Starter diets for broilers - taking into consideration the immaturity of the gut microbiome Richard Ducatelle
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Starter diets for broilers
taking into consideration the immaturity of the gut microbiome
Richard Ducatelle
Department of Pathology, Bacteriology and Avian Medicine
CHALLENGES:
1. Reduce antibiotic use in livestock production – decrease antimicrobial resistance
Antibiotics in Poultry – EU Antibiotics in Poultry - US
Ban of growth promoters in • Retailers are driving the reduction in antibiotic use.
Animal feed since 01-01-2006 –McDonalds: 2017 – only chicken raised without medically
important antibiotics.
–Chick-fil-A: 2019 – No antibiotics ever.
–Subway: 2016–No antibiotics ever
2
Celine De Maesschalck, University Ghent 3
Earth worm
Microbes have shaped the development of the GI-tract
106 CFU/g
Co-evolution
6
Clostridium perfringens is the most succesful bacterial pathogen in this planet
Viability (dashed line) and culturability (full line) of Butyricicoccus pullicaecorum after 2h at pH = 2
(Geirnaert et al., 2014)
The viable but non-culturable state
Initial colonization of the caeca
(Leurs et al., unpublished)
Trace minerals
Effect of zinc speciation on broiler performance in the starter phase
Starter diet Grower diet Finisher diet
Ingredient (%)
Feed formula:
Wheat 49.29 55.62 59.00
Rye 5.00 5.00 5.00
Soybean meal (48) 29.37 23.16 20.11
Soybeans 7.50 7.50 7.50
Rapeseed meal 2.00 2.00 2.00
Animal fat 2.50 2.60 2.70
Soy oil 1.00 1.00 1.00
Vitamin + trace (vitamix)§ 1.000 1.000 1.000
CaCO3 0.820 0.908 0.826
Di-Ca-phosphate 0.650 0.361 0.107
NaCl 0.264 0.226 0.268
Na-bicarbonate 0.104 0.157 0.101
L-Lys-HCl 0.160 0.175 0.154 + 50 ppm Zn-sulphate
DL-Methonine 0.256 0.208 0.167 or
L-threonine 0.071 0.064 0.049 + 50 ppm ZnAA-chelate
Phytase 0.020 0.020 0.020
(Degrande et al., 2019)Effects of Zinc source on digestibility
ZnS ZnAA P-value
Parameters
Feed intake (FI) (g) 1256 ± 49 1168 ± 90 0.059
Wet excreta (WE) (g) 1797 ± 72 1720 ± 146 0.271
WE/FI* 0.300 ± 0.02 0.304 ± 0.01 0.731
Digestibility coefficients (%)
Gross energy 72.0 ± 2.3 71.8 ± 1.7 0.878
Crude protein 58.2 ± 2.9 57.1 ± 2.2 0.512
Crude fat 78.8 ± 2.5 77.0 ± 4.3 0.423
Zinc 32.7 ± 1.3 36.4 ± 2.7 0.020
(Degrande et al., 2019)Effect of zinc speciation on broiler performance in the starter phase
Period BW BWG FI FCR
(g/animal) (g/animal/day (g/animal/
) day)
0-10 days ZnSO4 284.7 ± 5.6 24.4 ± 0.5 28.6 ± 0.9 1.172 ± 0.018
Zn-AA 290.5 ± 5.8 25.0 ± 0.5 28.7 ± 0.9 1.149 ± 0.012*
(Degrande et al., 2019)Duodenum ZnS ZnAA
Day 10
Villus length (VL) 1206 ± 221 1308 ± 155
*
Crypt depth (CD)) 314.5 ± 76.6 301.6 ± 62.1
Ratio (VL:CD) 3.99 ± 1.05 4.52 ± 1.04
*
Thickness T. muscularis 94.8 ± 30.4 91.3 ± 21.8
(Degrande et al., 2019)(Degrande et al., unpublished)
Metabolomics: negative
ionization mode
Pathway identificiation Pathway Hits.total Hits.sig Gamma
total adjusted Pathway identification Pathway Hits.total Hits.sig Gamma
p-value total adjusted
Amino sugar and nucleotide sugar 88 10 7 0.0005 p-value
metabolism
Pentose phosphate pathway 32 18 6 0.0042
Galactose metabolism 41 11 7 0.0005
Terpenoid backbone biosynthesis 33 3 2 0.0083
Valine, leucine & isoleucine 40 5 3 0.0023
degradation Vitamin B6 metabolism 32 8 3 0.0093
Starch and sucrose metabolism 50 5 3 0.0023 Cysteine and methionine metabolism 56 13 4 0.0098
Glycolysis or Gluconeogenesis 31 6 3 0.0037 Tryptophan metabolism 79 9 3 0.0124
Valine, leucine & isoleucine 27 10 4 0.0037 Taurine and hypotaurine metabolism 20 5 2 0.0188
biosynthesis
Histidine metabolism 44 7 2 0.0339
Phenylalanine,tyrosine & 27 11 4 0.0053
tryptophan biosynthesis
Pyrimidine metabolism 60 14 3 0.0389
Pantothenate and CoA 27 16 5 0.0068 Valine, leucine and isoleucine 27 8 2 0.0429
biosynthesis biosynthesis
Aminoacyl-tRNA biosynthesis 75 21 6 0.0081 Glycolysis or Gluconeogenesis 31 15 3 0.0463
Glycerophospholipid metabolism 39 8 3 0.0086 Nicotinate and nicotinamide 44 9 2 0.0527
Pentose phosphate pathway 32 10 3 0.0175 metabolism
Sphingolipid metabolism 25 5 2 0.0187 Pantothenate and CoA biosynthesis 27 9 2 0.0527
Sulfur metabolism 18 5 2 0.0187
Alanine, asparate and glutamate 24 10 2 0.0631
Fructose and mannose 48 16 4 0.0233
metabolism
metabolism
Inositol phosphate metabolism 39 10 2 0.0631
Methane metabolism 34 6 2 0.0275
Lysine degradation 47 17 4 0.0294 Pentose and glucuronate 53 12 2 0.0852
Propanoate metabolism 35 7 2 0.0380 interconversions
Glycine, serine & threonine 48 19 4 0.0442 Glycine, serine and threonine 48 12 2 0.0852
metabolism metabolism
Cysteine and methionine 56 22 4 0.0721 Phenylalanine, tyrosine and tryptophan 27 13 2 0.0967
metabolism biosynthesis
Glutathione metabolism 38 10 2 0.0771 Pyruvate metabolism 32 13 2 0.0967
Vitamin B6 metabolism 32 11 2 0.0918
(Degrande et al., unpublished)Essential oils Terpenes and terpenoids are the primary constituents of the essential oils of many types of medicinal plants. Terpenes are the major components of rosin. Rosin is a solid form of resin obtained from pine trees. It mainly consists of various resin acids, especially abietic acid.
Effect of resin acids on broiler performance in 13 institutional trials.
* *
* *
*
* * *
*
* *
(Vuorenmaa et al., 2019)In vivo
• Ross 308 broilers
• Control diet vs challenge diet
• + 0.2% resins
• Effect on intestinal microbiota and activity of host collagenases
In vitro
• Cell culture-based model of A549 cells, which express high levels of
matrix metalloproteinase-7 (MMP-7) was used
(Aguirre et al., 2019)In vivo:
DAY 1-14
*
1.4
***
FCR
1.3
Avg FCR
1.2
1.1
1.0
et
et
2
AC
di
di
is
R
al
os
m
+
or
et
bi
/N
di
ys
ol
is
D
tr
os
on
bi
C
ys
D
(Aguirre et al., 2019)In-feed resin acids prevent dysbiosis
diet-induced changes in duodenal villi
Villus height CD3 area
2500
Dysbiosis diet 20 *
* Dysbiosis diet
Control/Normal diet *
** Control/Normal diet
Villus height (µm)
2000 Dysbiosis diet + RAC-250
15 Dysbiosis diet + RAC-250
CD3 area (%)
1500 10
1000 5
500 0
et
et
et
50
et
50
di
di
di
di
-2
-2
is
AC
al
is
AC
al
os
m
os
m
R
R
or
bi
or
bi
+
+
/N
ys
/N
ys
et
et
ol
D
ol
di
D
di
tr
tr
on
is
on
is
os
C
os
C
bi
bi
ys
ys
D
D
Dysbiosis diet: shorter villi, more infiltration of CD3+ T-cells, and loss of performance (weight
gain)
Resins acids prevent dysbiosis diet-induced changes in duodenal villi (Aguirre et al., 2019)Zymography
Inhibition of mmp-7 expression in ileal tissue
Ileum MMP7 gene expression
1.4
1.2
1
0.8
0.6
0.4
*
0.2
0
Control Progres 3000
Partially g/tn
purified FOR100
pure 264.9
resin g/tn250 g/tn
acids
Resins 3 kg/tn
MMP7
Pre-pro-MMP7: 30 kDa (incl. signal peptide)
Pro-MMP7: 28 kDa (without signal peptide)
Mature MMP7: 18 kDa
(Aguirre et al., 2019)Resin acids break down collagen types I and IV in the ileum of broilers
Ileum
FIGURE 1. Breakdown of gelatin (A) and collagen types I and IV (B and C, respectively) in the ileum of broilers
with or without in-feed RAC (N=10, *p < 0.05, *p< 0.01)
(Aguire et al., 2019)Matrix metalloproteinases and wound repair
Martins et al., 2012
• Wound healing requires the
controlled activity of MMPs at
all stages of the wound healing
process
• The loss of MMP regulation is a
characteristic of chronic
wounds and contributes to the
failure to healWound healing assays • CaCo-2 cells • Collagen-coated plates • Scratch induction: woundmaker • Washed 2x with PBS • The pH of the cell medium was measured beforehand. • 48h-72h measurement • One image per 2 hours • Repeated 3 times
Resin acids: cell scratch wound healing
Wound healing assay
0 hrs 12 hrs 24 hrs
Resin acids (FOR-100 ultra)
120 Control
Resin acids (0,05%) ns
100 *** ***
wound extension %
80
0 hrs 12 hrs 24 hrs 60
40
20
Control
0
rs
s
s
hr
hr
H
12
24
0
0.01% resin acids promoted cell migration in a wound healing assayFiber
Cellulose Starter: day 1-13
▪ 310 male Ross 308 broiler
chicks
▪ Standard feed formula
▪ + 0.5% Cellulose (amorphous)
▪ 5h darkness
▪ Body weight
▪ Feed conversion ratio
De Maesschalck et al., 2019Day 10:
390 1.5
1.48
380
1.46
1.44
Body weight (g)
370
1.42
FCR
360 1.4
1.38
350
1.36
340 1.34
1.32
330 1.3
control cellulose control cellulose
De Maesschalck et al., 2019• Caecum:
De Maesschalck et al., 2019Alistipes genus
*Belongs to the phylum Bacteroidetes and is part of the chicken core microbiome
*Obligate strict anaerobe
(Pfleiderer et al., 2014)
*Gram-negative, non-spore-forming, non-motile
(Shkoporov et al., 2015)
*Contains a powerful cellulase and is bile-resistant
(Pfleiderer et al., 2014)
*Main metabolic end product is succinate
(Shkoporov et al., 2015)Effect of cellulose on growth of Alistipes in nutrient-poor medium
A. B.
p = 0.0095
growth (log1 0 cfu Alistipes /ml)
8
Succinate concentration (mM)
1.5
** p = 0.019
*
7 1.0
6 0.5
5 0.0
Blank Cellulose Blank CelluloseFate of succinate production in the caeca
*SUCNR1 = GPR91 = succinate receptor
(Ariza et al., 2012)
*Expressed on the surface of epithelial cells of small and large intestine in the mouse
(Diehl et al., 2016)Microbiota-Produced Succinate Improves Glucose Homeostasis via Intestinal Gluconeogenesis Filipe De Vadder, Petia Kovatcheva-Datchary, Carine Zitoun, Adeline Duchampt, Fredrik Bäckhed, Gilles Mithieux Cell Metabolism, Volume 24, Issue 1, 2016, 151–157
Conclusions *The starter phase is characterized by successive waves of different microbial families colonizing the caeca *The caecal lumen gradually becomes anaerobic between 1 and 10 days post hatch *Nutrional tools may help establishing a healthy ecosystem in the gut *Nutritional tools acting through stimulation of certain microbial families depend on the presence of these microbes *Some nutritional tools act through direct supporting of epithelial cell metabolism *Other nutritional tools act through preventing oxidative stress of the epithelial cells *Other nutritional tools again act through damage repair of the intestinal mucosa *Few nutritional tools act through supporting antibacterial defense mechanisms at the site of nutrient absorption
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