POTENTIAL HORMONAL AND
NUTRITIONAL STRATEGIES TO IMPROVE
EMBRYO SURVIAL IN DAIRY CATTLE
J.E.P. Santos, R. Cerri, S. Juchem, K. Galvão and R. Chebel
Veterinary Medicine Teaching and Research Center
University of California - Davis
INTRODUCTION
 Reproductive performance affects the profitability of dairy
operations.
 Pregnancy rates is the most meaningful measurement of
reproductive efficiency (Ferguson and Galligan, 1993)
 Pregnancy rate is the product of estrous detection and
conception rates (Macmillan, 1992).
 PR  Speed at which eligible cows become pregnant
Pregnancy Rate
(Survival Graph Analysis)
 Examine effective VWP
VWP
 Area above & below the
curve
Slope = PR
Pregnant
 Estimate PR
 Estimate proportion
remaining open
Open
30%
open
Overton, 2002
Value/Cow and Pregnancy Rate
Annuity Value - The accumulated value
corrected for the time value of money.
Anuity Value/cow/year
1000
900
Different combinations of
Heat and CR for a given PR.
800
y = 232.06Ln(x) + 1063
700
2
R = 0.9554
600
500
400
300
0
0.2
0.4
0.6
0.8
1
Pregnancy Rate
Galligan, Penn. Vet Conf., 1999
Costs Associated with Changes in
Reproductive
Efficiency
Costs of Improved Reproduction
90
Milk (lbs) and Cost ($)
85
80
75
70
65
60
55
50
45
40
8%
10% 12% 14% 16% 18% 20% 22% 24% 26% 28% 30%
Pregnancy Rate
Cost per cow
Cost per preg
Note: 8% PR ~ 18,000 RHA, Milk is per milking cow/day
Overton, 2002
Control of Reproductive Indices
Voluntary waiting period
Effect of TAI on DIM at First Postpartum AI in a Dairy Herd
VWP
VWP
TAI
Control of Reproductive Indices
Voluntary waiting period
Estrus detection rate
High Estrus Detection Rate Can be Achieved with
Aggressive Breeding Protocols
Control of Reproductive Indices
Voluntary waiting period
Heat detection rate
Conception rate
Pregnancy
Rate
High Estrus Detection Rate Can be Achieved with
Aggressive Breeding Protocols
CR = 35%
Control of Reproductive Indices
Voluntary waiting period
Heat detection rate
Conception rate
Pregnancy loss
Pregnancy
Rate
Rate of Late Embryonic Loss is Greater between
Days 27 and 45
Cows
Days 1st
Check
Days last
Check
Days
Loss
%
Loss/
Day
Chebel et al., 2002a
195
28
42
14
17.9
1.28
Moreira et al., 2000a
139
27
45
18
20.7
1.15
Chebel et al., 2002b
1,503
31
45
14
13.2
0.94
Stevenson et al., 2000
203
28
45
17
15.8
0.93
Santos et al., 2002b
360
31
45
14
11.1
0.79
Santos et al., 2002a
220
27
41
14
10
0.71
Cerri et al., 2002
176
31
45
14
9.7
0.70
Juchem et al., 2002
167
28
39
11
11.4
1.03
Fricke et al., 1998
89
28
56
28
13.5
0.48
Moreira et al., 2000b
211
32
74
42
13.7
0.33
Santos et al., 2000
171
28
90
62
17.4
0.28
1,601
28
98
70
19.6
0.28
285
30
280
250
22
0.09
Reference
Vasconcelos et al., 1997
Pursley et al., 1998
0.9%
Oocyte Quality
Uterine
Environment
Embryonic
Development
bST
Progesterone
Secretion
hCG
bST
Gossypol
Gossypol
IFN-t
secretion
PGF2
secretion
Successful
Implantation
PUFAs
Effects of bST on Reproductive
Functions in Dairy Cows
Follicle development
CL differentiation/P4 secretion
Fertilization/embryo development
Uterine environment
PGF2α secretion (bst vs IGF-I)
Oviduct vs Endometrium
Experimental Design
Donor Cows
Superovulation
d0
Artificial
Insemination
bST
d7
Recipient Cows
d 0
Estrus
d1
Treat
recipients
bST Control
Control
Flushing
Record results
Freeze embryos
d7
Embryo Transfer
Pregnancy
diagnosis
(rectal palpation)
Number of Ova/Embryos per Flush for Control (n = 26)
and bST (n = 26) Treated Donors (LSM + SE).
10
Control
9
bST
8
7
6
* P < 0.05
5
4
3
2
1
9.4
per
flush
9.3
per
flush
5.4
per
flush
7.4
per
flush
0
Total
Unfertilized
Degenerate
Transferable
Frequency of distribution of transferable
embryos among stages of development
60
60
50
50
40
40
30
30
20
20
10
10
0
Morula
Early Blast
Blast
Exp Blast
bST
Control
P < 0.001
Percentage of transferable embryos
relative to total ova/embryos flushed
80
70
60
50
40
30
20
10
0
56.4 %
(150/266)
77.2 %
(206/267)
Control
bST
P < 0.001
Pregnancy rates following embryo transfer for
treatment groups
Pregnancy rates (%)
Control recipient
60
bST recipient
56.1 %
(23/41)
50
40
30
43.2 %
(16/37)
43.3 %
(26/60)
25.6 %
(11/43)
20
Control embryo
bST embryo
Experimental Design
Oocytes
Maturation
Control
GH
(100
ng/ml)
Fertilization
Potential Zygotes
IgG
Anti-IGF-I
(10 g/ml)
(10 g/ml)
Control
Trt 1
Trt 4
GH (100 ng/ml)
Trt 2
Trt 5
IGF-I (100 ng/ml)
Trt 3
Trt 6
Control
Trt 7
Percentage of cleaved oocytes that developed to
blastocyst at d 8 of embryo culture
40
P < 0.05
P < 0.01
35
30
P < 0.02
P > 0.10
25
20
15
10
5
0
Control
IgG
GH
IgG
IGF
Anti-IGF
GH
Anti-IGF
IGF
FLORIDA STUDY DESIGN
63
DIM
70
DIM
72
DIM
73
DIM
147
DIM
GnRH
PGF2
GnRH
TAI
Pregnancy
diagnosis
bST-63
bST-73
Control
Moreira et al. (2001)
Pregnancy Rates at 74 d after TAI for
Cyclic Cows (LSM + SE; n = 375).
70
58.1%
56.1%
60
42.6%
50
40
34.2%
33.7%
25.3%
30
20
10
0
bST-63 bST-73 Control
No Presynch
bST-63
bST-73 Control
Presynch
California Study Design
bST +/500 mg/14 d
PGF2α
25 mg
PGF2α
PGF2α
GnRH
25 mg
25 mg
100μg
AI/Detected Estrus
d 70
PGF2α
Presynch
25 mg
d 37
d 51
d 77
GnRH
TAI
100 µg
d 63
Blood P4
Ovsynch / TAI
d 70
d 72 d 73
Blood P4
Timing of the Ovsynch/Select Synch Injections
Estrus Detection
8
6
4
P4
2
0
d 5 to d 12
GnRH
HIGH
PGF2
+7 d
HIGH
GnRH
TAI
+9.5 d +10 d
DIESTRUS COWS
Pregnancy Diagnosis and
Reinsemination
AI
Palpate
Ultrasound
End
Pregnant
Estrus Detection / AI
d0
d 31
Open
PGF2α
AI
Effect of Reproductive Management (RM) and bST
on Conception at day 45 after AI in Cycling Cows
bST: P < 0.04
RM: P < 0.03
bST * RM: P < 0.30
55
CR d 45, %
50
45
40
35
30
25
20
ED
OV
bST
Control
Effect of Reproductive Management (RM) and bST
on Pregnancy Rate at day 45 after AI in Cycling
Cows
bST: P < 0.04
RM: P < 0.03
bST * RM: P < 0.30
55
PR d 45, %
50
45
40
35
30
25
20
ED
OV
bST
Control
Effect of Reproductive Management (RM) and bST on
Losses of Pregnancy between 31 and 45 d after First
Postpartum AI in Cycling Dairy Cows
Preg. Loss, %
20
bST: P < 0.07
RM: P < 0.32
bST*RM: P < 0.17
16
12
8
4
0
ED
bST
OV
Control
Study in Mexico with Cows Considered
to Have Low Fertility
Effect of bST treatment on pregnancy rates in dairy cows
Treatment
No. AI
Control
bST
3 to 4
31.5a (n = 206)
37.4a (n = 117)
5 to 7
15.2b (n = 76)
25.3ab (n = 54)
>8
3.9b (n = 27)
25.3ab (n = 30)
16.9b (n = 309)
29.3a (n = 201)
Overall
a, b
Different superscripts in the same row differ (P < 0.05)
Morales-Roura et al.(2001)
12
20000
9
15000
6
10000
3
5000
**
0
0
2
4
6
8
10
Day of cycle
Mann et al., 1999
12
14
16
Interferon- (units per uterus)
Progesterone (ng.ml-1)
Progesterone Concentration and Embryo
Production of Interferon-
0
Large Small
embryo embryo
Day 16
interferon
** P<0.01
EXPERIMENTAL DESIGN
GnRH,
100  g
D0
Experimental day
hCG, 3,300 IU (i.m.) or
3 ml of saline + BCS
D5
Days after AI
AI on morning of detected
PGF 2  ,
estrus
25 mg
D 7……..……….…………………..12
Estrus detection
Blood for P4 +
ultrasound for CL
# and location
D 11…………16
Preg. by
ultrasound
+ BCS
D 28
Preg. by
palpation +
BCS
D 45 and 90
Conception
% rates (%)
Effect of Treatment on Conception Rates at days 28,
45 and 90 post AI
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
7.1 %
4.1 %
6.5 %
d 28
hCG: P < 0.05
d 45
Control
hCG
d 90
Nutritional Manipulation to
Improve Embryo Survival
Track Record of Fat Supplementation
Effects on Lactating Dairy Cows
Supplementation with fatty acids
Caloric
• High energy content (often improves milk production)
Extra-caloric effects
• Acetyl CoA for cholesterol synthesis
• Alters steroid clearance
• Influences plasma progesterone concentrations
• alters ovarian follicle dynamics , CL function and uterine
secretion of PGF2
Diet
1.68 Mcal/kg DM
0% Ca-LCFA
Size of
Dominant follicle
16.5 mm
1.68 Mcal/kg DM
2.2% Ca-LCFA
18.7 mm
Lucy et al., 1993 JDS 76:1014
Effect of Fat Supplementation on
Plasma Progesterone Concentration
Reference
Time of
measurement
Control
(ng/ml)
Fat
(ng/ml)
Carroll et al., 1990 d 9 - 15 of cycle
6.6a
7.7b
a
b
Lucy et al., 1993
d 1 - 12 of cycle
4.2
5.2
a,b
Sklan et al.,1991 d 8 - 20 of cycle Greater accumulation
4.5a
4.2a
6.0b
4.8b
Spicer et al.,1993
wk 5 - 12 PP
Son et al., 1996
wk 2 - 12 PP
Garcia et al., 1998
wk 1 - 7 PP
Greater accumulation a,b
Adams, 1998
wk 2 - 9 PP
Greater accumulation a,b
Targeting Specific FA
Ca Salts of Long Chain Fatty Acids
Ca
Ca
Ca
FA
FA
FA
FA
Ca
Ca
Ca
Scott and Ashes (1993)
Manipulating PGF2 Synthesis
Effects on the uterus and corpus luteum
Beneficial effects early postpartum on uterine
immunity and involution
• Stimulation early postpartum might improve
uterine involution and resumption of cyclicity
Inhibition during maternal recognition of
pregnancy
• might improve embryo survival
n-6 Family
n-3 Family
Linolenic acid
(C18:3)
Linoleic acid
(C18:2)
6 desaturase
 Linolenic acid
(C18:3)
Stearidonic acid
(C18:4)
Elongase
Dihomo- Linolenic acid
(C20:3)
Eicosatetraenoic acid
(C20:4)
5 Desaturase
Arachidonic Acid
(C20:4)
Eicosapentaenoic acid
(C20:5)
Synthesis of PGF2α
Arachidonic acid
Cyclooxygenase
PGHS
Peroxidase
PGES
PGE2
PGFS
PGF2
Prostaglandin F 2 Response in Postpartum
Beef Heifers Fed Rumen Bypass Fat
Fed Calcium Salts of Fatty Acids (CSFA) at 3% of
estimated dry matter intake (.23 kg/heifer/d) vs isocaloric
control supplement (.72kg ground barley) from time of
calving.
12 heifers per treatment
Blood samples collected every other day from day 1-15
postpartum.
Filey S.J.et al, 2000
3000
2500
*
2000
*
1500
1000
500
0
1
3
5
7
9
11
Day postpartum
Control
13
15
CSFA
Filey S.J.et al, 2000
Concentrations of PGFM in Holstein Cows
3
Control
PGFM, ng/mL
2.5
Infected and Treated
2
Infected and Not Treated
1.5
1
0.5
0
0
10
Seals et al., 2002
20
30
40
Days postpartum
50
60
Improving Embryo
Survival
Pregnancy, day 12 to 19
r
Ovary
IFN - 
CL
PGF 2 
Embryo
Progesterone
Pregnant Uterus
10
9
8
7
6
5
4
3
2
1
0
15
16
17
18
19
20
Day
21
22
23
24
25
PGHS

series
Responses of PGFM in Cows Fed or
Not Fed Fish Meal
140
-240 min:
120
Estradiol (3 mg, iv)
100
80
Oxytocin
(100 IU iv)
60
*
*
*
* P<0.05
40
20
0
-60
-30
0
30
Control
60
90
120
Time (min)
150
Fish Meal
180
210
240
Experimental Design
>90% Confluence
24 h Starvation
0
3
Add EPA
0, 20, 100 µM
Cells Grown
to Confluence
6h
Extraction of RNA
Northern blot analysis
of PGHS-2
Wash Cells
Add PDBu
(100 ng/ml)
IFN-: 0, 0.5 or 50 ng/ml
24 h Starvation
0
3
6h
Effect of EPA and IFN-tau on Secretion of
PGF2α from Bend Cells Treated with PDBU
7000
IFN-tau: P < 0.01
EPA:
P < 0.01
PGF2α (pg/ml)
6000
5000
4000
3000
2000
1000
0
EPA 0
EPA 20 EPA 100
IFN 0
Treatment
IFN 0.5
IFN 50
Effect of EPA and IFN-tau on Concentrations of PGHS2 mRNA in Bend Cells Treated with PDBU
1.4
EPA: P>0.1
IFN-: P<0.01
1.2
1
0.8
0.6
0.4
0.2
0
0
20
EPA (µM)
PGHS-2 mRNA
18S rRNA
100
0
0.5
50
IFN- (ng/ml)
Pregnancy, day 12 to 19
r
Ovary
IFN - 
CL
PGF 2 
Embryo
Progesterone
Pregnant Uterus
10
9
8
7
6
5
4
3
2
1
0
15
16
17
18
19
20
Day
21
22
23
24
25
Experimental Design
Determine
PGF2α in
medium
Remove EPA
Add PDBu and
IFN-
IFN-: 0, 50 or 100 pg/ml
24 h Starvation
Add EPA
0, 3 or 20 µM
0
3
6h
3 x 3 Factorial
Effect of Combined Treatment with IFN- and EPA on
Secretion of PGF2α from BEND Cells Stimulated with PDBu
PGF2α (pg/ml)
3000
EPA: <0.01
IFN-: <0.01
EPAxIFN - : NS
2500
2000
EPA (µM)
0
3
20
1500
1000
500
0
0
50
IFN- (pg/ml)
100
Pregnancy, day 12 to 19
+
EPA
Ovary
r
IFN - 
CL
PGF 2 
Embryo
Progesterone
Pregnant Uterus
10
9
8
7
6
5
4
3
2
1
0
15
16
17
18
19
20
Day
21
22
23
24
25
Fatty Acid Study - California
 710 multiparous Holstein cows
 Commercial dairy farm in Central CA (RHA = 12,026 kg)
 September 2001 to October 2002
 Two treatments:
400 g of fatty acids from tallow
400 g of fatty acids from palm and fish oil in a Ca salt
form
Nutrient content of experimental diets
Treatment
Nutrient
Ca Salts
Tallow
NEL,1 Mcal/kg
1.62
1.61
CP, %
18.1
18.1
RUP,1 % CP
40.6
40.6
NDF, %
30.5
30.5
NFC, %
39.0
39.2
Crude fat, %
6.1
6.1
EPA, g
15.0
3.0
DHA, g
15.0
3.0
1Based
on NRC (2001) and adjusted for 26 kg of DM intake.
Reproductive Management for First
Postpartum AI
PGF2α
PGF2
25 mg
25 mg
GnRH
PGF2α
GnRH
100μg
25 mg
100μg
Presynch
d 33
Ovsynch / TAI
d 47
d 61
Ovsynch
Blood /PTAI
4
d 68
d 70
d 71
TAI
Effect of Source of Dietary FA on Pregnancy
Rate at Day 28 After TAI in Dairy Cows
TRT: P < 0.15
Season: P < 0.74
TRT * Season: P < 0.29
Pregnancy Rate, %
44
41
38
35
N=50
N= 172
N=94
32
29
26
N=50
23
20
TN
HS
Season
Ca Salts
Tallow
Effect of Source of Dietary FA on Losses of Pregnancy
from 28 to 39 d after TAI During Thermoneutrality in
Multiparous Dairy Cows
Pregnancy loss, %
14
12
TRT: P < 0.09
10
8
N=75
6
4
N=68
2
0
Ca Salts
Tallow
Feeding Fish Meal Improved
Pregnancy Rates of Beef Cows
Experimental Methods
82 lactating, primiparous beef cows
Cows were penned; fed a corn silage-based diet;
diets were isonitrogenous & isocaloric
Dietary Supplements: 5% fish meal or 8.7% corn
gluten meal
Diets fed 25 d prior to and thru a 90-d breeding
season over a 2-year study
Bonnette et al., 2000
Feeding Fish Meal Effects on
Pregnancy Rates of Beef Cows
80
P < 0.14
75
PR, %
70
65
60
55
50
Fish Meal
CGM
Bonnette et al. (2000)
D 17
Embryo
Successful Early
Pregnancy
Embryonic loss due to
abnormally high
secretion of PGF2α
High
IFN-
Endometrium
Inhibits
PGHS-2
Outcome
Reduced
PGF2α
CL and
Pregnancy
Maintained
High
PGF2α
CL Lysed
Pregnancy
Lost
AA
Low
IFN-
Active
PGHS-2
AA
Successful Early
Pregnancy
EPA and DHA
compensate for low
secretion of IFN-
Low
IFN-
PGHS-2
DHA, EPA
Reduced
PGF2α
CL and
Pregnancy
Maintained
High
PGF3α
(inactive)
(exogenous)
Embryo
Endometrium
Outcome
Cottonseed and Gossypol
 Cottonseed is generally fed to lactating dairy cows at
10 to 15% of the diet
 Provides a unique blend of protein, fat and fiber for
inclusion in diets of dairy cows
 Cottonseed contains gossypol (0.5 to 1.2%), which is
a toxin present throughout the cotton plant that helps
to control pests
 However, gossypol can be toxic to mammals and it
disrupts reproduction in males and possibly in
female ruminants
Experimental Protocol
Diets: 10% WUP vs 10% of a 1:2 blend of WUP & cracked Pima
813 cows
day postpartum
D 3 - 24 PP
30 ± 3
44 ± 3
63 ± 3
93 ± 3
183
BCS
PGF
PGF
Blood +
BCS
Blood + BCS
BCS
Heat detection & AI
Pregnancy Diagnosis
- 3 dairy farms
- 5 pens/trt
Nutrient Composition of Diets
Cottonseed
Item
WUP
BWUPCP
SEM
P<
15
15
--
--
OM, %
91.1
91.3
0.45
0.43
NEL, Mcal/kg
1.70
1.71
0.04
0.36
CP, %
18.4
18.5
0.34
0.87
Fat, %
7.0
6.9
0.13
0.48
ADF, %
21.8
21.8
0.93
0.99
NDF, %
33.1
32.3
0.32
0.05
TG, mg/kg
743
1,000
--
--
Samples, n
Effect of Type of Cottonseed on Performance of Dairy Cows
Cottonseed
Item
WUP
BWUPCP
SEM
P<
Cows, n
402
401
--
--
DMI, kg/d
24.4
24.3
0.71
0.91
FG, g/d
17.8
23.4
0.70
0.01
Milk, kg/d
39.9
39.7
0.35
0.78
3.5% FCM, kg/d
40.0
40.2
0.36
0.61
Santos et al. (2002)
Plasma Total Gossypol in Primiparous and Multiparous Cows
Fed WUP or a Blend of WUP and Cracked Pima
Plasma gossypol, ug/ml
14
12
10
8
6
4
2
0
10
33
WUP-P
Santos et al. (2002)
61
WUP-M
91
DIM
BUPCP-P
120
BUPCP-M
152
Effect of Type of Cottonseed and Gossypol Intake on
Conception and Pregnancy Rates of Lactatin Dairy Cows
P < 0.01
34
31
28
25
22
19
16
13
10
%
%
P < 0.72
First AI, CR
WUP
BWUPCP
Santos et al. (2002)
90
85
80
75
70
65
60
55
50
PR, 170 DIM
WUP
BWUPCP
Effect of Type of Cottonseed and Gossypol Intake
on Days Open in Lactating Dairy Cows
Whole Upland:
Blend of cracked Pima and WUP:
1.0
Cottonseed: P < 0.01
Proportion of non-pregnancy
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
60
80
100
Days Open
120
140
160
180
Effect of Plasma Gossypol Concentrations
on Days Open in Dairy Cows
Q1
Q2
Q3
Q4
1.0
P < 0.05
0.9
0.8
Probability
0.7
0.6
0.5
0.4
0.3
0.2
0
20
40
60
80
100
Time to Failure
120
140
160
180
Plasma Gossypol Concentration in Lactating Dairy
Cows
P < 0.04
9
9
8
8
7
7
6
6
ug/ml
ug/ml
P < 0.01
5
5
4
4
3
3
2
2
Total Gossypol
Pregnant
Santos et al. (2002)
Open
Total Gossypol
No Abortion
Abortion
Trasferable embryos, %
Effect of Free Gossypol Intake on Embryo Quality in
Superovulated Holstein Dairy Heifers
50
45
40
35
30
25
20
15
10
Gossypol: P < 0.04
0 mg/kg
20 mg/kg
40 mg/kg
FG intake (mg/kg of BW)
Oocyte Quality
Uterine
Environment
Embryonic
Development
bST
Progesterone
Secretion
hCG
bST
Gossypol
Gossypol
IFN-t
secretion
PGF2
secretion
Successful
Implantation
PUFAs
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