7. Friday Monogastric Sessions dr peter selle university of sydney - phytase changes the hepatic flow of amino acids

1. Phytase impacts on the post-enteral
availability of amino acids
Amy Moss and Peter Selle,
Poultry Research Foundation within the University of Sydney

3. Post-enteral availability
of amino acids
DIGESTION of protein by several endogenous enzymes
ABSORPTION of amino acids and oligopeptides by an array
of Na+-dependent and Na+-independent transport systems
plus PepT-1
TRANSITION of amino acids across enterocytes to enter the
portal circulation or enter anabolic and catabolic pathways

4. The PRF curiosity in the black-
box of the gut mucosa stems
from our interest in
Phytate and phytase
Starch-protein digestive
dynamics
Reduced-crude protein diets
Amino acids
and glucose
Catabolism
for energy provision
Amino acids
Anabolism
Mucin
Digestive enzymes
Gut integrity, NPNs
Net portal ammonia (NH4
+)
outflow accounts for 18% of
dietary intake of amino acid In
weaner pigs
(Stoll et al., 1998)
“Catabolic ratio”

5. Rerat A, Simoes-Nunes C, Mendy F, Vaissade P, Vaugelade P.
Splanchnic fluxes of amino acids after duodenal infusion of carbohydrate solutions containing free amino acids or
oligopeptides in the non-anaesthetized pig. Br J Nutr 1992; 68: 111–38.
Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominates the first-pass
intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J Nutr 1998;
128: 606–14.
van der Meulen J, Bakker JG, Smits B, de Visser H. Effects of source of starch on net portal
flux of glucose, lactate, volatile fatty acids and amino acids in the pig. Br J Nutr 1997; 78:
33–44.
Reeds PJ, Burrin DG, Jahoor F, Wykes L, Henry J, Frazer EM. Enteral glutamate is almost
completely metabolized in first pass by the gastrointestinal tract of infant pigs. Am J Physiol
1996; 270: E413–18.
Peter J Reeds, Douglas G Burrin, Barbara Stoll, Johannes B van
Goudoever (2000) Role of the gut in the amino acid economy of the
host. In: Proteins, Peptides and Amino Acids in Enteral Nutrition
Vol. 3, pp. 25–46.
Peter J Reeds

6. -10
40
90
140
190
240
Arg Ile Leu Lys Met Phe Thr Val Ala Asp GluX Gly Pro Ser Tyr
Portalappearance%Intake
100%
Net portal availability of amino acids in
pigs as a percentage of intake
(Reeds et al. 2000)

7. 0
50
100
150
200
Arg Ile Leu Lys Met Phe Thr Val Ala Asp GluX Gly Pro Ser
Portalappearance%Intake
100%
Gross portal availability of amino acids in
poultry as a percentage of intake
(Truong et al. 2017; Selle et al. 2016)

8. Alanine increased by about 200% in both species
– attributed to alanine synthesis in the gut mucosa from valine and glutamic acid
The net and gross patterns are reasonably similar in both species with two
notable exceptions
Glutamic acid/glutamine – zero in pigs but > 100% in poultry
Glutamate and glutamine are catabolised extensively in the gut mucosa so zero in
pigs is sensible, that this is not the case in poultry may be due to gross portal
values and entry of glutamine into enterocytes from the arterial circulation
Threonine 47% in pigs but 148 - 183% in poultry – a real conundrum?
Collectively, threonine, serine
and glycine comprise 22.3% of
amino acids in feathers
(Stilborn et al. 1997)
Hepatic threonine
dehydrogenase activity?
(Lee et al. 2014)

9. 0
200
400
600
800
1000
1200
1400
1600
Arg His Ile Leu Lys Met* Phe Thr* Val* Cys* Gly Ser Tyr
Systemic plasma threonine levels increased by
87% (1635 vs 879 nmol/mL) with the transition from 18.3% to
15.9% protein corn-soy diets for female broilers at 42 days
Both diets contained 7.2 g/kg threonine (analysed)
[Fancher & Jensen, 1989]

10. Threonine concentrations in plasma taken from
the anterior mesenteric vein were significantly correlated to
both weight gain and FCR (Selle et al. 2016)
R² = 0.8368
1425
1475
1525
1575
1625
40 50 60 70 80
Weightgain(g/bird)
Threonine in portal bloodstream (mg/ml)
R² = 0.5976
1.4
1.45
1.5
1.55
1.6
1.65
40 50 60 70 80
FCR
Threonine in portal bloodstream (mg/ml)

11. Post-enteral availability of amino acids is ultimately
determined by the ‘black-box’ –
the enterocytes of the gut mucosa
Exogenous PHYTASE?
Phytase is generating a ‘proximal shift’ in sites of
amino acid absorption which may spare amino acids
from catabolism in the gut mucosa
Harada and Inagaki (2012)

12. Ca:avail P Nil 1000 FTU/kg Response
1.43
2.14
2.86
3.57
Main effect
0.780
0.723
0.770
0.718
0.748
0.864
0.849
0.824
0.821
0.840
10.8%
17.4%
7.01%
14.3%
12.3%
As a main effect, 1000 FTU/kg phytase increased the ileal
digestibility of 17 amino acids by an average of 12.3%
Amerah et al. (2014)
Quadratic relationship (r2 = 0.736) between phytate degradation
and AA digestibilities
Exogenous phytase enhances digestion
of protein and absorption of amino acids

13. 0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
DI PI DJ PJ
7.2% 9.1% 20.4% 49.5%
Phytase increases apparent digestibility coefficients
of amino acids (n = 16) in four small intestinal segments
Truong et al. (2015)
Truong HH, Bold RM, Liu SY, Selle PH (2015) Standard phytase inclusion in maize-
based broiler diets enhances digestibility coefficients of starch, amino acids and
sodium in four small intestinal segments and digestive dynamics of starch and
protein. Animal Feed Science and Technology 209, 240-248.

14. Phytase ‘retrieves’ Na along the small intestine
with a linear relationship (r = 0.825; P = 0.002) between
digestibility coefficients of Na and protein (16 amino acids) in
the distal ileum.
-3.6
-3.1
-2.6
-2.1
-1.6
-1.1
-0.6
-0.1
DI PI DJ PJ
36.1% 47.8% 34.1% 43.4%

15. Exogenous phytase enhances
digestion of protein by preventing and/or disrupting binary
protein-phytate complex formation at < iP of protein and
absorption of amino acids via Na+-dependent transporters
driven by the ‘sodium pump’ (Na+/K +-ATPase)
Phytate has been shown to depress
Na pump activity in rats and enhance
Na pump activity in broiler chickens
but the underlying mechanisms are
not clear
Na concentrations in enterocytes?
Rephosphorylation of the sodium
pump?

16. The BioPlatforms Project

17. Glutamate/glutamine and glucose are the principal
substrates for energy provision in the avian gut mucosa
(Watford et al., 1979).
The post-enteral availability of amino acids is
determined by their transition across the enterocyte
Catabolism of amino acids within the
gut mucosa is important.
If the ‘catabolic ratio’ within enterocytes can be
manipulated perhaps more amino acids would be
available within the portal circulation.

18. 1) The apparent ileal digestibility coefficients and disappearance
rates (g/bird/day) of amino acids
2) The concentrations of amino acids and glucose within the
portal circulation
Objective
Therefore, the present study will determine the influence of
exogenous phytase inclusions in poultry diets on:

19. Experimental Design
• Four wheat-soybean meal diets with 0, 500, 1000 and 2000 FTU/kg
phytase inclusion (Buttiauxella sp. expressed in Trichoderma reesei;
Axtra® PHY, Danisco Animal Nutrition, Marlborough, UK)
• Diets were formulated to Aviagen recommendations
• The accuracy of the phytase inclusions were confirmed by analysis:
Treatment Analysed phytase activity
(FTU/kg)
Control
500 FTU/kg formulated
1000 FTU/kg formulated
2000 FTU/kg formulated
274
744
1107
2308

20. Method
• Each dietary treatment was offered to eight replicate cages (six birds
per cage) with a total of 192 male Ross 308 chicks from 7 to 28 days
post-hatch
• Experimental data were analysed as a one-way ANOVA and Pearson
correlations were performed using IBM® SPSS® Statistics 20 program
(IBM Corporation. Somers, NY USA)
• At day 28 birds were euthanised and anterior mesenteric vein blood
samples collected and distal ileal digesta samples expressed and
pooled by cage

21. Results
• Overall, birds outperformed 2014 Ross 308
objectives:
• The overall mortality rate of 4.68% was not influenced
by dietary treatment.
Weight Gain
(g/bird)
Feed Intake
(g/bird)
FCR
(g/g)
This Study 1436 2045 1.426
Ross Objective 1387 2052 1.479

22. Growth Performance
• Phytase inclusions significantly increased weight gain
• 500 FTU phytase significantly improved FCR
• Feed intake was not influenced by dietary treatment
b
a
ab ab
1.36
1.38
1.4
1.42
1.44
1.46
1.48
0 500 1000 2000
Feedconversionratio(g/g)
Phytase inclusion (FTU/kg)
a
b
b
b
1320
1340
1360
1380
1400
1420
1440
1460
1480
1500
0 500 1000 2000
Weightgain(g/bird)
Phytase inclusion (FTU/kg)
8.4% 5.1%
Weight gain FCR

23. Ileal AA digestibility coefficients
2000 FTU/kg phytase increased the digestibility in 15 of 16
amino acids by an average of 5.29%
In agreement with Cowieson et al. (2017) who reported an average
improvement of 5% to phytase inclusion across 24 studies
0
2
4
6
8
10
Arg His Ile Leu Lys Met Phe Thr Val Ala Asp Glu Gly Pro Ser Tyr
%Change
% Change from 0 to 2000 FTU phytase
4.8%
6.1%
5.7%
5.1% 5.1%
2.8%
4.3%
7.3%
6.1% 6.3%
7.4%
9.5%
5.5%
4.3%
6.0%

24. Ileal AA disappearance rates
2000 FTU/kg phytase increased disappearance rates in 13 of 16
amino acids by an average of 6.91%
AA disappearance rates correlated to weight gain (r = 0.480; P = 0.005)
0
2
4
6
8
10
12
Arg His Ile Leu Lys Met Phe Thr Val Ala Asp Glu Gly Pro Ser Tyr
%Change
6.9% 7.1%
6.1%
9.1%
8.1% 8.2%
9.4%
11.5%
% Change from 0 to 2000 FTU phytase
6.8%
8.3%
7.8%
7.3%
8.1%

25. Portal blood AA concentration
500 FTU/kg phytase significantly increased portal blood
concentration of 7 amino acids
-20
-10
0
10
20
30
40
Arg His Ile Leu Lys Met Phe Thr Trp Val Ala Asp
+
Asn
Glu
+
Gln
Gly Pro Ser
%Change
% Change from 0 to 500 FTU phytase
18% 18%
40%
14%
28%
13%
18%

26. phytase inclusions reduced glutamic
acid and glutamine concentrations in
the portal circulation
(R2 = 0.132; P = 0.049)
100
150
200
250
300
350
400
450
0 500 1000 1500 2000
Glu+Glnconcentration(µg/mL)
Phytase (FTU/kg)
10
12
14
16
18
20
22
24
26
28
30
0 500 1000 1500 2000
Glucoseconcentration(µg/mL)
Phytase (FTU/kg)
phytase inclusions increased
plasma glucose concentration in
the portal circulation
(R2= 0.127; P = 0.045)
Glutamic acid + Glutamine
concentration in portal blood
Glucose concentration
in portal blood
Glutamic acid/glutamine and glucose
may both be catabolised for energy
Logical to look at them in
tandem

27. 0
5
10
15
20
25
0 500 1000 1500 2000
Ratio(µg/mL/µg/mL)
Phytase inclusion
(R2 = 0.252; P <0.02)
The ratio of
(Glutamic acid + Glutamine)/ Glucose
in portal blood to phytase inclusion
Therefore, it appears that the energy substrate utilised by the intestinal
mucosa is subject to manipulation by phytase inclusion
Does this influence performance?

28. 1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
5 10 15 20 25
Feedconversionratio(g/g)
Ratio (µg/mL / µg/mL)
The ratio of
(Glutamic acid + Glutamine)/ Glucose
in portal blood to FCR
(R2 = 0.374; P < 0.002)
Manipulating the ratio to favour relatively higher glucose concentrations in the
portal blood was significantly related to improvements in FCR

29. • Phytase improved amino acid digestibility and
disappearance rates
• Phytase has the capacity to influence the post-
enteral availability of amino acids
• The likelihood is phytase inclusion contributed to
an improved feed efficiency by manipulating the
energy substrate of the gut mucosa
• Additional studies are required to confirm this
important observation
Conclusions

30. Thank-you!