining %ash of the bones.In this experiment, the %ash of bone is gradually increased in group 1 (46.35), 2 (46.54), 3 (48.93), 4 (49.93) and 5 (55.07).But in group 6, there is an average percentage of ash (46.92) in the bone because of deficiency of vitamin and mineral in their diet.Ash% is higher in group 5 (55.07) and lower in group 1 (46.35).So, it can be said that with increasing the level of Spirulina in broiler diet, ash% was increased and the best result was obtained when supply the 100% Spirulina [4].

Blood analysis
The plasma of birds from different dietary groups showed that they had significant differences (p<0.05)among them.Blood cholesterol was higher (42) in group 5 and triglycerides level was higher (128) in group 1 in this experiment.Other groups of birds from group 2, 3, 4 were not shown any significant differences (p>0.05)among them.But group 1, 5 and 6 had significant differences among them [5].Cholesterol level was gradually increased from group 1(36) to group 5 (42) and lower in group 6 (26).In case of triglycerides, the highest level (128) in group 1 and lowest level (49) in group 6.There were no significant differences (p>0.05) between group 2 and 3.There were great significant differences (p<0.05)among the group 1, 4, 5 and 6 [7].
From Table 12, it can be shown that both the level of cholesterol and triglycerides of blood were lower in group 6; 26 and 49 respectively.So, it could be assumed that Spirulina can provide an adequate amount of cholesterol and triglycerides in the blood of the birds in the Spirulina treated groups [19].

Mortality and Economic analysis of using Spirulina
No bird belongs to any group died during 28 days of feeding trial.Production cost was calculated by considering the cost of feed [19].The cost per kilogram feed was lowest in group 5 and 6 than control.Production cost per kilogram live weight of broiler was lowest (P<0.05) in group 5 (100% Spirulina) followed by group 4 (99.81), 3 (99.88), 2 (99.94) and control (100).Chicken health can improve with low dietary Spirulina levels of 10 g/kg in the ration, indicating greater production cost efficiency [20].
From the above discussion, it is clear that the diet which contains no vitamin-mineral premix might be compensated by the addition of 100% Spirulina as an efficient alternative organic source of vitamin-mineral for the supplementation in broiler diet to produce natural organic products [21,23,24,25,].Moreover, Spirulina enhances to increase feed intake without toxicity [26,27,28,29,30].Further research is needed for the confirmation about of economic and feasibility to recommend in diets of different poultry species [31][32][33].

Conclusion
It may be concluded that diets containing no vitamin-mineral premix with 100% Spirulina improve the performance of broiler.Further research is needed for the confirmation of economic and feasibility to recommend in diets of poultry.

Figure 2 .
Figure 2. Live weight gain (LWG) of birds at different age.

Figure 3 .
Figure 3. Cumulative weight gains of birds at different age.

Figure 4 .
Figure 4. Feed intake of birds at different age.

Figure 5 .
Figure 5. Cumulative feed intake of birds at different age.

Figure 6 .
Figure 6.FCR of birds at different age.

Figure 7 .
Figure 7. Cumulative FCR of birds at different age.

Figure 9 .
Figure 9. Production cost per kg live weight gain.

Table 2 .
The layout of the experiment (Level of premix + Spirulina).

Table 3 .
Formulation of diet (kg/100kg) in different dietary groups.

Table 4 .
Nutrient composition (g/100 kg air dry sample) of different treatment groups

Table 5 .
Vaccination schedule followed for the experimental broilers

Table 8 .
Weight (g/bird) of birds at different age.

Table 9 .
Live weight gain of birds at different age.

Table 10 .
Feed intake of birds at different age.

Table 11 .
FCR of birds at different age.