Published December 4, 2014
Administration of human chorionic gonadotropin 7 days after
fixed-time artificial insemination of suckled beef cows1
C. R. Dahlen,* S. L. Bird,† C. A. Martel,‡ KC Olson,‡ J. S. Stevenson,‡
and G. C. Lamb§2
*Northwest Research and Outreach Center, University of Minnesota, Crookston 56716;
†North Central Research and Outreach Center, University of Minnesota, Grand Rapids 55744;
‡Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201;
and §North Florida Research and Education Center, University of Florida, Marianna 32446
ABSTRACT: We determined the effects of administering hCG 7 d after a fixed-time AI (TAI) on ovarian
response, concentrations of progesterone, and pregnancy rates in postpartum suckled beef cows. Cows at 6
locations received 100 µg of GnRH (Fertagyl, Intervet
Animal Health, Millsboro, DE) and a controlled internal drug release (CIDR) device (CIDR EAZI-Breed,
Pfizer Animal Health, New York, NY), followed in 7 d
by 25 mg of PGF2α (Lutalyse, Pfizer Animal Health)
and CIDR removal. At 64 h after CIDR removal, cows
received an injection of GnRH and AI (d 0), and then
were stratified by days postpartum and parity and assigned randomly to 2 treatments administered 7 d after
TAI: 1) 1 mL of saline (saline; n = 252); or 2) 1,000 IU
of hCG (Chorulon, Intervet Animal Health; n = 254).
Blood samples were collected on d −21, −10, and 33
relative to TAI (d 0) at all locations, on d 7 and 68
at 5 locations, and on d 14 at 1 location to determine
concentrations of progesterone. Transrectal ultrasonography was used to determine pregnancy status on d 33
and 68 at all locations, to monitor response of follicles
and corpora lutea (CL) in response to treatment at 1
location (n = 106) on d 7 and 14, and to determine
the number of CL present in pregnant cows on d 33
in 3 locations (n = 130). Pregnant cows had greater
(P < 0.05) concentrations of progesterone at the time
of treatment (d 7) compared with nonpregnant cows
(3.7 ± 0.1 vs. 2.6 ± 0.2 ng/mL, respectively). On d
14, hCG-treated cows had a greater (P < 0.05) volume of luteal tissue (12.1 ± 0.5 vs. 7.3 ± 0.5 cm3, respectively) and greater concentrations of progesterone
(6.8 ± 0.4 vs. 5.4 ± 0.5 ng/mL, respectively) compared
with saline-treated cows. A greater (P < 0.01) percentage of hCG-treated cows (90.6%) had multiple CL on
d 14 compared with saline-treated cows (0%), and a
greater percentage of pregnant cows treated with hCG
(74.6%) had multiple CL on d 33 compared with salinetreated cows (3.0%). Pregnancy rates of hCG-treated
cows (56.3%) tended (P = 0.07) to differ from those of
saline-treated cows (50.0%). Concentrations of progesterone in pregnant hCG-treated cows were greater (P
< 0.05; 7.7 ± 0.3 vs. 5.8 ± 0.3 ng/mL, respectively) on
d 33 than for pregnant saline-treated cows, but were
similar between treatments on d 68 (7.2 ± 0.3 vs. 6.7
± 0.4 ng/mL, respectively). We conclude that treatment with hCG increased the volume of luteal tissue
on d 14 and concentrations of progesterone on d 14 and
33 after TAI. Treatment with hCG tended to increase
pregnancy rates at 5 of 6 locations from 1.1 to 27 percentage points (average = 10.2) compared with saline,
but cumulative pregnancy rates determined on d 68
after TAI were similar between treatments.
Key words: beef cow, human chorionic gonadotropin, luteal function, ovulation synchronization
©2010 American Society of Animal Science. All rights reserved.
1
Sincere appreciation is expressed to Bethany Funnell (North
Central Research and Outreach Center, University of Minnesota,
Grand Rapids) and Jamie Gardner (Department of Animal Sciences
and Industry, Kansas State University, Manhattan) for assistance
in data collection and laboratory analyses. The authors thank Intervet Animal Health (Millsboro, DE) for their donation of hCG
J. Anim. Sci. 2010. 88:2337–2345
doi:10.2527/jas.2009-2596
(Chorulon) and GnRH (Fertagyl), and Pfizer Animal Health (New
York, NY) for their donation of PGF2α (Lutalyse) and CIDR inserts
(CIDR EAZI-Breed).
2
Corresponding author: [email protected]
Received October 20, 2009.
Accepted February 25, 2010.
2337
2338
Dahlen et al.
Table 1. Characteristics of cows at locations where the study was conducted
Location
Item
No. of cows
State1
Breed2
Parity
DPP3
BCS4
Cycling,5 %
1
81
KS
AN × HH
3.3 ± 0.2y
66 ± 1.6x
5.4 ± 0.05z
91.4z
2
68
KS
AN × HH
1.4 ± 0.3w
90 ± 1.7z
5.4 ± 0.06z
94.1z
3
103
MN
AN
3.5 ± 0.2y
64 ± 1.4x
5.5 ± 0.05z
41.3w
4
6
Mean
or total
KS
AN × HH
4.8 ± 0.2z
72 ± 1.3y
5.2 ± 0.04y
93.3z
506
—
—
3.2
71
5.3
74.4
5
79
56
KS
AN, SM, HH
2.4 ± 0.2x
75 ± 1.6y
5.2 ± 0.05y
58.2x
KS
AN, SM, HH
2.6 ± 0.3x
63 ± 1.9x
4.8 ± 0.06x
69.6y
119
w–z
Means within a row lacking common superscripts differ (P < 0.10).
KS = Kansas; MN = Minnesota.
2
AN = Angus; HH = Hereford; SM = Simmental.
3
DPP = days postpartum at the time of controlled internal drug release device (CIDR; EAZI-Breed CIDR containing 1.38 g of progesterone,
Pfizer Animal Health, New York, NY) insertion.
4
BCS, 1 = emaciated, and 9 = obese (Whitman, 1975), was assessed at the time of GnRH (Fertagyl, Intervet Animal Health, Millsboro, DE)
injection and CIDR insertion.
5
Percentage of cows cycling based on changes in concentrations of progesterone in blood serum collected at the time of CIDR insertion and 10
d prior.
1
INTRODUCTION
Research in the area of estrus and ovulation synchronization has produced programs that achieve pregnancy rates that meet the demands of most beef producers
while handling cattle only 3 times (Lauderdale, 2009).
The time and labor associated with implementing these
programs and the resulting variable fertility are concerns that have limited adoption and application of AI
by beef producers (National Animal Health Monitoring
Service, 2009). Development of additional synchronization programs should identify methods that may further improve pregnancy rates obtained using the current protocols.
Administration of hCG from 5 to 7 d after estrus or
AI increased concentrations of progesterone in lactating dairy cows (Santos et al., 2001; Hanlon et al., 2005;
Stevenson et al., 2007), dairy heifers (Diaz et al., 1998;
Chagas e Silva and Lopes da Costa, 2005), beef cows
(Nishigai et al., 2002; Machado et al., 2008), and beef
heifers (Funston et al., 2005; Walker et al., 2005). Increased concentrations of progesterone have been associated with increased conceptus growth rates (Garrett
et al., 1988). Interferon-τ (INFτ), the factor associated
with maternal recognition of pregnancy in cattle, is
produced by the trophoblast of the growing conceptus
(Demmers et al., 2001), and concentrations of INFτ are
closely related (R2 = 0.83) to embryo length (Mann
et al., 2006). Increased concentrations of progesterone
may result in increased conceptus growth rates and a
greater proportion of pregnancies that are maintained
to term.
Administration of hCG has the potential to increase
the fertility of cows when administered after AI by inducing formation of ancillary luteal tissue. Approximately 1 wk after estrus, a large percentage of firstwave dominant ovarian follicles ovulate in response to
GnRH (Vasconcelos et al., 1999). Further, 1,000 IU of
hCG was as effective as GnRH in ovulating follicles
in suckled beef cows (Burns et al., 2008). The goal of
this research was to determine whether administration
of hCG to suckled beef cows 7 d after a fixed-time AI
(TAI) could alter ovarian response, concentrations of
progesterone, and increase pregnancy rates.
MATERIALS AND METHODS
All cows in this experiment were managed according to guidelines set forth in the Guide for the Care
and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999), and all procedures
were approved by the Institutional Animal Care and
Use Committees at the University of Minnesota and at
Kansas State University.
Animals and Treatments
Spring-bred cows from 6 locations in 2 midwestern
states were used in this study. Herd size ranged from
57 to 119 cows. Mean BCS assessed at the onset of the
ovulation synchronization program (scale of 1 to 9; 1
= emaciated, 9 = obese; Whitman, 1975) was 5.3, with
a range of 4.0 to 7.0. Mean days postpartum was 71.3,
with a range of 18 to 110 d. Mean parity was 3.2, with
a range of 1 to 14. Differences existed among locations
for all traits summarized except d 33 pregnancy rates
(Table 1).
Ovulation was synchronized in cows by administering 100 μg of GnRH intramuscularly (i.m.; 2 mL of
Fertagyl, Intervet Animal Health, Millsboro, DE) and a
controlled internal drug release device (CIDR; EAZIBreed CIDR containing 1.38 g of progesterone, Pfizer
Animal Health, New York, NY), followed in 7 d by 25
mg of PGF2α i.m. (5 mL of Lutalyse, Pfizer Animal
Health) and CIDR removal, followed in 64 h by TAI
and a second injection of GnRH (CO-Synch + CIDR
protocol; Larson et al., 2006). Cows were stratified by
days postpartum, BCS, and parity before random as-
Human chorionic gonadotropin after artificial insemination
2339
Figure 1. Schematic diagram of the experimental protocol for suckled beef cows treated with hCG or saline 7 d after fixed-time AI (TAI).
GnRH = 100 µg of intramuscular (i.m.) injection of GnRH (Fertagyl, Intervet Animal Health, Millsboro, DE); CIDR = treatment with a controlled internal drug release insert containing 1.38 g of progesterone (EAZI-Breed CIDR, Pfizer Animal Health, New York, NY); PG = 25 mg
i.m. injection of PGF2α (Lutalyse, Pfizer Animal Health); hCG = 1,000 IU i.m. injection of hCG (Chorulon, Intervet Animal Health); saline = 1
mL i.m. injection of physiological saline.
signment to receive 1 of 2 treatments, 7 d after TAI: 1)
1 mL of physiological saline i.m. (n = 252), or 2) 1,000
IU of hCG i.m. (1 mL of Chorulon, Intervet Animal
Health; n = 254; Figure 1).
At locations 1, 2, 4, 5, and 6, exposure to live bulls
(cleanup bulls) was not initiated until a minimum of
10 d after AI to ensure a detectable difference between
pregnancies initiated by AI and those resulting from
natural matings by cleanup bulls. At location 3, cows
were visually observed a minimum of 3 times daily (at
least 45 min each) for estrus beginning immediately
after TAI until the completion of a 67-d breeding season. Inseminations were performed using the morningevening rule and were performed 9 to 14 h after the
first detected estrus.
centrations of progesterone at locations 1, 2, 4, 5, and
6 were measured via RIA (Skaggs et al., 1986). Interand intraassay CV were 10.0 and 6.2%, respectively.
Repeated pool samples averaged 3.96 ± 0.39 ng/mL
in 16 assays (n = 48). Concentrations of progesterone
at location 3 were analyzed by RIA using progesterone
kits (Coat-A-Count, Siemens Medical Solutions Diagnostics, Los Angeles, CA). The assay kit was validated
for bovine serum (Kirby et al., 1997) using an assay
volume of 100 μL. Assay tubes for the standard curve
contained 0.01, 0.025, 0.05, 0.2, 0.5, 1, 2, and 4 ng of
progesterone/tube. Assay sensitivity for a 100-μL sample was 0.1 ng/mL. Pooled samples produced intra- and
interassay CV of 9.3 and 10.5%, respectively.
Blood Samples and RIA
Ultrasound of Ovarian Structures
and Pregnancy Diagnosis
Blood samples were collected in 10-mL Vacutainer
tubes (BD Worldwide, Franklin Lakes, NJ) that did
not contain additive, via tail venipuncture on d −21
and −10, and on the day of treatment (d 7) at 5 locations (locations 1, 2, 4, 5, and 6), on d 14 at 1 location
(location 3), on d 33 at all locations, and on d 68 at 5
locations (locations 1, 2, 4, 5, and 6). Blood was refrigerated for up to 24 h after collection and centrifuged at
1,500 × g for 10 to 15 min at 3°C; serum was decanted
into storage vials and stored at −20°C until analyzed
for concentrations of progesterone. Concentrations of
progesterone in blood samples collected on d −21 and
−10 were used to determine the percentage of cows cycling before initiation of the ovulation-synchronization
breeding program. When either of the 2 blood samples
had concentrations of progesterone ≥1 ng/mL, the cow
was considered to be cycling (Lamb et al., 2001). Con-
The ovaries of cows at location 3 were examined
by transrectal ultrasonography (7.5-MHz linear array
transducer, Aloka 900V, Corimetrics Medical Systems
Inc., Wallingford, CT) before treatment and 7 d later
(7 and 14 d after TAI), and all ovarian structures were
mapped to monitor changes in corpora lutea (CL) and
follicles in response to treatment. The vertical and horizontal diameter of the largest follicle on each ovary and
all CL were measured on d 7, and the vertical and horizontal diameter and location of all CL were measured
on d 14. Volume of CL tissue was calculated using the
formula V = 4/3πr3, where r was one-half the average
value for vertical and horizontal CL measurements. In
cases in which CL had fluid-filled cavities, the volume
of the cavity was subtracted from the total volume of
the CL, resulting in a value that reflected the estimated
luteal tissue volume of each CL. Progesterone per cubic
2340
Dahlen et al.
centimeter of luteal tissue on d 14 was calculated by
dividing concentrations of progesterone (ng/mL) by the
total volume of luteal tissue (cm3) present at the time
of ovarian scan and concurrent blood collection.
Because ovulation in all cows had previously been
synchronized, the majority were expected to have a CL
present at the time of treatment. Ovulation occurred if
a new CL was detected in either ovary 7 d after treatment in the same ovarian location where a follicle was
previously identified before treatment, or when a single
CL was detected 7 d after treatment when no CL was
present at treatment. The ovulatory follicle was defined
as the largest follicle present in either ovary at treatment that was subsequently replaced by a CL detected
7 d later on the same ovary.
Transrectal ultrasonography (7.5-MHz linear array
transducer, Aloka 900V, at location 3 or 5.0-MHz linear array transducer, Aloka 500, at locations 1, 2, 4,
5, and 6, Corimetrics Medical Systems Inc.) was used
on d 33 and 68 to determine the presence of a viable
conceptus at all locations and on d 33 and to determine
the number of CL in pregnant cows at locations 3, 4,
and 5 (n = 130).
Statistical Analyses
The GENMOD procedure (SAS Inst. Inc., Cary, NC)
was used to analyze all binomial data, and the GLM
procedure (SAS Inst. Inc.) was used to analyze noncategorical data. Means were separated by using the least
significant difference in the GLM procedure when a
protected F-test (P ≤ 0.05) was detected by ANOVA.
Concentrations of progesterone at the time of treatment were analyzed using the GLM procedure. The
model consisted of location, cycling status, pregnancy
status determined on d 33, and all 2-way interactions.
Days postpartum and BCS at initiation of the breeding
program were regression covariables.
Effects of treatment on volume of luteal tissue and
concentrations of progesterone on d 14 at location 3
were analyzed using the GLM procedure. The model
consisted of treatment (saline or hCG), cycling status,
pregnancy status determined on d 33, and treatment
interactions with cycling status and pregnancy status.
In addition, days postpartum and BCS at initiation of
the breeding program were regression covariables.
Pregnancy rate on d 33 and 68, pregnancy loss from
d 33 to 68, and incidence of multiple CL on d 33 were
analyzed using the logistic regression procedures GENMOD and GLM. The model included treatment (saline
vs. hCG), cycling status before onset of the breeding
program, location, treatment × location, treatment ×
cycling status, sire nested within location, AI technician nested within location, as well as BCS and days
postpartum as regression variables. Three cows at location 3 were not included in models containing cycling
status and BCS terms because 2 were missing data for
cycling status and 1 was missing BCS data. Between
Table 2. Pregnancy rates on d 33 and 68 after a fixedtime AI in suckled beef cows treated with saline or hCG
on d 7 after AI
Treatment,1 %
(total No. of cows)
Item
Saline
Pregnancy rate, d 33
Location 1
Location 2
Location 3
Location 4
Location 5
Location 6
Cumulative pregnancy rate, d 68
50.0
40.0
45.7
58.8
46.3
59.3
50.0
78.3
hCG
x
(252)
(40)
(35)
(51)
(41)
(27)
(58)
(249)
56.3
51.2
72.7
50.0
47.4
62.1
59.0
80.2
(254)y
(41)
(33)
(52)
(38)
(29)
(61)
(253)
x,y
Means within a row lacking common superscripts tended (P =
0.07) to differ.
1
Cows received either 1 mL of saline or 1,000 IU of hCG (Chorulon,
Intervet Animal Health, Millsboro, DE) on d 7 after a fixed-time AI.
d 33 and 68 after TAI, 4 cows were removed from the
study and were excluded from any analyses performed
using fertility data from d 68.
The chi-squared analysis of SAS (FREQ procedure)
was used to evaluate the effects of treatment on percentage of pregnant cows having 1 or 2 CL on d 33.
Concentrations of progesterone on d 33 and 68 in pregnant cows were evaluated using the GLM procedure.
The model included the effects of treatment (saline and
hCG), cycling status, location, treatment interactions
with location and cycling status, as well as BCS, and
days postpartum as regression covariables.
RESULTS
Fertility
Pregnancy rates on d 33 tended (P = 0.07) to be
greater for cows treated with hCG (56.3%) compared
with saline (50.0%; Table 2). In 5 of the 6 locations,
pregnancy rates ranged from 1.1 to 27.0 percentage
points (average = 10.2%) greater for cattle treated with
hCG compared with saline. In contrast, no differences among treatments were detected when cumulative
pregnancies were confirmed on d 68. For each whole
unit increase in BCS (range 4 to 7), pregnancy rate
on d 68 increased 9.9% (P < 0.05). Compared with
cows that were cycling at the initiation of the breeding
protocol, noncycling cows had similar pregnancy rates
on d 33 but lesser pregnancy rates (P < 0.01) on d 68
(Figure 2). A treatment × cycling status interaction
(P < 0.05) was detected for pregnancy loss from d 33
to 68. Cows treated with hCG that were not cycling
(8.8%; 3/34) had greater (P < 0.05) loss than cycling
hCG-treated cows (0.9%; 1/109), whereas no difference
in pregnancy loss was detected for cows treated with
saline that were noncycling (3.4%; 1/29) and for salinetreated cows that were cycling (3.1%; 3/97).
2341
Human chorionic gonadotropin after artificial insemination
Figure 2. Pregnancy rates of cycling and noncycling cows on d 33
and 68 after fixed-time AI. x,yMeans on d 68 differ (P < 0.01).
Concentrations of Progesterone and CL
At location 3, a total of 102 of 106 cows evaluated
via ultrasound before treatment on d 7 had visible CL
tissue (96.2%). Based on the appearance of new luteal
structures on d 14, a greater (P < 0.01) proportion of
cows ovulated in response to hCG (90.6%) than after
saline (0%; Table 3). Of the cows treated with hCG
that ovulated, accessory CL were present on d 14 on
the ovary ipsilateral to the original CL in 56% (27/48)
of cows, and on the ovary contralateral to the original CL in 44% (21/48) of cows. No differences (P =
0.11) were detected between treatments in volume of
the largest CL present on d 14 on the ovary ipsilateral
to the original CL observed on d 7. Although hCGtreated cows had greater (P < 0.05) total luteal tissue
volume and concentrations of progesterone 7 d posttreatment, saline-treated cows had greater (P < 0.05)
concentrations of progesterone per cubic centimeter of
luteal tissue (Table 3). At location 3, all cows treated
with hCG that developed accessory CL and became
pregnant had accessory CL visible via ultrasound on d
33 (25 of 25 cows).
Cows that were pregnant had greater (P < 0.01) concentrations of progesterone at the time of treatment
Figure 3. Concentrations of progesterone on d 33 and 68 in pregnant cows treated with saline or hCG (Chorulon, Intervet Animal
Health, Millsboro, DE) 7 d after fixed-time AI. x,yMeans on d 33 differ
(P < 0.01).
than cows that were not pregnant. For every whole unit
increase in BCS, concentrations of progesterone on d 7
increased (P < 0.05) by an average of 0.35 ± 0.15 ng/
mL. Of the cows that became pregnant, concentrations
of progesterone were greater (P < 0.01) on d 33 for
those treated with hCG compared with those treated
with saline. This difference was no longer detectable
on d 68 (Figure 3). Concentrations of progesterone in
pregnant cows on d 33 were affected by BCS; for each
whole unit increase in BCS, concentrations of progesterone increased by 0.66 ± 0.34 ng/mL. This relationship between BCS and progesterone, however, was not
present on d 68.
The proportion of pregnant cows with multiple CL
present on d 33 was greater (P < 0.01) for cows treated
with hCG (74.6%) compared with cows treated with
saline (3.0%; Table 4). One hCG-treated pregnant cow
(1.6%) evaluated on d 33 had 3 CL, and thus may have
experienced a double-induced ovulation in response to
hCG. In addition, a treatment × location interaction
was present (P < 0.05); a greater (P < 0.05) proportion
of pregnant cows from location 3 (92.3%) that were
treated with hCG had multiple CL compared with cows
from location 4 (66.7%) and location 5 (55.5%) that
were treated with hCG. Of cows treated with hCG that
Table 3. Effects of treating suckled beef cows with saline or hCG 7 d after AI on ovarian dynamics and concentrations of progesterone 7 d posttreatment (d 14)
Treatment1
Item
No. of cows
Volume of luteal tissue on d 7, cm3
Induced ovulation,2 %
No. of corpora lutea on d 14
Largest corpus luteum volume on d 14, cm3
Total corpus luteum volume on d 14, cm3
Progesterone on d 14, ng/mL
Progesterone per unit of luteal tissue,3 (ng/mL)/cm3
x,y
Saline
hCG
51
8.8 ± 0.53
0.0x
1.0 ± 0.03x
7.4 ± 0.46
7.3 ± 0.51x
5.4 ± 0.46x
0.79 ± 0.05x
51
9.0 ± 0.51
90.6y
1.9 ± 0.03y
8.4 ± 0.44
12.1 ± 0.48y
6.8 ± 0.43y
0.58 ± 0.05y
Means within a row lacking common superscripts differ (P ≤ 0.05).
Cows received either 1 mL of saline or 1,000 IU of hCG (Chorulon, Intervet Animal Health, Millsboro, DE)
on d 7 after fixed-time AI.
2
Ovulation resulting from treatment administration 7 d after AI; includes 3 cows treated with hCG that did
not have corpora lutea present at the time of treatment.
3
Concentration of progesterone on d 14 divided by total volume of luteal tissue determined via transrectal
ultrasound at the time of blood collection.
1
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Dahlen et al.
Table 4. Location and number of corpora lutea (CL)
present in pregnant, suckled beef cows on d 33 after
treatment with saline or hCG on d 7 after fixed-time
AI
Treatment,1 % (total No. of cows)
No. of CL
One
Right ovary
Left ovary
Two or more
Right ovary
Left ovary
Both ovaries
Saline
hCG
x
25.4y (16)
9.5 (6)
15.9 (10)
74.6y (47)
38.1 (24)2
6.3 (4)
30.2 (19)
97.0 (65)
70.1 (47)
26.9 (18)
3.0x (2)
0.0 (0)
0.0 (0)
3.0 (2)
x,y
Means within a row lacking common superscripts differ (P <
0.01).
1
Cows received either 1 mL of saline of 1,000 IU of hCG (Chorulon,
Intervet Animal Health, Millsboro, DE) on d 7 after fixed-time AI.
2
One cow had 3 CL.
ovulated, accessory CL were present at d 14 on the
ovary ipsilateral to the original CL in 56% (27/48) of
cows, and on the ovary contralateral to the original CL
in 44% (21/48) of cows.
DISCUSSION
Fertility
Pregnancy rates on d 33 of the current study tended
to be greater for cows treated with hCG on d 7 compared with those treated with saline. Effects of administration of hCG after AI on pregnancy rates have varied. Increased 28-, 45-, and 90-d pregnancy rates were
observed when lactating dairy cows were treated with
hCG on d 5 after estrus (Santos et al., 2001). In addition, administration of hCG between d 4 and 9 after
AI increased conception rates, but only in 3 of 5 herds
(Stevenson et al., 2007). When administered on d 6 of
the estrous cycle to Japanese Black beef cattle, 1,500
IU of hCG increased pregnancy rates of frozen-thawed
embryos transferred on d 7 (68 vs. 45%, respectively;
Nishigai et al., 2002). In contrast, treating lactating
dairy embryo transfer recipients with hCG 5 d after
ovulation failed to increase d 28 conception rates of
embryos transferred on d 7 (Galvão et al., 2006). No
differences in pregnancy rates were observed after hCG
treatment of anestrous dairy cows or beef heifers after AI compared with untreated females (Breuel et al.,
1990; Funston et al., 2005; Hanlon et al., 2005). Pregnancy rates in the current study also were variable,
with improvements occurring at 5 of 6 locations.
Pregnancy rates of cycling and noncycling cows were
similar at d 33 but were greater for cycling cows on d
68. In our previous reports (Thompson et al., 1999;
Stevenson et al., 2000; Lamb et al., 2001; Larson et al.,
2006), we determined that ovulation synchronization
protocols that include progestogens, GnRH, or both
initiate the induction of estrous cycles in noncycling fe-
males that result in pregnancies. As a result, pregnancy
rates of noncycling cows after ovulation synchronization
with progestogens and GnRH are similar between cycling and noncycling cows (Larson et al., 2006; Wilson
et al., 2010). A large percentage of the cows that fail to
become pregnant to TAI are likely noncycling cows that
were not induced to resume estrous cycles, that fail to
return to estrous during the subsequent 30 d, and that
fail to conceive to cleanup bulls.
Induction of cyclicity by the CO-Synch + CIDR protocol may also be related to BCS at initiation of the
breeding season. For every unit increase in BCS, pregnancy rate on d 68 increased by 9.9%. In our previous
reports we noted that an increase in BCS of a single
unit resulted in an 11.5% (Larson et al., 2006) and
23% (Lamb et al., 2001) increase in the proportion of
cows pregnant to AI after ovulation synchronization. In
addition, the impact of inclusion of a CIDR into ovulation synchronization induced a greater percentage of
noncycling cows to initiate estrous cycles and enhanced
pregnancy rates when those noncycling cows were in
greater body condition or had longer postpartum intervals (Lamb et al., 2001).
Cows with multiple CL had greater d 28 conception rates than those having a single CL (Santos et al.,
2001). Moreover, hCG-treated heifers with an accessory CL had greater pregnancy rates than those without
an accessory CL (Chagas e Silva and Lopes da Costa,
2005).
Ovarian Dynamics and Concentrations
of Progesterone: d 7 to 14
Our hypothesis was that post-AI administration of
hCG would increase concentrations of progesterone either through induction of an accessory CL or stimulation of the original CL present at the time of hCG
treatment. Concentrations of progesterone in cows
monitored on d 14 were 1.4 ng/mL greater in cows
treated with hCG than in those treated with saline in
our experiment. Increased concentrations of progesterone have been observed when hCG was administered 5
to 7 d after estrus or AI in lactating dairy cows (Santos
et al., 2001; Hanlon et al., 2005; Stevenson et al., 2007),
dairy heifers (Diaz et al., 1998; Chagas e Silva and
Lopes da Costa, 2005), beef cows (Nishigai et al., 2002;
Machado et al., 2008), and beef heifers (Funston et al.,
2005; Walker et al., 2005).
As expected, more cows treated with hCG had multiple CL on d 14 than those treated with saline. Two
cows (3%) treated with saline having multiple CL may
have double ovulated in response to the GnRH injection administered concurrent with the TAI. Likewise,
a similarly small proportion of hCG-treated cows may
have double ovulated before treatment. Lactating
dairy cows that received hCG on d 5 after estrus had
a greater number of CL present between d 11 to 16
(86%) compared with those treated with saline (23%;
Human chorionic gonadotropin after artificial insemination
Santos et al., 2001). Accessory CL developed in 11 of
12 recipient cows treated 7 d after embryo transfer on
d 7 postestrus (Nishigai et al., 2002). Lactating dairy
cattle used as embryo transfer recipients were treated
5 d after GnRH-induced ovulation with hCG, resulting
in 93% of cows with accessory CL compared with 1.5%
of untreated cows (Galvão et al., 2006).
In the present experiment, none of the cows evaluated on d 7 and 14 developed multiple induced luteal
structures in response to treatment. In contrast, multiple induced luteal structures were identified in 24% of
lactating dairy cows ovulating in response to treatment
with GnRH or hCG between d 4 to 9 after AI (Stevenson et al., 2007) and in 25% of lactating dairy cows
treated with hCG on d 5 after estrus (Galvão et al.,
2006). Frequency of multiple induced ovulations after
treatment with hCG on d 5 were greater in lactating
dairy cows having dominant follicles ≤10 mm (9/17;
53%) at the time of treatment compared with cows
having dominant follicles >10 mm in diameter (22/108;
20%; Galvão et al., 2006). In the current study, 7 of the
cows monitored on d 7 had dominant follicles ≤10 mm
at the time of treatment (range 8 to 10 mm), 6 cows
ovulated a follicle, and none had double ovulations.
Discrepancies among reports regarding the incidence of
multiple ovulations may result from a greater incidence
of codominant follicles and corresponding multiple ovulations in lactating dairy cattle compared with suckled
beef cows. Alternatively, it could be due to the larger
dose of hCG administered (3,300 IU) compared with
that in the current report (1,000 IU).
Total volume of luteal tissue was greater in cows
treated with hCG than in cows treated with saline.
Accessory CL induced with hCG were smaller than the
original CL on the ovary in the study by Fricke et al.
(1993). In the present study, no difference in volume
of the largest CL on d 14 was detected between treatments. Dairy cows treated with hCG between d 4 and
9 after AI had greater total luteal volume and a greater
change in diameter of the original CL 7 d after treatment with hCG than untreated controls (Stevenson
et al., 2007). Area and volume of the largest CL and
original CL area also were increased by treatment with
hCG on d 5 after estrus (Santos et al., 2001) or induced
ovulation (Galvão et al., 2006). In addition, diameter of
the original CL, induced CL, and concentrations of progesterone were greater in crossbred beef heifers treated
with hCG than in those treated with GnRH or LH
(Binelli et al., 2001).
Although the total volume of luteal tissue and concentrations of progesterone were increased in the current
study, progesterone production per unit of luteal tissue
(ng per mL/cm3) was less in cows treated with hCG
than with saline. Although not reported by Santos et
al. (2001), calculations made using their data indicated
that both primiparous (3.2 vs. 3.8 ng per mL/cm3, for
hCG and saline-treated cows, respectively) and multiparous (1.3 vs. 1.6 ng per mL/cm3, for hCG and sa-
2343
line-treated cows, respectively) dairy cows treated with
hCG had decreased concentrations of progesterone per
unit of luteal tissue compared with saline-treated cows.
Therefore, although hCG may induce an increased
quantity of luteal tissue, progesterone secretion did not
increase at the same rate. Similarly, luteal tissue from
cows treated with hCG had less basal hormone and
LH-induced in vitro secretion of progesterone than produced by luteal tissue from untreated cows (Veenhuizen
et al., 1972; Fricke et al., 1993).
Of the cows treated with hCG that ovulated, accessory CL were present on d 14 on the ovary ipsilateral
to the original CL in 56% (27/48) of cows and on the
ovary contralateral to the original CL in 44% (21/48)
of cows. These proportions are similar to those found in
dairy heifers when 56% (52/93) developed CL ipsilateral to the original CL and 44% (41/93) developed accessory CL contralateral to the original CL after treatment with hCG (Chagas e Silva and Lopes da Costa,
2005). The proportion of lactating dairy cattle receiving hCG or GnRH 4 to 9 d after AI that had accessory
CL develop on the ovary ipsilateral to the original CL
was 32%, the proportion contralateral was 41%, and
28% developed accessory CL on both ovaries (Stevenson et al., 2007).
Cows from 5 locations that were pregnant on d 7
(n = 213) had greater concentrations of progesterone
than those that were not pregnant (n = 190). Concentrations of progesterone were greater on d 6 or 7
in pregnant, lactating dairy cows than in those that
were not pregnant (Mann et al., 1999; Demetrio et al.,
2007). In contrast, beef heifers that were not pregnant
had greater concentrations of progesterone on d 7 compared with those that were pregnant; moreover, pregnant heifers had greater concentrations of progesterone
compared with nonpregnant heifers beginning only on
d 17 (Breuel et al., 1989).
CL Dynamics and Concentrations
of Progesterone: d 33 to 68
Cows that became pregnant and were treated with
hCG 7 d after AI had greater concentrations of progesterone on d 33 compared with those treated with saline;
however, the effects of treatment were temporary because differences did not persist to d 68. Effects of hCG
stimulation of luteal cells, the additional progesterone
production, or both of the induced accessory CL that
were first observed on d 7 after treatment apparently
diminished between d 33 and 68 of pregnancy. Pregnant
Japanese Black embryo recipients that were treated
with 1,500 IU of hCG on d 6 had greater concentrations
of progesterone when evaluated between d 40 to 50 after embryo transfer (Nishigai et al., 2002). Treatment
of lactating dairy cows with hCG at the time of positive
pregnancy (d 29 to 42 after AI) determination failed to
have greater concentrations of progesterone compared
with those treated with saline (Stevenson et al., 2008).
2344
Dahlen et al.
Although concentrations of progesterone were greater
in cows with induced luteal structures during the first
2 wk after treatment at pregnancy determination (d 29
to 42), no differences were present 4 wk after treatment
(Stevenson et al., 2008).
Administration of 100 mg of progesterone from d 1
to 4 of pregnancy increased concentrations of blood
progesterone and fetal development compared with
untreated females (Garrett et al., 1988). In addition,
supplemental progesterone via CIDR insert increased
embryo development and INFτ secretion when administered from d 5 to 9 after AI, but not when administered
from d 12 to 16 (Mann et al., 2006). Previous reports
indicated that concentrations of progesterone were elevated within 2 d of hCG administration and were initially ascribed to the effects of hCG on existing luteal
tissue and later to supplemental progesterone from the
induced CL (Lewis et al., 1990; Fricke et al., 1993). In
the current study, concentrations of progesterone were
elevated after treatment with hCG from at least d 14
after AI until d 33, but not until d 68 after TAI. This
sustained increase in concentrations of progesterone
that occurred from maternal recognition of pregnancy
until early postimplantation stages of pregnancy may
result in increased pregnancy survival.
Of the cows at location 3 treated with hCG that developed accessory CL and became pregnant, accessory
CL were visible by transrectal ultrasound on d 33 in all
cases monitored (25 of 25 cows). When lactating dairy
cattle received hCG 5 d after estrus and had double
ovulations in response to treatment (determined on d
14), 7% regressed 1 CL by d 28 (Santos et al., 2001).
In addition, when lactating dairy cows were treated
with hCG at the time of pregnancy diagnosis (29 to 42
d), 39% experienced regression of induced luteal structures by 4 wk posttreatment (57 to 70 d; Stevenson et
al., 2008). From d 28 to 42, a total of 62% (18 of 29)
of Holstein heifers that developed accessory CL contralateral to the original CL after treatment with hCG
experienced regression of the induced CL, whereas no
heifers experienced regression of the accessory CL during this period that developed on the ovary ipsilateral
to the original CL (Chagas e Silva and Lopes da Costa,
2005).
In the present study, concentrations of progesterone
were increased in pregnant cows after treatment with
hCG, and treatment with hCG tended to increase pregnancy rates. Pregnant cows had greater concentrations
of progesterone on d 7 after AI than cows that were
not pregnant. Therefore, we conclude that treatment
with hCG increased the volume of luteal tissue on d
14 and concentrations of progesterone on d 14 and 33
after TAI. Treatment with hCG tended to increase
pregnancy rates, improving pregnancy rates at 5 of 6
locations from 1.1 to 27 percentage points (average =
10.2) compared with saline; however, cumulative pregnancy rates determined on d 68 after TAI were similar
between treatments.
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