The Role of Protein and Amino Acid Nutrition in ... - Adisseo.biz

THE ROLE OF PROTEIN AND AMINO
ACID NUTRITION ON REPRODUCTION
OF DAIRY COWS
José Eduardo P. Santos and Charles Staples
Department of Animal Sciences
University of Florida

Protein and Reproduction
• Negative association between urea N
concentration and pregnancy in dairy cows when
urea N is excessive
• Studies evaluating the effects of protein on
embryo quality and pregnancy usually fed cows
diets with excessive concentrations of true
protein or urea

Influence of Dietary Ratio of DIP to UIP on MUN
MUN, mg/100 ml
20
15
10
5
5.6
a
11.6
b
13.4
c
14.4
c
17.8
d
JDS 76:525
0
80:80 100:100 120:80 100:120 120:120
Intake of DIP & UIP as a % of NRC

Relationship of Bulk Tank MUN and Days to First AI
Days to first AI
140
120
100
80
60
40
20
0
Live. Prod. Sci. 37:91
85
78 78
86
103
128
10 12 14 16 18 20
Milk Urea N, mg/100 ml

Overfeeding Protein delays 1st Ovulation
First ovulation, days
60
50
40
30
20
10
0
**
25
39
** 50 * P = 0.10
34
22 26 17
22
Florida Utah Maine Maine
*
Control
Excess DIP/CP
** P = 0.05
*

Overfeeding Protein Delays Conception
Days open
120
100
80
60
40
20
0
**
96
106
* P = 0.10
** P < 0.06
** *
72
82
Control
Excess CP
71 80
Oregon '79 Maine '88 Maine '96

Effect of Dietary CP% on Fertility
Concep/Preg Rate, %
100
90
80
70
60
50
40
30
20
10
0
Oregon
*
Israel
Israel
*
OK
Maine
Israel
*
13-17% CP diets
19-21% CP diets
NY
*
NY
* *
Maine
LA

Dietary CP% on Blood Urea N
Reference Animal No. 13-17% CP 19-21% CP
- - - - BUN, mg% - - - -
Oregon, 1979
Israel, 1981
Israel, 1983
OK, 1987
Maine, 1988
Israel, 1989
NY, 1990
NY, 1993
Maine, 1996
LA, 1999
30
39
250
109
57
139
65
80
64
119
--
9
9
15
10
25
12
14
9
20
18
15
17
25
24
32
19
24
21
25
Average 14 22

MUN or PUN and Pregnancy per AI in Dairy Cows
Adapted from Butler et al. J. Anim. Sci. (1996) and Ferguson et al. J. Dairy Sci. (1993)
Pregnancy/AI, %
55
50
45
40
35
49.2
320/650
41.6
119/286
< 19 mg/dL
> 19 mg/dL
30

MUN and Predicted Pregnancy at 1 st AI in
Lactating Dairy Cows
Negative association only in the first
postpartum AI
No effect on subsequent AI
Guo et al. J. Dairy Sci. 87:1878–1885 (2004)

Potential Mechanisms for Reduced
Fertility in Lactating Dairy Cows

Stages of the Reproductive Process Important for
Establishment of Pregnancy in Cattle
42 d 42 d
Follicular development and oocyte growth
Oocyte maturation
Fertilization
Preimplantation development
IFN-τ
x
Prevention of Luteolysis
PGF-2α

Effect of Dietary Protein on PUN and Uterine
pH on d 7 of the Estrous Cycle of Heifers
P < 0.05 P < 0.05
25
7.2
20
7.1
mg/dl
15
10
pH
7.0
6.9
6.8
5
6.7
0
PUN, mg/dl
6.6
Uterine pH
Balanced High RUP High RDP
Balanced High RUP High RDP
Elrod et al. (1993)

Effect of Dietary Protein on PUN and
Conception Rate (CR) in Heifers
mg/dl
25
20
15
10
5
P < 0.05 P < 0.05
%
90
80
70
60
50
40
0
Peak PUN
30
CR
Balanced
High RDP
Balanced
High RDP
Elrod e Butler (1993)

Effect of Urea/Saline Infusion on Uterine
pH on d 7-8 of the Estrous Cycle
7.4
Uterine pH
PUN
30
Uterine pH
7.3
7.2
7.1
7.0
6.9
6.8
6.7
25
20
15
10
PUN, mg/dL
6.6
6.5
5
6.4
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Saline
Urea
Rhoads et al. J. Dairy Sci. (2004)

Effect of Urea Concentration on in Vitro Embryo Production
Added to the oocyte maturation medium
Added to the embryo culture medium
Ocon and Hansen J. Dairy Sci. 86:1194-1200 (2003)

Effect of Dimethadione on pH and in Vitro Embryo Production
Ocon and Hansen J. Dairy Sci. 86:1194-1200 (2003)

Pyruvate Metabolism of Ovine Blastocyst Produced by
IVF after Maturation with Granulosa Cells Previously
Exposed to NH 4 Cl
30
Blastocyst
P < 0.05 P = 0.06
Cell
0.6
pMol/3h/blastocyst
25
20
15
0.5
0.4
0.3
0.2
pMol/3h/cell
10
0mM 5mM 10mM 0 Mm 5 mM 10 mM
0.1
NH4Cl (granulosa cells)
Rooke et al. Anim. Reprod. Sci. (2000)

Effect of CP and RDP on Embryo Quality from
Superovulated Cows
CP (RDP)
Cows
Transferable Embryos
Yes
No
Oocytes
Transferable
%
Viable, vital
stain
%
Reference
12.3
(59.7)
27.4
(70.7)
22 4.0 1.6 1.8 49.7 53.1 b Garcia-Bojalil et al.
(1994)
22 4.9 2.0 1.8 54.0 66.7 a
15.7
(ND)
21.9
(ND)
12 3.4 0.3 0.5 82.0 ND
11 5.0 0.6 0.5 83.0 ND
Rhoads et al. (2006)
16.0
(73.0)
16.1
(64.0)
19 4.5 4.0 3.1 44.2 b ND
Blanchard et al.
(1990)
19 5.5 3.3 2.3 66.9 a ND
a, b P < 0.10

Effect of level of protein feeding on embryonic survival
Rhoads et al. Anim. Reprod. Sci. 91: 1 (2006)
15.5 mg/dL vs 24.4 mg/dL 7.7 mg/dL vs 25.2 mg/dL

Protein Can Also Influence
Energy Metabolism

Effect of DIP & Fat on Reproduction
of Dairy Cows (Garcia-Bojalil et al., 1998)
• 45 multiparous Holstein cows
• Dietary treatments were
– 1) 11.1% DIP, 0% CaLCFA
– 2) 11.1% DIP, 2.2% CaLCFA
– 3) 15.7% DIP, 0% CaLCFA
– 4) 15.7% DIP, 2.2% CaLCFA
• Diets fed for the first 17 weeks PP

Effect of DIP & Fat on Reproduction
of Dairy Cows (Garcia-Bojalil et al., 1998)
Dietary Protein Sources
• 11.1% DIP diets
– Corn gluten meal
– Fish meal
– Blood meal
– Meat and bone meal
• 15.7% DIP diets
– Soybean meal and urea

Effect of DIP and Fat on Body
Weight Change of Lactating Cows
Body weight, kg
620
600
580
560
540
520
DIP; P = 0.01
11.1% DIP
" + fat
15.7% DIP
" + fat
0 2 4 6 8 10 12 14
Week of lactation

Effect of DIP and Fat on
Accumulated Plasma Progesterone
Progesterone, ng/ml
70
60
50
40
30
20
10
0
DIP by FAT interxn; P = 0.001
11.1% DIP
" + fat
15.7% DIP
" + fat
1 8 15 22 29 36 43 50
Day of lactation

Some Protein Sources Have
Been Linked with Improved
Fertility

Pregnancy/Conception
Rates Due to Feeding Fish Meal
% conception/pregnancy
90
80
70
60
50
40
30
20
10
0
*
**
**P< 0.05; *P

What are Reasonable
MUN or BUN Values?

Typical MUN Values of Cows Fed
Diets Balanced for N and Energy
Reference
MUN, mg%
Oltner and Wiktorsson, 1983 13.8
Roseler et al., 1993 11.6
Jonker et al., 1998 13.5

Urea
Detoxification by liver
Ammonia
Deamination of Amino Acids
Rumen
Small
Intestine
Muscle

Diet Containing 17% CP and Balanced for Met
(2.17% MP) & Lys (6.8% MP) Requirements
• Lactating cow producing 45 kg of milk with 3.15%
true protein and consuming 25 kg of DM
– Metabolizable protein required = 3,050 g/d
– Metabolizable protein intake = 3,150 g/d
– Predicted PUN = 21 mg/dL
3% surplus of MP
• Lactating cow producing 20 kg of milk with 3.30%
true protein and consuming 16 kg of DM
– Metabolizable protein required = 1,600 g/d
– Metabolizable protein intake = 1,935 g/d
– Predicted PUN = 14 mg/dL
20% surplus of MP

N Metabolism by Rumen Bacteria
Peptides
Peptides and AA
Mono & Disaccharides
Proteins
deaminase
Peptides & AA
Microbial cells
& VFAs
NH 3 + Keto-acid
ATP
ADP
Portal
System
NH 3 + H NH 4
Deamination of AA and NH 3
production results from lack of ATP
available for microbial growth

Effect of Dietary Protein Concentration and
Quality on Performance of Dairy Cows
50
45
40
LoCP = 17.0%
HiCP = 18.3%
40.8
46.2
b
42.9
46.6
b
kg/d
35
30
a
a
25
20
15
10
a
b b b
HiCP-LoDRUP HiCP-HiDRUP LoCP-HiDRUP LoCP-
HiDRUP+Met
DM intake, kg/d Milk, kg/d Noftsger and St. Pierre J. Dairy Sci. (2003)

Excess of
dietary
protein
NH 3
urea
protein
AA
?
NH 3 /NH 4
+
Urea + NH 3
?
Follicle
development
Toxic effects on
oocytes and
granulosa cells
Enhances
blastocyst
development
Delays embryo
transport
Impairs early
embryonic development

Summary - Protein
‣ Only excessive amounts of protein/N might influence embryo
quality and pregnancy
alter the uterine environment (pH)
‣ Associated with increased urea-N in reproductive tissues
Lack of ATP for adequate microbial growth relative to N availability in the rumen
‣ Recommendations
Formulate diets based on metabolizable protein needs of the
animal
Lactating cows: reduce the CP content to ~ 16 to 17% when
high quality protein sources are utilized and balance for
amino acids

Amino Acids and Reproduction
• Bovine conceptus requires amino acids for development
– Embryo growth
– Placental development
• Some amino acids have physiological functions beyond
building blocks for tissue deposition
– Signaling molecules (Arginine -> NO, polyamines)
– Energy metabolism (Glycine)
– Neurotransmiter (GABA)
– DNA methylation (Methionine)

Bovine Peri-Implantation Events
Conceptus
position
Conceptus
Development
Estradiol
(pg/ml)
10
5
Fimbria
Oviduct
Utero-tubal
junction
Shedding of
zona pellucida
425µ
150µ 170µ 205µ 340µ
IFN τ production
by trophoblast
3
mm
Implantation
250 mm
Progesterone
Estradiol
6
3
Progesterone
(ng/ml)
0
4
8
Days after mating
12 16
20

Some Amino Acids are Neurotransmiters
‣ Aspartate, glycine, glutamate, and gamma aminobutyric acid
(GABA)
‣ Binding of N-Methyl-D-Aspartate to receptors results in
activation of ion channels in neurons and allows for entry of Na +
and Ca ++ and exit of K +
Stimulates the release of GnRH

Hormonal relationship between the hypothalamus,
anterior pituitary and ovary
Senger, 2003

DNA Methylation
• Embryos up to the 8 cell stage:
– DNA is de-methylated
• 8 cell to Morula:
– DNA is methylated
– Modifies and add epigenetic information to the
genome of the embryo. This process “epigenetic
reprograming” is complete in the early embryonic
stages

Methylation Pathways
(DNA, proteins, lipids)
Phosphatidyl
Choline
Phosphatidil-
Etanolamine
CH 3
S-adenosyl-Met
S-adenosylhomocysteine
CDP-Choline
Dimethylglycine
Met
Tetrahydrofolate
B 12
Choline
Betaine
Homocystheine
CH 3 -Tetrahydrofolate
Acetylcholine
Cysteine
Glutathione

Amino Acid Concentrations in the Oviduct
and Uterus of Dairy Heifers
Amino acid concentration, µM
2,200
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
Oviduct
Uterus
Asp Glu Asn Ser His Gln Gly Thr Arg Tau Ala Tyr Met Trp Val Phe Ile Leu Lys
Hugentobler et al. Mol. Reprod. Dev. 94: 445-454 (2007

Methionine Concentrations in Different
Body Compartments of Dairy Cows
• Plasma:
– 16 to 35 µMol/L
• Uterine fluid:
– 31 to 46 µMol/L
• Oviduct:
– 31 to 49 µMol/L
Hugentobler et al. Mol. Reprod. Dev. 94: 445-454 (2007

2 Experiments
• Bovine embryo development cultured in presence
of different concentrations of methionine
– What is the adequate concentration in vitro for early
embryo development?
– Can embryo development be improve if
concentrations are greater than normally found in
reproductive tissues?

Effect of Methionine Concentration on in Vitro
Development to Blastocyst in Cattle
40
35
Day 7
a,b
P < 0.05
40
35
b
Day 8
b
b
a,b
P < 0.01
b
b
%
30
25
20
15
10
a
b
b
b
b
b
%
30
25
20
15
10
a
5
5
0
0 35 50 100 200 400
µM
0
0 35 50 100 200 400
µM
Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

Effect of Methionine Concentration on
Blastocyst Cell Number
250
Blastocyst
250
Expanded Blastocyst
200
200
Cell number
150
100
Cell number
150
100
50
50
0
0 35 50 100 200 400
µM
0
0 35 50 100 200 400
µM
Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

Effect of Methionine Concentration on in Vitro
Development to Blastocyst in Cattle
Blastocyst/oocyte, %
50
40
30
20
10
a
b
Day 7
c
c
a,b, c
P < 0.05
bc
bc
Blastocyst/oocyte, %
50
40
30
20
10
a
b
Day 8
b
b
a,b
P = 0.01
b
b
0
0 7 14 21 28 35
µM
0
0 7 14 21 28 35
µM
Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

Effect of Methionine Concentration on Glutathione
Concentration and % of Advanced Blastocyst
Glutathione, pMol on d 7
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Glutathione on D 7
a
a,b
P < 0.005
b
b
b
b
b
0 7 14 21 28 35
µM
Advanced Blastocyst, %
90
80
70
60
50
40
30
20
10
0
Day 7
a,b, c
P < 0.001
c
bc bc
bc
b
a
0 7 14 21 28 35
µM
Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

Metabolic fates of arginine in mammalian cells
Wu and Morris Jr. Biochem. J. (1998) 336, 1-17

NO and Polyamines Enhance Angiogenesis and
are Potent Vasodilator in the Lungs and Placenta
• Increase blood flow
and nutrient supply
to the fetus (Sladek
et al., 1997)
• Reverse some of the
effects of intrauterine
growth
retardation in
humans and sheep
(de Boo et al. 2005;
Thureen et al., 2002)

Approximate Embryonic and Fetal Losses
When Calving Rate is 30%
100
80
10-15%
fertilization
failure
25% of the pregnancies
are lost after placentation
Two-cell Bovine Embryo
Embryo
Cow
Number
60
40
20
0
SURVIVAL DEPENDS ON
--Genetic inheritance
--Non-genetic inheritance
(from egg & sperm)
--Environment
57%
early
embryo
mortality 15%
late
embryo
mortality
10%
fetal loss
AI Fertilization Day 28 Day 45 Term
US
Palpation

Calcium-Independent iNOS Activity in Murine Placenta
on Different Days of Gestation
Baylis et al. Mol. Human Reprod. vol.5 pp. 277–286, 1999

In Situ Hybridization for Inducible Nitric Oxide Synthase (iNOS)
mRNA in Human Placenta
A
B
A = staining in the syncytioblast and cytotrophoblast
B = negative control
C = light photomicroscopy of a placental arteriole
D = dark photomicroscopy of a placental arteriole
C
D
Baylis et al. Mol. Human Reprod. vol.5 pp. 277–286, 1999

In Situ Hybridization for Cationic AA Transporter in
Ovine Endometrium

Effect of Daily Arginine i.v. Infusion on Progesterone and
Ovarian Artery Resistance Index
Progesterone, ng/mL
9
8
7
6
5
4
3
2
AUC: P < 0.004
Arginine
Control
Resistance index
0.6
0.5
0.4
0.3
0.2
P < 0.05
1
0.1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Day of the cycle
0
Control
Arginine
Arginine infused at 27 mg/kg of BW from estrus to d 15
Luther et al. unpublished

Number of CL and Fetuses in Ewes Treated
or Not with i.v. Arginine
Number per ewe
2.0
1.5
1.0
0.5
Control
Arginine
P < 0.05 P < 0.03
0.0
CL D 25 D 45
Luther et al. unpublished

Number of CL and Fetuses in Sows Fed a Diet
with 1% Arginine
Number or Kg
18.0
15.0
12.0
9.0
6.0
3.0
Control
Arginine
P < 0.05
P < 0.05
0.0
Piglets/liter Live/liter Liter birth weight
Mateo et al. J. Nutr. 137: 652-656 (2007)

Summary
• Glycine is the main amino acid in the reproductive tract of
bovine
– Energy source for the early embryo; synthesis of other AA; tissue deposition
• Arginine
– Might mediate improvements in CL function and placentation through gaseous
signaling, angiogenesis and blood flow
• Methionine
– Tissue deposition
– Phospholipid synthesis
– DNA methylation
– Concentrations in the oviduct and endometrium seem adequate for optimum
early embryo development

THANK YOU
José Eduardo P. Santos
Department of Animal Sciences
University of Florida
Jepsantos@ufl.edu