BRIOLERS: THE EFFECT OF PAPAYA LEAF MEAL (PLM) | CHAPTER 3-4-5



CHAPTER THREE
MATERIALS AND METHODS
3.1       Experimental Animals:
           The animals used for this experiment were  90 day old Anak 2000 chicks, which were obtained from a reputable dealer in Abakaliki.

3.2       Experimental Site
            The experiment was carried out at Poultry Unit of the Teaching  and  Research farm,  Department of Animal Science, Ebonyi State University Abakaliki, Nigeria.  The analyses were done at Department of Medical  Labouratory Technology and Research Institute, Ebonyi State University Abakaliki, Nigeria.


3.3       Experimental Diet.
            Papaya leaves were harvested from around the compounds and villages around Abakaliki town. They were dried at room temperature then crushed to a finer state. The diet was formulated, using locally sourced materials that were cheap and affordable. Table 1 shows the percentage composition of the diet.

Table 1: The percentage composition of the experimental starter diets (%).

TRT 1
TRT 2
TRT 3
TRT 4
TRT 5
Maize
50
50
50
50
50
Wheat offals
6
8
6
5
8
SBM
10
10
10
10
10
GNC
19
16
14
10
18
PKC
6
4
3
3
7
PLM
0
5
10
15
0
FISH
3
3
3
3
3
LIMESTONE
3
0
0
0
0
BONE MEAL
2
3
3
3
3
PRE MIX
0.25
0.25
0.25
0.25
0.25
SALT
0.25
0.25
0.25
0.25
0.25
LYSINE
0.25
0.25
0.25
0.25
0.25
METHIONIME
0.25
0.25
0.25
0.25
0.25
ORGACID
-
-
-
-
22g
TOTAL
100
100
100
100
100
ME(kg)
12.2
12.53
12.79
12.97
12.2
% CPV
22.12
22.15
22.2
22
21.7
% CF
4.2
4.3
4.2
4.2
4.4

TABLE 2: FINISHER MASH
TRT 1
TRT 1
TRT 2
TRT 3
TRT 4
TRT 5
Maize
52
52
52
52
52

Wheat Offals
8
8
8
5
8

SBM
6
6
6
6
6

GNC
15
14
12
12
15

PKC
12
8
5
3
12

PLM
0
5
10
15
0

Fish
3
3
3
3
3

PLM
0
0
0
0
0

Limestone
0
0
0
0
0

Bone meal
3
3
3
3
3

Premix
0.25
0.25
0.25
0.25
0.25

Salt
0.25
0.25
0.25
0.25
0.25

Lysine
0.25
0.25
0.25
0.25
0.25

Methionine
0.25
0.25
0.25
0.25
0.25

Orgacid @ 1.5 kg/ton


45g

Total
100
100
100
100
100

ME (kJ)
12.25
12.60
12.65
13.26
12.31

% CPV
19.69
20.35
18.20
21.15
19.72

% CF
4.16
4.12
3.32
3.75
4.25


3.         Experimental Design
            The birds were randomly allocated to five experimental diets namely; T1(control), T2(5% PLM), T3(10% PLM), T4(15% PLM), and T5( 22g or orgacid).

3.4       Management of the Animals.
            The poultry unit was cleared of cobwebs, swept and disinfected using diazintol (Diazinon dimphlate) and formaldehyde. The pen was allowed to rest for 2 days. On arrival of the birds they were randomly assigned to their replicates according to treatments used.
 The cleaning of the house was repeated at regular intervals till the end of the experiment. Water and feed troughs were also cleaned on daily basis.

Table 3: The proximate analysis of the diets
Starter diets

%CP
%CF
ME(kg)
EE
T1
22.29
4.25
12.16
3.64
T2
22.32
4.31
12.51
3.72
T3
22.37
4.36
12.62
3.79
T4
22.20
4.21
12.98
3.88
T5
22.31
4.25
12.16
3.64

Table 4 Finisher diets

%CP
%CF
ME(kg)
EE
T1
19.69
4.16
12.25
3.67
T2
20.35
4.12
12.60
3.79
T3
18.20
3.32
12.65
3.85
T4
21.15
3.75
13.26
3.90
T5
19.72
4.25
12.31
4.25

3.6       Sample Collection
            Faecal droppings were collected on the Day 1  of the trial and weekly thereafter till the last week of the experiment.
At the 8th week 15 birds, one per replicate were slaughtered and the small intestines and caecum  taken for microbial  count. The small intestine and caecum were preserved on ice  in a cold flask till it reached the laboratory.

3.7 Materials Used in Culture
            The media used were Eosinmethylene blueagar (EMB) and Salmonella – Shigella agar (SSA). Eosin methylene blue Agar (EMB) is used for the primary isolation of, and for differentiating between enterobacteria. The media contains (W/V), peptone (1%), lactose and sucrose (each at 0.5%), or lactose only at (1%), K2HPO4 (0.2%), eosin Y (0.04%), methylene blue (0.0065%), agar (91.5%).
            Salmonella –Shigella Agar (SSA) contains Agar (13.5 g/l), lactose (10.0g/l), bite salts (8.5g/l), Na2S2O3 (8.5g/l), sodium citrate (8.5g/l), beef extract (5.0g/l), pancreatic digest of casein (2.5g/l), peptic digest of animal tissue (2.5g/l).
            All media were prepared according to manufacturer’s instructions and were poured into a sterile culture plate and allowed to gel.
Faecal (weekly) droppings and the intestines and caecum from each representative repricate (bird) were used.
3.8 Stages of the culture process.
3.8.1. Macroscopy: This is the physical examination of the stool sample to ascertain is colour and consistency.
Colour – whitish /blackish
Consistency – formed.
3.8.2. Preparation of Media
The media were prepared according to the manufacturer’s instructions.
3.8.4 Inoculation
      The last diluent was inoculated using 0.1 ml from the last test tube and inoculated using the media for the experiment and the content was incubated for 24 hours.
3.8.5 Colony Count
            The colonies were counted and recorded, using light microscope to view the stained slides and data recorded for each replicates.
3.9 Intestine and Caecum Content Culture.
            0.5g each of digest from the small intestinal or caecum  was collected and underwent the same procedure as  in the faecal culture. The results were recorded also.
  3.10 Data Analysis.
            The weekly faecal samples and digesta from small intestinal and caecum  were subjected to One-Way Analysis of Variance (ANOVA). Where differences existed,their means were separated using Fishers Least Significant Difference (F-LSD) (One tail test) (Snedecor and cochran, 1980).
The linear additive model for CRD experiment (Obi, 2002) is as follows:
Xij = μ + ∑I + ∑ij
Where Xij            = individual observation
μ                            = population or overall mean
∑I                          = treatment effect
∑ij                         = experimental error of residual

CHAPTER FOUR
RESULT
4.1 THE MEAN COUNT (cfu/g) of Escherichia coli of BROILER DROPPINGS
The Result of The Mean Count (cfu/g) of Escherichia coli  of Broiler Droppings as presented in table 5.

TABLE 5:     Mean Count (cfu/g) 0f Escherichia coli of  broiler
droppings.
Escherichia Coli
T1
T2
T3
T4
T5
Week 1
6.4 x 105
7.2x 105
5.3 x 104
3.1 x 104
3.1 x 104
Week 2
4.2 x 104
3.7 x 104
6.4 x 104
5.6 x 104
1.6 x 104
Week 3
1.0 x 105
6.7 x 104
8.8 x 104
5.7 x 104
3.7 x 104
Week 4
9.3 x 105
3.2 x 104
6.7 x 104
4.4 x 104
3.1 x 104
Week 5
8.3 x 105
6.7 x 104
7.8 x 104
8.8 x 104
1.6 x 104
Week 6
1.0 x 105
6.2 x 104
4.1 x 104
4.1 x 104
1.0 x 104
Week 8
1.1 x 105
5.2 x 104
2.5 x 104
2.5 x 104
9.3 x 103
Mean
1.5 x 105
1.3 x 105
5.2 x 104
4.3 x 104
1.9 x 104
Mean count  SEM*   4.9 x 104      4.7 x 109 cfu/g. There were no significant different (p>0.05) among the treatment


The population of Escherichia coli is highest on T2 (7.2 x 105 cfu/g) and least on the T4 (3.1 X 104cfu/g) and T5 ( 3.1 x 104cfu/g) at week 1. Week 8 had the highest population on T1 (1.5 x 105cfu/g) and least on T5 ( 1.9 x 104cfu/g).
   
4.2       THE MEAN COUNT (cfu/g) of Salmonella enteritidis of BROILER DROPPINGS.

The Result of The Mean Count (cfu/g) of Salmonella enteritidis of Broiler Droppings.

TABLE 6: Mean Count (cfu/g) of Salmonella enteritidis of  Broiler  
Droppings.
Salmonella enteritidis
T1
T2
T3
T4
T5
Week 1
6.4 x 105
3.4x 104
2.9 x 104
4.4 x 104
2.9 x 104
Week 2
7.0 x 104
1.9 x 104
3.7 x 104
6.3 x 104
6.5 x 104
Week 3
2.7 x 104
2.5 x 104
3.1 x 104
2.0 x 104
6.8 x 104
Week 4
3.6 x 104
1.0 x 104
2.9 x 104
1.2 x 104
4.3 x 104
Week 5
6.4 x 104
2.2 x 104
1.5 x 104
2.5 x 104
1.2 x 104
Week 6
7.2 x 104
2.0 x 104
1.6 x 104
1.8 x 104
9.3 x 103
Week 8
7.8 x 104
1.5 x 104
9.3 x 103
1.3 x 104
1.0 x 104
Mean
1.1 x 105
1.8 x 104
2.1 x 104
2.4 x 104
2.1 x 104
Mean count  SEM*   2.4 x 104      6.7 x 108 cfulg. There were no significant different (p>0.05).

The population of Salmonella enteritidis  is highest on T1 ( 6.4 X 105cfu/g) and least on T3 and T4 ( 2.9 X 104cfu/g) at week 1.
4.3       THE MEAN COUNT (cfu/g) OF Shigella of BROILERS
DROPPINGS.
      The Result of The mean count (cfu/g) of Shigella of Broiler Droppings.
TABLE 7: Mean Count (cfu/g) of Shigella of  Broilers Droppings.
Shigella
T1
T2
T3
T4
T5
Week 1
8.3 x 104
3.1x 104
6.2 x 104
3.1 x 104
6.7 x 104
Week 2
1.3 x 105
8.8 x 104
9.9 x 104
7.2 x 104
6.2 x 104
Week 3
2.0 x 104
3.1 x 104
2.0 x 104
4.1 x 104
2.0 x 104
Week 4
3.6 x 104
3.1 x 104
2.0 x 104
3.1 x 104
2.0 x 104
Week 5
3.8 x 104
1.7 x 104
3.1 x 104
6.2 x 104
4.1 x 104
Week 6
9.3 x 104
1.7 x 104
2.0 x 104
3.1 x 104
2.4 x 103
Week 8
9.9 x 104
1.5 x 104
1.3 x 104
2.0 x 104
1.2 x 104
Mean
6.2 x 104
2.9 x 104
3.3 x 104
3.6 x 104
3.1 x 104
Mean count  SEM*   2.4 x 104      7.3 x 108 cfulg. There were no significant (p>0.05) different among the means.


The population of Shigella  is highest on T1 ( 8.3 X 104cfu/g) and least on T2 ( 3.1 X 104cfu/g)
4.4       THE MEAN COUNT (cfu/g) of Lactobacillus spp of BROILER
DROPPINGS.

 The Result of The Mean of COUNT (cfu/g) of Lactobacillus spp of Broilers Droppings.

TABLE 8: Mean Count (cfu/g) of Lactobacillus spp of  broilers
droppings.
lactobacillus spp
T1
T2
T3
T4
T5
Week 1
6.7 x 104
7.8x 104
1.0 x 105
9.3 x 104
1.0 x 105
Week 2
8.2 x 105
1.3 x 105
1.2 x 105
1.2 x 105
1.3 x 105
Week 3
8.2 x 104
2.1 x 105
2.1 x 105
2.6 x 105
1.3 x 105
Week 4
8.6 x 104
1.6 x 105
2.1 x 105
2.6 x 105
2.5 x 105
Week 5
7.9 x 104
2.1 x 105
2.5 x 105
2.5 x 105
2.6 x 105
Week 6
8.9 x 104
2.0 x 105
2.1 x 105
2.5 x 105
2.0 x 104
Week 8
9.3 x 104
1.9 x 105
2.3 x 105
1.6 x 105
1.6 x 105
Mean
7.2 x 104
1.5 x 105
1.7 x 105
1.7 x 105
1.3 x 105
Mean count  SEM*   8.6 x 104      8.7 x 108 cfulg. There were no significant (p>0.05) different at all treatments.

The mean count (cfu/g) of Lactobacillus spp of broilers droppings were found to be no significant at (p>0.05) for all treatments considered. The mean count (cfu/g) of Lactobacillus spp of broilers droppings showed the highest count at T3 (10% PLM) and T4 (15% PLM) respectively. While the least mean count of Lactobacillus spp showed at T1(control).

4.5 THE MEAN COUNT (cfu/g0 of Staphylococcus aureus  of BROILER DROPPINGS.

The Result of The Mean Count (cfu/g) of Staphylococcus aureus Broiler Droppings.

TABLE 9: Mean Count (cfu/g) of Staphylococous aureus of  broilers
        droppings.
Staphylococous aureus
T1
T2
T3
T4
T5
Week 1
5.2 x 104
4.9 x 104
1.2 x 105
5.0 x 104
8.3 x 104
Week 2
6.2 x 104
4.2 x 104
3.0 x 104
3.2 x 104
7.4 x 104
Week 3
7.4 x 104
1.0 x 104
1.3 x 105
1.6 x 105
1.2 x 105
Week 4
7.8 x 104
1.6 x 105
1.3 x 105
1.8 x 105
2.1 x 105
Week 5
8.4 x 104
1.6 x 105
2.1 x 105
1.7 x 105
1.7 x 105
Week 6
9.2 x 104
1.6 x 105
1.8 x 105
1.8 x 105
1.6 x 104
Week 8
8.8 x 104
1.6 x 105
2.4 x 105
1.4 x 105
1.9 x 105
Mean
6.6 x 104
9.3 x 104
1.3 x 105
1.1 x 105
1.3 x 105
Mean count  SEM*   6.6 x 104      6.3 x 109 cfulg. There were significant different (p>0.05) at the means.

The mean count (cfu/g) of Staphylococcus aureus of broilers droppings was found to be significant different at (p>0.05) for the means considered. The mean count showed the highest value at T3(10% PLM) and T5 (22g of orgacid) of  Staphylococcus aureus  of broiler droppings. The least mean count was showed at T1(control) and T2 (5% PLM).  


4.6 THE MEAN COUNT (cfu/g) of Lactobacillus spp and THE ENTEROPATHOGENS in THE DIGESTA of BROILERS CAECUM and SMALL INTESTINE at WEEK 8 of AGE.

The Result of Lactobacillus spp  and The Enteropathogens Mean Counts in The Digesta of Broilers Caecum and Small Intestine at Week 8 of Age.

TABLE 10: Mean Count (cfu/g) of Lactobacillus spp and the enteropathogens in the digesta of broilers caecum and small intestine at week 8 of age.
Caecum microbes
T1
T2
T3
T4
T5
Esotherichia coli
1.1 x 105
5.2 x 104
2.0 x 104
4.6 x 104
4.1 x 104
Salmmomella enteristidis
4.1 x 104
4.6 x 104
3.1 x 104
3.6 x 104
4.1 x 104
Shigella
7.8 x 104
3.6 x 104
2.5 x 104
3.1 x 104
3.1 x 104
Staphylococcus aureus
8.8 x 104
1.8 x 105
2.0 x 105
1.6 x 105
1.5 x 105
Lactobacillus spp
8.8 x 104
2.0 x 105
2.1 x 105
2.1 x 105
1.9 x 105
SEM
5.7 x 104
9.2 x 105
9.8 x 104
8.8 x 104
7.9 x 105
Small intestine microbes





Escherichia coli
7.8 x 104
3.6 x 104
3.8 x 104
2.3 x 104
3.1 x 104
Salmonella enteritidis
3.6 x 104
3.6 x 104
1.5 x 104
2.5 x 104
2.5 x 104
Shigella
5.7 x 104
2.5 x 104
2.5 x 104
2.0 x 104
2.5 x 104
Staphylococcus aureus
3.6 x 104
7.2 x 104
9.3 x 104
1.0 x 105
6.2 x 104
Lactobacillus spp.
4.6 x 104
9.9 x 104
2.1 x 105
1.4 x 105
1.1 x 105
There is significant different (p<0.05)

The mean count of Lactobacillus spp and the enteropathogens in the digesta of broilers caecum showed the highest mean count at T3( 10% PLM) and T4 (15% PLM) respectively. While the least mean count of Lactobacillus showed at T1 (control). The enteropathogens in the digesta showed that at caecum the highest value were recorded at T3(10%PLM) showed by Staphylococcus aureus. While the least count of enteropathogens were recorded at T3 (10% PLM)  showed by Escherichia coli.
The mean count of Lactobacillus spp and enteropathogens in the digesta of broilers small intestine showed the highest mean count at T3(10% PLM). While the least mean count of  Lactobacillus spp showed at T1(control). The enteropathogens in the digesta showed that at small intestine the highest value were recorded at T4(15% PLM) showed by Staphlococcus aureus . While the least count of enteropathogens in the small intestine were recorded at T3(10% PLM) showed by  Salmonella enteritidis followed by T4(15% PLM) showed by Shigella.  

CHAPTER FIVE
5.0                                                                                                             DISCUSSION
In table 5, Escherichia coli in broilers droppings were found to be no significant different (p>0.05). The mean count (cfu/g) of Escherichia coli of broilers droppings showed the highest mean value at T1(control)( 1.5x 105cfu/g) followed by T2 (5% PLM) (1.3 x 105cfu/g). While those on T5(orgacid) showed the least mean count (1.9 x 104cfu/g). This could be as result of the effect of papaya leaf meal (PLM) which may be resistant to Escherichia coli. This is in line with Mohanery and Mahzorieh (2005) stated a decrease in Escheichia coli population in the intestines of broiler chicken with malic acid.
The mean count (cfu/g) of Salmonella enteritidis of broiler droppings  was found to be no significant (p>0.05) different. The result showed the highest mean count of Salmonella enteritidis at T1(control) (1.1 x 105cfu/g) followed by T4 (15% PLM) (2.4 x 104+cfu/g). Showed the least mean count at T2 (5% PLM) (1.8 x 104cfu/g) of Salmonella enteritidis in broiler droppings. Which is agreement with (Krishna et al., 2008) which states that  herbal containing papaya leaf meal (PLM) or roots as one of the constituents has antibacterial activity against Salmonella tyhii, Salmonella enteritidis, etc.
The mean count (cfu/g) of Shigella in broiler droppings were found to be no significant (p>0.05) different. The result showed that at T1(control) ( 6.2 x 104cfu/g) has the highest value followed by T4(15% PLM) (3.6 x 104cfu/g) of  Shigella in broiler droppings. While the least count was seen at T2 (5% PLM)(2.9 X 104cfu/g) of Shigella in broiler droppings. This agreed with (Nwofia et al., 2002) which said that purified extracts from matured papaya leaf also produces very significant antibacterial activity on Staphlococcus aureus, Bacillus cereus, Escherichia coli and Shigella flexneri.
In table 8, the result reviewed the mean count (cfu/g) of Lactobacillus spp in broiler droppings. It showed that at T3(10% PLM) (1.7 X 105cfu/g)  and T4(15% PLM) (1.7 X 105cfu/g) had the highest mean count of Lactobacillus spp in broiler droppings. While the least mean count was seen at T1(control)( 7.2 x 104cfu/g) of Lactobacillus spp in broiler droppings. This agreed with  Amit-Romach et al., (2004) that the predominant population of Lactobacillus spp as a result of probiotic effect . This showed thath there were increase in Lactobacillus species more at T3(10% PLM) and (T4 (15% PLM) as a result of the effect of papaya leaf meal (PLM) fed to the birds.
The result showed on table 9 reviewed that the mean count (cfu/g) of Staphylococcus aureus of broilers droppings were found to be significant different (p<0.05). The mean count showed the highest value of T3(10% PLM)(1.3 X 105cfu/g) and T5(orgacid)(1.3 x 105cfu/g) respectively of staphylococcus aureus in broilers droppings. The least mean count showed at T1(control) (6.6 x 104cfu/g) and T2(5% PLM) (9.3 X 104cfu/g). This is also in agreement with Nwofia et al, (2002) purified extracts from matured papaya leaf also produces very significant antibacterial activity on Staphylococcus aureus, Bacillus cereus, Escherichia coli.
   The mean count of Lactobacillus spp and the enteropathogens in the digesta of broilers caecum showed the highest mean count at T3(10% PLM) (2.1 x 105cfu/g) and T4 (15% PLM) (2.1 x  105cfu/g) respectively. While the least mean count of Lactobacillus spp  in broiler droppings was seen at T1(control) (8.8 x 104cfu/g). The enteropathogens in the digesta showed that at caecum the highest value was recorded at T3(10% PLM) (2.0 x 105cfu/g) showed by Staphylococcus aurues. While the least count of enteropathogens was showed by  Escherichia coli at T3(10% PLM) (2.0 x104cfu/g). These result corroborate with soumaya et al, (2011) and Barnes  et al, (1972) that in the caecum the effect of lactic acid increased thus decreasing the pathogenic baterial in the caecum.
 The mean count of  Lactobacillus spp and the enteropathogens in the digesta of broilers small intestine showed that there was an increased at T3(10% PLM) (2.1 x 105cfu/g). Showing a least mean count of Lactobacillus spp  in the digesta of broilers at T1( control) (2.0 x 104cfu/g). The enteropathogens in the highest value were recorded at T4 (15% PLM) ( 1.0 x 105cfu/g) showed by Staphylococcus aureus. While the least mean count recorded at T3(10% PLM) (1.5 x 104cfu/g) showed by Salmonella enteritidis  followed by T4(15% PLM) (2.0 x 104cfu/g) showed by Shigella. This corroborated with Brzokal  et al,( 2007) microbiology analyses of the small intestine digesta showed that lactic acid bacteria increased the intestinal counts of Lactobacillus spp  compared with control group and with antibiotic group.                                                                                

5.1       Conclusion
            The research showed that in the digesta of broilers  the mean count of  Lactobacillus spp increased significantly  (P < 0.05) with both T3 and T4 having the highest mean count of  1.7 x 105 cfu/g.The population of Lactobacillus spp  was observed to increase in T4(15% PLM) at week 4 having a mean count of 1.7 x 104 cfu/g; and that of T5( orgacid) at week 5 having a mean count of 2.6 x 105 .
            Therefore from all indication T3 (10% PLM) had the best lactobacillus spp count. It is also observed that T3 and T4 had the lowest mean counts of enteropathogens examined. Therefore if at T3 (10% PLM) and T4 (15% PLM) that it does not have any physiological implication on the broilers then in summary, T3 (10% papaya leaf meal) and T4 (15% papaya leaf meal (PLM) is the best level of inclusion.

5. 2      Recommendation
            It is therefore recommended that at T3 (10% PLM) and T4 (15% PLM)  has the best microbicidal property. It can be use since the lowest mean count of bacteria was found in them and also the population of lactobacillus spp  increased significantly in the same inclusion levels.

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Serial Dilution
Procedure:
·        Five test tube of equal measurement was set on a test tube rack.
·        5 mls of normal saline solution was added in the first test tube, that is, the stock and 4 mls of normal saline was added in the subsequent four test tubes.
·        0.5g of the sample and latter digesta from   measured with automatic measuring device.
·        The  5 mls of the normal saline was added to the sample and was thoroughly mixed.
·        1 ml of the solution from the stock was transferred to the next test tube that contain 4 mls of normal saline solution.
·        The 2nd test tube was shaken thoroughly 2 mins  and  then 1 ml was collected from it and added to the 3rd test tube.
·        The  mixture in the 3rd test tube was thoroughly shaken for 2 mins then 1ml was collected from it and placed in the 4th test tube. The process was repeated also in the fourth and fifth test tubes.
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