THE ROLE OF PROTEIN AND AMINO ACID NUTRITION ON REPRODUCTION

OF DAIRY COWS

José Eduardo P. Santos and Charles StaplesDepartment 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

5.6

11.613.4 14.4

17.8

0

5

10

15

20M

UN

, mg/

100

ml

80:80 100:100 120:80 100:120 120:120

Intake of DIP & UIP as a % of NRC

Influence of Dietary Ratio of DIP to UIP on MUN

a

cb

JDS 76:525

dc

85 78 78 86103

128

020406080

100120140

Day

s to

firs

t AI

10 12 14 16 18 20Milk Urea N, mg/100 ml

Relationship of Bulk Tank MUN and Days to First AI

Live. Prod. Sci. 37:91

25

3934

50

2226

1722

0102030405060

Firs

t ovu

latio

n, d

ays

Florida Utah Maine Maine

Overfeeding Protein delays 1st Ovulation

* *

ControlExcess DIP/CP

**** * P = 0.10

** P = 0.05

96106

7282

7180

020406080

100120

Day

s op

en

Oregon '79 Maine '88 Maine '96

Overfeeding Protein Delays Conception

** *

ControlExcess CP**

* P = 0.10** P < 0.06

0102030405060708090

100C

once

p/Pr

eg R

ate,

%

Ore

gon

Isra

el

Isra

el OK

Mai

ne

Isra

el NY NY

Mai

ne LA

Effect of Dietary CP% on Fertility13-17% CP diets19-21% CP diets

*

**

*

* *

Dietary CP% on Blood Urea NReference 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 CowsAdapted from Butler et al. J. Anim. Sci. (1996) and Ferguson et al. J. Dairy Sci. (1993)

49.2

41.6

30

35

40

45

50

55

Preg

nanc

y/A

I, %

< 19 mg/dL> 19 mg/dL

320/650

119/286

MUN and Predicted Pregnancy at 1st AI in Lactating Dairy Cows

Guo et al. J. Dairy Sci. 87:1878–1885 (2004)

Negative association only in the first postpartum AI

No effect on subsequent AI

Potential Mechanisms for Reduced Fertility in Lactating Dairy Cows

Follicular development and oocyte growth

42 d 42 d

Oocyte maturation

Fertilization Preimplantation development

IFN-τ

PGF-2α

x

Prevention of Luteolysis

Stages of the Reproductive Process Important for Establishment of Pregnancy in Cattle

Effect of Dietary Protein on PUN and Uterine pH on d 7 of the Estrous Cycle of Heifers

0

5

10

15

20

25

mg/

dl

PUN, mg/dl

Balanced High RUP High RDP

6.6

6.7

6.8

6.9

7.0

7.1

7.2

pHUterine pH

Balanced High RUP High RDP

P < 0.05 P < 0.05

Elrod et al. (1993)

Effect of Dietary Protein on PUN and Conception Rate (CR) in Heifers

0

5

10

15

20

25

mg/

dl

Peak PUN

Balanced High RDP

30

40

50

60

70

80

90

%CR

Balanced High RDP

P < 0.05 P < 0.05

Elrod e Butler (1993)

Effect of Urea/Saline Infusion on Uterine pH on d 7-8 of the Estrous Cycle

6.46.56.66.76.86.97.07.17.27.37.4

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

Uterine pH PUN

Saline Urea

Ute

rine

pH

PUN

, mg/

dL

5

10

15

20

25

30

Rhoads et al. J. Dairy Sci. (2004)

Ocon and Hansen J. Dairy Sci. 86:1194-1200 (2003)

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

Pyruvate Metabolism of Ovine Blastocyst Produced by IVF after Maturation with Granulosa Cells Previously

Exposed to NH4Cl

Rooke et al. Anim. Reprod. Sci. (2000)

10

15

20

25

30

0mM 5mM 10mM 0 Mm 5 mM 10 mM

NH4Cl (granulosa cells)

pMol

/3h/

blas

tocy

st

0.1

0.2

0.3

0.4

0.5

0.6

pMol/3h/cell

Blastocyst CellP < 0.05 P = 0.06

Effect of CP and RDP on Embryo Quality from Superovulated Cows

Transferable EmbryosCP (RDP) Cows

Yes No

Oocytes Transferable %

Viable, vital stain

%

Reference

12.3 (59.7)

22 4.0 1.6 1.8 49.7 53.1b

27.4 (70.7)

22 4.9 2.0 1.8 54.0 66.7 a

Garcia-Bojalil et al.(1994)

15.7 (ND)

12 3.4 0.3 0.5 82.0 ND

21.9 (ND)

11 5.0 0.6 0.5 83.0 ND

Rhoads et al. (2006)

16.0 (73.0)

19 4.5 4.0 3.1 44.2 b ND Blanchard et al.(1990)

16.1 (64.0)

19 5.5 3.3 2.3 66.9 a ND

a, b P < 0.10

Effect of level of protein feeding on embryonic survivalRhoads 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 Reproductionof 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 Reproductionof Dairy Cows (Garcia-Bojalil et al., 1998)

• 11.1% DIP diets – Corn gluten meal– Fish meal– Blood meal– Meat and bone meal

• 15.7% DIP diets– Soybean meal and urea

Dietary Protein Sources

Effect of DIP and Fat on Body Weight Change of Lactating Cows

520

540560

580600

620

0 2 4 6 8 10 12 14

Week of lactation

Bod

y w

eigh

t, kg

11.1% DIP " + fat15.7% DIP " + fat

DIP; P = 0.01

Effect of DIP and Fat on Accumulated Plasma Progesterone

010203040506070

1 8 15 22 29 36 43 50

Day of lactation

Pro

gest

eron

e, n

g/m

l

11.1% DIP " + fat15.7% DIP " + fat

DIP by FAT interxn; P = 0.001

Some Protein Sources Have Been Linked with Improved

Fertility

0102030405060708090

Oregon Oregon Florida Florida Israel Ireland

No FishPlus Fish

Pregnancy/ConceptionRates Due to Feeding Fish Meal

% c

once

ptio

n/pr

egna

ncy

Location

*

*

** **

**P< 0.05; *P<0.10

**

What are ReasonableMUN 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

Ammonia

Rumen SmallIntestine Muscle

Urea

Deamination of Amino Acids

Detoxification by liver

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

• 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

3% surplus of MP

N Metabolism by Rumen Bacteria

Peptides

Proteins

Peptides and AA

Peptides & AA

Mono & Disaccharides

Microbial cells& VFAs

ATP ADPNH3 + Keto-acid

deaminase

NH3 + H NH4Portal System

Deamination of AA and NH3production results from lack of ATPavailable for microbial growth

Effect of Dietary Protein Concentration and Quality on Performance of Dairy Cows

40.8

46.242.9

46.6

10

15

20

25

30

35

40

45

50

kg/d

HiCP-LoDRUP HiCP-HiDRUP LoCP-HiDRUP LoCP-HiDRUP+Met

DM intake, kg/d Milk, kg/d Noftsger and St. Pierre J. Dairy Sci. (2003)

LoCP = 17.0%HiCP = 18.3%

ab b b

b b

aa

Excess of dietary protein

protein

NH3

Toxic effects on oocytes and

granulosa cells

ureaAA

NH3/NH4+?

Follicle development

?

Enhances blastocyst

development

Urea + NH3

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

RecommendationsFormulate diets based on metabolizable protein needs of the

animal

Lactating cows: reduce the CP content to ~ 16 to 17% whenhigh quality protein sources are utilized and balance foramino 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)

Days after mating

Fimbria

Oviduct Utero-tubaljunction

Implantation

Progesterone

Estradiol

150µ 170µ 205µ 340µ250 mm

3mm425µ

840

5

10

1612 20

3

6

Con

cept

uspo

sitio

n C

once

ptus

Dev

elop

men

t Es

trad

iol

(pg/

ml)

Prog

este

rone

(ng /

ml)

IFN τ productionby trophoblast

Shedding ofzona pellucida

Bovine Peri-Implantation Events

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)

CH3

S-adenosyl-Met

Met

Homocystheine

Cysteine Glutathione

PhosphatidylCholine

Choline

Acetylcholine

Betaine

Dimethyl-glycine

Phosphatidil-Etanolamine

Tetrahydrofolate

CH3-Tetrahydrofolate

B12

CDP-Choline

S-adenosyl-homocysteine

Amino Acid Concentrations in the Oviduct and Uterus of Dairy Heifers

0

200

400

600

800

1,000

1,200

1,400

1,600

1,800

2,000

2,200

Asp Glu Asn Ser His Gln Gly Thr Arg Tau Ala Tyr Met Trp Val Phe Ile Leu Lys

Amin

o ac

id c

once

ntra

tion,

µM

Oviduct Uterus

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 earlyembryo development?

– Can embryo development be improve ifconcentrations are greater than normally found inreproductive tissues?

Effect of Methionine Concentration on in Vitro Development to Blastocyst in Cattle

0

5

10

15

20

25

30

35

40

0 35 50 100 200 400

%

µM

Day 7

0

5

10

15

20

25

30

35

40

0 35 50 100 200 400

%

µM

Day 8

Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

a

bb

bb

b

b

a

bb

bba,b P < 0.05

a,b P < 0.01

Effect of Methionine Concentration on Blastocyst Cell Number

0

50

100

150

200

250

0 35 50 100 200 400

Cel

l num

ber

µM

Blastocyst

0

50

100

150

200

250

0 35 50 100 200 400

Cel

l num

ber

µM

Expanded Blastocyst

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

0

10

20

30

40

50

0 7 14 21 28 35

Bla

stoc

yst/o

ocyt

e, %

µM

Day 7

0

10

20

30

40

50

0 7 14 21 28 35

Bla

stoc

yst/o

ocyt

e, %

µM

Day 8

Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

a,b, c P < 0.05

a,b P = 0.01

a

b

c cbc

bc

bb

a

bb

b

Effect of Methionine Concentration on Glutathione Concentration and % of Advanced Blastocyst

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 7 14 21 28 35

Glu

tath

ione

, pM

olon

d 7

µM

Glutathione on D 7

0

10

20

30

40

50

60

70

80

90

0 7 14 21 28 35

Adv

ance

d B

last

ocys

t, %

µM

Day 7

Bonilla et al. J. Dairy Sci. vol. 92, E-Suppl. 1 (Abstr.) pp. 69 (2009)

a,b P < 0.005

a,b, c P < 0.001a

bb

bb

bbc bc

a

bbc

c

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 (Sladeket al., 1997)

• Reverse some of the effects of intra-uterine growth retardation in humans and sheep (de Boo et al. 2005; Thureen et al., 2002)

SURVIVAL DEPENDS ON--Genetic inheritance--Non-genetic inheritance (from egg & sperm)

--Environment

Embryo

Cow

AI Fertilization Day 28 Day 45 Term

Num

ber0

20

40

60

80

10010-15%

fertilizationfailure

57%early

embryomortality 15%

late embryomortality

10%fetal loss

US Palpation

Approximate Embryonic and Fetal LossesWhen Calving Rate is 30%

Two-cell Bovine Embryo

25% of the pregnancies are lost after placentation

Calcium-Independent iNOS Activity in Murine Placenta on Different Days of Gestation

Baylis et al. Mol. Human Reprod. vol.5 pp. 277–286, 1999

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

B = negative control

A = staining in the syncytioblast and cytotrophoblast

C = light photomicroscopy of a placental arteriole

D = dark photomicroscopy of a placental arteriole

A B

DC

In Situ Hybridization for Cationic AA Transporter in Ovine Endometrium

Effect of Daily Arginine i.v. Infusion on Progesterone and Ovarian Artery Resistance Index

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Pro

gest

eron

e, n

g/m

L

Day of the cycle

ArginineControl

0

0.1

0.2

0.3

0.4

0.5

0.6

Control Arginine

Res

ista

nce

inde

x

P < 0.05AUC: P < 0.004

Luther et al. unpublished

Arginine infused at 27 mg/kg of BW from estrus to d 15

Number of CL and Fetuses in Ewes Treated or Not with i.v. Arginine

0.0

0.5

1.0

1.5

2.0

CL D 25 D 45

Num

ber p

er e

we

Control Arginine

Luther et al. unpublished

P < 0.05 P < 0.03

Number of CL and Fetuses in Sows Fed a Diet with 1% Arginine

Mateo et al. J. Nutr. 137: 652-656 (2007)

0.0

3.0

6.0

9.0

12.0

15.0

18.0

Piglets/liter Live/liter Liter birth weight

Num

ber o

r Kg

Control Arginine

P < 0.05

P < 0.05

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 YOUJosé Eduardo P. SantosDepartment of Animal Sciences University of FloridaJepsantos@ufl.edu