Effects of Minazel Plus on Aflatoxin B1 and Ochratoxin A mycotoxicosis in broiler chickens

EFFECTS OF MINAZEL PLUS ON AFLATOXIN B1 AND OCHRATOXIN A MYCOTOXICOSIS IN BROILER CHICKENS

Jog Raj1*, Panagiotis Tassis2, Vasilios Tsiouris2 and Marko Vasiljević1 1Patent

Co, DOO., Misicevo, Serbia of Veterinary Medicine, Aristotle University of Thessaloniki,

2School

Greece. *jog.raj@patent-co.com

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The aim of this study was to document the efficacy of mycotoxin absorbent Minazel® Plus (MP) (modified clinoptilolite) on broiler chickens ingesting feed containing AFB1 and OTA from the 1st day up to 42nd day of age, registering alterations in health status and performance parameters.

The trial was performed in the Unit of Avian Medicine experimental unit, at the Farm Animals Clinic, of the School of Veterinary Medicine of the Aristotle University of Thessaloniki, Greece in 2019. The duration of the experiment was 42 days.

The treatment groups were:

T1: negative control group fed with control feed.

T2: Positive control group receiving control feed contaminated with 0.1 ppm AFB1 and 1 ppm OTA. T3: Group receiving the T2 group feed (0.1 ppm AFB1 and 1 ppm OTA) with the addition of Minazel® Plus (1 kg/ton of feed).

T4: Group receiving the T2 group feed (0.1 ppm AFB1 and 1 ppm OTA) with the addition of Minazel® Plus (3 kg/ton of feed).

On the 25th and 42nd day of age 4 and 6 birds, respectively, with 4 replicates (from each experimental group) per sampling day were blood sampled and removed.

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The following parameters were calculated: body weight (BW), feed intake (ADFI), average daily weight gain (ADWG), feed conversion ratio (FCR), as well as adjusted AFCR that was calculated based on a standard reference weight of 2,500g.

Furthermore, mortality and morbidity were investigated, as well as blood biochemical parameters tests, and gross lesions scoring of the intestine, gizzard and liver were performed.

Additionally, investigation of gut microbiota was also performed.

SAMPLES

PARAMETERS ANALYSED

T1 Production parameters • Body weight (BW) • Feed intake (ADFI) • Average Daily Weight Gain (ADWG) • Feed Conversion Ratio (FCR) • Adjusted AFCR

Control Feed 25 days

T2 Contaminated feed (0,1 ppm AFB1 and 1 ppm OTA)

T3 Contaminated feed (0,1 ppm AFB1 and 1 ppm OTA) + Minazel® Plus (1 kg/ton of feed)

4 birds from each group

Mortality & Morbidity Blood Blood biochemical parameters

42 days Gross lesions scoring (intestine, gizzard and liver)

T4 Contaminated feed (0,1 ppm AFB1 and 1 ppm OTA) + Minazel® Plus (3 kg/ton of feed)

6 birds from each group

Blood

Gut microbiota

RESULTS The data presented in Table 1 showed the better FCR for Minazel® Plus treated groups.

Absence of ADWG improvement was recorded for the total trial period.

A significant improvement of AFCR was observed in the T4 group when compared with T2 group, whilst comparison of AFCR between T3 and T2 was only marginally not significant.

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Table 1. Performance Parameters. Values expressed as means (standard deviation in parentheses).

Group T1 (Feed)

Group T2 (Feed+0.1 ppm AFB1+1 ppm OTA)

Group T3 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 1 kg/ton)

Group T4 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 3 kg/ton)

BW (g) day 1

38,4 (2,55)

38,83 (2,7)

38,98 (2,4)

38,4 (2,55)

BW (g) day 7

131,28 (10,07)a

131,83 (8,5)a

128,2 (7,52)a#

132,78 (8,06)a#

BW (g) day 15

384,1 (40,35)a#

370,25 (27,15)a#

371,46 (39,42)a#

382,73 (31,19) a#

BW (g) day 25

1086,68 (101,05)a

1002,8 (74,49)b

1069,35 (120,56)a

1007,85 (106,1)b

BW (g) day 32

1762,71 (125,49)

1610,13 (112,1)

1637,33 (173,28)

1638,21 (156,21)

BW (g) day 42

2605,25 (217,44)

2439,58 (198,68)

2429,67 (256,49)

2467,67 (230)

ADWG (g) 1-25

41,93(1,46)a

38,56 (1,30)b

41,22 (3,52)ab#

38,78 (1,69)a#b

ADWG (g) 25-42

89,33 (1,28)a#

84,52 (0,94)b

80,02 (5,49)c

86,46 (2,35)a#b

ADWG (g) 1-42

61,12 (1,24)a

57,16 (0,99)b

56,92 (0,52)b

57,84 (0,77)b

FCR 1-25

1,619 (0,042)

1,724 (0,039)

1,627 (0,13)

1,721 (0,052)

FCR 25-42

1,234 (0,081)

1,396 (0,04)

1,270 (0,092)

1,214 (0,082)

FCR 1-42

1,411 (0,026)

1,502 (0,04)

1,429 (0,04)

1,424 (0,024)

AFCR

1,390 (0,018)a

1,514 (0,048)b

1,443 (0,037)ab

1,431 (0,027)a

# Means with such superscript in the same row suggest trend towards statistical significance (0.05<p<0.1) a,b Means with different superscripts in the same row differ significantly (p<0.05) P Values BW day 7 (Treatment X Time interaction observed): T3VsT4=0.060 P Values BW day 15 (Treatment X Time interaction observed): T1VsT2=0.068; T1VsT3=0.077; T2VsT4=0.078; T3VsT4=0.089 P Values BW day 25 (Treatment X Time interaction observed): T1VsT2<0.001; T1VsT4<0.001; T2VsT3=0.001; T3VsT4=0.002.

P Values BW day 32 and day 42 (Absence of Treatment X Time interaction observed): T1VsT2=0.002; T1VsT3=0.003; T1VsT4=0.010 Absence of BW improvement for the total trial period, only group3 on the 25th day showed the greatest value at that time point.

P Values ADWG days 1 - 25 (Treatment X Time interaction observed): T1VsT2=0.041; T1VsT4=0.055; T2VsT3=0.10 P Values ADWG days 25 - 42 (Treatment X Time interaction observed): T1VsT2=0.005; T1VsT3<0.001; T1VsT4<0.08; T2VsT3=0.008; T3VsT4<0.001 P Values ADWG days 1 - 42 (Treatment X Time interaction observed): T1VsT2=0.018; T1VsT3=0.012; T1VsT4=0.047 Absence of ADWG improvement for the total trial period FCR statistics suggested absence of treatment X time interaction (therefore no superscripts can be used).

Thus, an overall effect for the whole time period is suggested with the following significant P Values T1VsT2<0.001; T2VsT3=0.001; T2VsT4=0.003. Better FCR for both Minazel® Plus-treated groups

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The data in Table 2 showed a possible liver-protective effect of the test product in the T4 group against mycotoxin-induced liver injury as suggested by differences observed in total proteins and ALT levels.

1st Blood Sampling Table 2. Performance Parameters. Values expressed as means (standard deviation in parentheses).

Group T1 (Feed)

Group T2 (Feed+0.1 ppm AFB1+1 ppm OTA)

Group T3 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 1 kg/ton)

Group T4 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 3 kg/ton)

Total Proteins

2,48 (0,26)a#

3 2,39 (0,28)a

2,45 (0,27)a#

2,21 (0,42)a#

Albumins*

1,14 (0,14)

1,06 (0,14)

1.11 (0,15)

0,97 (0,24)

ALP*

384,1 (40,35)a#

370,25 (27,15)a#

371,46 (39,42)a#

382,73 (31,19) a#

ALT

1086,68 (101,05)a

1002,8 (74,49)b

1069,35 (120,56)a

1007,85 (106,1)b

AST

1762,71 (125,49)

1610,13 (112,1)

1637,33 (173,28)

1638,21 (156,21)

2605,25 (217,44)

2439,58 (198,68)

2429,67 (256,49)

2467,67 (230)

Glucose*

41,93(1,46)a

38,56 (1,30)b

41,22 (3,52)ab#

38,78 (1,69)a#b

Cholesterol

89,33 (1,28)a#

84,52 (0,94)b

80,02 (5,49)c

86,46 (2,35)a#b

-gt*

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2nd Blood Sampling

Group T1 (Feed)

Group T2 (Feed+0.1 ppm AFB1+1 ppm OTA)

Total Proteins

2,96 (0,69)a#b

2,64 (0,71)a#

2,7 (0,44)a

3,16 (0,95)b

Albumins*

1,4 (0,3)

1,25 (0,23)

1,23 (0,17)

1,35 (0,38)

ALP*

1727 (568,42)

1761,44 (637,72)

1973 (463,89)

1761,44 (637,72)

ALT

5,19 (1,76)a

5,8 (1,61)a#

8,25 (2,93)a#

4,69 (1,7)b

AST

126,25 (13.82)a

229,6 (39,01) b

235,25 (34,69)b

219,13 (41,49)b

7,56 (2,16)

8,27 (2,4)

8,31 (3,05)

9,88 (5,03)

Glucose*

202,13 (24,25)

207,73 (30,93)

208,88 (17,03)

204,81 (25,74)

Cholesterol

112,13 (17,96)

113 (13,76)

112,31 (10,93)

114,63 (20,76)

-gt*

Group T3 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 1 kg/ton)

Group T4 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 3 kg/ton)

*Comparison of mean values of Albumins, glucose, A:P -GT are presented without superscripts as differences for both time periods due to absence of Treatment X Time interaction in the statistical evaluation. Statistically significant differences and trends towards P Values Albumins at both blood samplings (Absence of Treatment X Time interaction observed): T1Vs.T4=0.021 P Values Glucose at both blood samplings (Absence of Treatment X Time interaction observed): T2Vs.T3=0.070 P Values ALP at both blood samplings (Absence of Treatment X Time interaction observed): T1Vs.T3=0.048; T3Vs.T4=0.010; P Values -GT at both blood samplings (Absence of Treatment X Time interaction observed): T1Vs.T4=0.065; Absence of Treatment X Time interaction observed in cholesterol mean values comparisons among groups a,b Means with different superscripts in the same row differ significantly (p<0.05) and Treatment X Time interaction at the specific parameter statistical evaluation was observed P Values Total Proteins at 1st blood sampling (Treatment X Time interaction observed): T1VsT4=0.053; T3VsT4=0.076.

P Values Total Proteins at 2nd blood sampling (Treatment X Time interaction observed): T1VsT2=0.074; T2VsT4=0.012; T3VsT4=0.043 P Values ALT at 1st blood sampling (Treatment X Time interaction observed): T1Vs.T4=0.003; T2Vs.T4=0.011; T3Vs.T4<0.001 P Values ALT at 1st blood sampling (Treatment X Time interaction observed): T1Vs. T4=0.001; T2Vs.T3=0.077; T2Vs.T4=0.028; T3Vs.T4<0.001 P Values AST at 1st blood sampling (Treatment X Time interaction observed): All comparisons among groups with different superscripts<0.001 P Values AST at 2nd blood sampling (Treatment X Time interaction observed): T1 Vs all groups<0.001

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A significant increase in pH in duodenum and a lower pH at the jejunum was observed in the T3 group.

This reduction of the pH may inhibit the growth of acid-sensitive bacteria, such as Salmonella spp., Campylobacter spp. and Clostridium perfringens.

Table 3. Macroscopic lesions and intestinal findings. Values expressed as means (standard deviation in parentheses).

Group T1 (Feed)

Group T2 (Feed+0.1 ppm AFB1+1 ppm OTA)

Group T3 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 1 kg/ton)

Group T4 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 3 kg/ton)

Intestinal lesions

1,44 (0,82)a#

1,63 (0,71)a

1,95 (0,9)a#

1,8 (0,72)a

Gizzard lesions

0,23 (0,42)

0,18 (0,5)

0,08 (0,27)

0,05 (0,22)

Liver lesions

0,1 (0,3)

0,15 (0,43)

0,28 (0,45)

0,23 (0,42)

pH_Duodenum

5,88 (0,53)b

5,95 (0,55)a

6,08 (0,2)c

6,01 (0,2)ac

pH_Jejunum

5,89 (0,17)b

6,08 (0,15)a

5,91 (0,2)b

5,98 (0,11)ab

pH_Ileum

6,39 (0,65)a

5,84 (0,88)ab

5,71 (0,56)ab#

5,68 (0,85)b

pH_Caeca

6,01 (0,36)a

5,97 (0,32)a#

6,29 (0,41)a#

6 (0,26)a

Viscosity_Jejunum

1,39 (0,46)

1,57 (0,8)

1,45 (0,4)

1,11 (0,25)

Viscosity_Ileum

1,47 (0,56)

1,58 (0,64)

1,48 (0,3)

1,22 (0,35)

# Means with such superscript in the same row suggest trend towards statistical significance (0.05<p<0.1). a,b Means with different superscripts in the same row differ significantly (p<0.05). pH Duodenum T1VsT2=0.006; T1VsT3<0.001; T1VsT4=0.018; T2VsT3=0.018. pH Jejunum T1VsT2<0.001; T2VsT3=0.042

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The data in Table 4 shows the reduction of E. coli counts in Minazel® Plus treated groups.

Table 4 Microbiological analysis of caecal content at the end of the study period. Bacterial counts are expressed as base-10 logarithm colony-forming units per gram of caecal digesta (n=16/treatment group).

Values expressed as means (standard deviation in parentheses).

Group T1 (Feed)

Group T2 (Feed+0.1 ppm AFB1+1 ppm OTA)

Group T3 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 1 kg/ton)

Group T4 (Feed+0.1 ppm AFB1+1 ppm OTA+ MP 3 kg/ton)

Caecal weight (g)

8,36 (2,54)

9,1 (3,07)

8,29 (2,81)

10,76 (5,28)

E. coli counts

4,58 (0,87)ab

5,44 (0,94)b

4,07 (1,12)a

4,92 (1,22)ab

Clostridium spp. counts

4,47 (0,81)

4,36 (0,74)

4,01 (0,85)

3,91 (0,98)

Lactobacillus spp. counts

6,36 (0,66)

6,47 (0,62)

6,30 (0,59)

6,47 (0,56)

Bifidobacterium spp. counts

5,34 (0,62)

4,91 (0,95)

5,10 (0,86)

5,11 (0,79)

# Means with such superscript in the same row suggest trend towards statistical significance (0.05<p<0.1). a,b Means with different superscripts in the same row differ significantly (p<0.05).

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CONCLUSIONS

Better FCR for both Minazel® Plus treated groups at 1 and 3 kg/ton as compared to mycotoxin-fed group T2.

Significantly increased protein levels and reduced ALT mean levels that could be associated with improved liver function in group T4.

Significantly increased pH in Duodenum and lower pH at the Jejunum in the 3rd group that could assist on the inhibition of growth of acid-sensitive bacteria, such as Salmonella spp., Campylobacter spp. and Clostridium perfringens.

Reduction of E. coli and counts in Minazel® Plus treated groups.

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