Comparison of the effects of gonadotropin-releasing hormone, human
chorionic gonadotropin or progesterone on pregnancy per artificial
insemination in repeat-breeder dairy cows
B. Khoramian1, N. Farzaneh*, M. Talebkhan Garoussi, M. Mohri
Department of Clinical Sciences, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, P.O. Box: 91775-1793, Iran
a r t i c l e i n f o
Received 14 January 2008
Accepted 22 May 2010
Post insemination treatment
Pregnancy per AI
a b s t r a c t
Three different treatments were compared to improve pregnancy per artificial insemination (P/AI) in
repeat-breeder (RB) dairy cows. All cows (n = 103) were assigned to one of four groups: (1) gonadotro-
pin-releasing hormone (GnRH); (2) human chorionic gonadotropin (hCG); (3) once-used controlled inter-
nal drug release (CIDR) device; and (4) control. All treatments performed 5–6 days after artificial
insemination (AI) and milk samples were collected just before treatment for progesterone assays. There
were no significant differences in milk fat progesterone concentration among trial groups. Cows were
observed for estrus signs thrice daily. Pregnancy per AI on day 45 in hCG and CIDR groups were signifi-
cantly higher than GnRH and control groups (60.0% and 56.0% vs. 26.9% and 29.6%, respectively), but there
were no differences in P/AI between GnRH and control groups. There were also no significant differences
between hCG and CIDR groups. Milk fat progesterone concentrations were compared between pregnant
and non-pregnant cows in each group and only in the hCG group it was significantly lower in pregnant
cows. In conclusion, treating repeat-breeder cows with hCG or once-used CIDR 5–6 days after AI
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Several investigators have demonstrated lower progesterone
concentrations in plasma of cows that failed to conceive and this
was evident as early as day 6 after insemination (reviewed by
Thatcher et al., 2001). Luteal deficiency during the very early stages
of pregnancy has been hypothesized as a cause of pregnancy fail-
ures (Shelton et al., 1990). Progesterone is essential for orchestrat-
ing the histotrophic environment for nourishment of the conceptus
(Santos et al., 2004). Progesterone also inhibits luteolysis by
decreasing sensitivity to oxytocin by binding to oxytocin receptors
(Grazzini et al., 1998) and enhancing conceptus development
which, stimulates secretion of interferon-s (Mann and Lamming,
2001). Mann et al. (2006) indicated that early progesterone supple-
mentation resulted in a fourfold increase in trophoblast length and
a six fold increase in uterine concentration of interferon-s.
In repeat-breeder cows, the time when the embryo enters the
uterus and undergoes blastocoele formation (day 6–8) has been
suggested to be a critical period during which embryo death occurs
(Shelton et al., 1990). Also a slower than normal rise in progester-
one concentration and a lower total progesterone concentration
have been reported in low-fertility cows and repeat-breeder heif-
ers in the first 6 days after estrus (Shelton et al., 1990; Bage
et al., 2002).
Several methods have been used aiming to increase P/AI during
the luteal phase but these methods have been conducted with con-
flicting results. This may be related to different hormones, doses or
products used by investigators but also to the precise day at which
the various treatments were initiated. A meta-analysis of 17 pro-
gesterone supplementation studies showed that while treatment
during the first week of pregnancy resulted in an overall increase
in pregnancy rate, treatment during the second and third weeks
of pregnancy gave no overall significant increase (Mann and Lam-
An alternative strategy to increase progesterone concentrations
is use of hCG or GnRH or its agonists a few days after artificial
insemination. Administration of hCG during early luteal phase in-
duce ovulation of the first-wave dominant follicle and formation
of an accessory corpus luteum (CL). The accessory CL increases
plasma progesterone concentrations and also could decrease the
estrogenic environment during the period of pregnancy recogni-
tion (Breuel et al., 1989; Fricke et al., 1993; Schmitt et al., 1996;
Diaz et al., 1998). Diaz et al. showed that in all hCG-treated heifers,
the dominant follicle of the third follicular wave did not reach 9 to
10 mm in size until approximately day 20. Thus the potential
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* Corresponding author. Tel.: +98 5118763853; fax: +98 5118763852.
E-mail address: firstname.lastname@example.org (N. Farzaneh).
1Present address: Department of Clinical Sciences, School of Veterinary Medicine,
University of Tehran, Tehran, Iran.
Research in Veterinary Science 90 (2011) 312–315
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estrogenic follicle for hCG-treated heifers would not occur until
day 20 versus day 14 in control heifers with a two-wave follicular
cycle. Therefore, hCG treatment would decrease the estrogenic
environment during the period of pregnancy recognition (Diaz
et al., 1998). Human chorionic gonadotropin has activity similar
to LH, and after binding to LH receptors, causes small luteal cells
to increase progesterone synthesis (Stevenson et al., 2007). The
GnRH and hCG were equally effective in inducing an accessory
CL, but the subsequent increase in progesterone concentrations
was greater in hCG-treated heifers (Schmitt et al., 1996). Adminis-
tration of synthetic GnRH after AI coincides with the rise or peak of
the first follicular wave had different results on pregnancy rates in
lactating cows (Leslie et al., 1986; Schmitt et al., 1996; Bartolome
et al., 2005; Howard et al., 2006; Sterry et al., 2006). However most
of these studies focused on embryonic mortality following concep-
tion at first service, therefore conclusions may not be applicable to
Based on previous studies we hypothesized that supplementing
repeat-breeding cows with exogenous progesterone or administra-
tion of GnRH or hCG during early luteal phase could increase P/AI.
2. Materials and methods
This study was conducted over a 5 month period from January
to May 2006 with a total of 103 repeat-breeder Holstein cows in
a large commercial dairy herd in Tehran suburb, Iran. All of the se-
lected cows had a history of 3 to 7 unsuccessful inseminations with
normal interestrus intervals. They were less than 10 years of age
and had no abnormal discharge from the genital tract or history
of recent reproductive diseases. Cows were observed for estrus
signs thrice daily and inseminated according to AM–PM rule. Cows
were housed in the loose-housing dairy barns; they were milked
three times a day and fed a total mixed ration three times a day.
Body condition was scored on scale from 1 to 5 (where 1 = emaci-
ated to 5 = extremely fat) once a month (Edmonson et al., 1989). All
reproductive, health and management records were computerized
using cow-side dairy herd software (Damshid Pars, Tehran, Iran).
All the selected cows were paired according to their parity, milk
yield, number of services and body condition scores (BCS) and as-
signed to four groups:
Cows in group 1 (n = 26) received 20 lg buserelin (Receptal;
Intervet International, Holland) intramuscularly (i.m.). Cows in
group 2 (n = 25) received an i.m. injection of 1500 IU hCG (Choru-
lon, Intervet International, Holland). In group 3 (n = 25) a once-
used CIDR (Inter Ag, New Zealand) that was previously used for
7 days applied to each cow for 10 days and cows in group 4
(n = 27) received 5 ml placebo (0.9% sterile saline solution), i.m.
All cows were treated 5–6 days after AI and pregnancy diagnosis
was performed 45 days after insemination by rectal palpation.
Milk samples (15–20 ml) were collected just before treatment
only from healthy quarters into plastic tubes and frozen at
?20 ?C until separation of milk fat and subsequent progesterone
analysis. Due to the noninvasive nature and simplicity of sample
collection, the determination of progesterone in milk has become
an important tool for progesterone assay. Progesterone concentra-
tions in milk fat are much higher than in blood plasma, skim milk
or whole milk. Consequently, analysis of milk fat progesterone will
provide a wider distribution of progesterone concentrations and
increase the diagnostic possibilities, especially in repeat-breeder
cows with lower progesterone levels.
Milk samples were thawed in the room temperature and centri-
fuged at 4 ?C at 1500g for 20 min. The cream was transferred to an-
other tube and placed in a 90 ?C water bath for 5–10 min. A clear
yellow supernatant of milk fat was formed. In the event that the
fat did not separate from the milk, the samples were centrifuged
at room temperature at 1500g for 5 min and the procedure re-
peated. Ten microliter aliquots of milk fat were extracted with
phase was discarded and the methanolic phase dried. The residues
were dissolved in 0.5 ml of PBS (Waldmann et al., 2001). Progester-
one concentration was measured using a commercial enzyme-
linked immunosorbent assay (ELISA) kit (progesterone ELISA,
DRG Ins., GmbH, Germany). The sensitivity of the assay was
0.05 ng/ml and the intra- and inter-assay coefficients of variation
were 3.7 and 8.3%, respectively. The suitability of the assay for
use with cow milk fat was tested as described previously (Wald-
mann et al., 1999; Fallah Rad and Ajam, 2008). Samples have been
tested twice to achieve more accurate results.
Comparison among groups in regard of milk production, parity,
number of services, progesterone concentrations and the BCS of
cows at enrollment were performed with ANOVA.
Pregnancy per AI among four groups was compared by chi-
square analysis. Concentration of progesterone in milk fat in preg-
nant and non-pregnant cows were compared among four groups
by Kruskal–Wallis and then compared separately in each group
by Mann–Whitney analysis.
Data were analyzed using SPSS package version 9 (SPSS Inc.,
Chicago, IL, USA) and differences between groups with a P-value
60.05 were considered significant.
One hundred and three cows were assigned to one of four
groups according to parity; AI numbers; milk production and
BCS, because these factors may have correlation with conception
rates. There were no significant differences among these factors
in four groups (P > 0.05). Also there were no significant differences
in milk fat progesterone concentration among trial groups
(P > 0.05) (Table 1).
Pregnancy per AI on day 45 was significantly lower in cows in
groups 1 and 4 compared to groups 2 and 3 (P < 0.05), but there
was no differences in pregnancy per AI between group 1 and group
4 (P > 0.05). Also, there was no significant differences between
groups 2 and 3 (P > 0.05) (Table 2).
Milk fat progesterone concentrations in pregnant cows was not
significantly different among four groups, also there was no signif-
icant differences in non-pregnant cows among four groups
(P > 0.05) (Table 3).
Milk fat progesterone concentrations between pregnant and
non-pregnant cows in each group separately compared and only
in the cows in the second group (hCG) progesterone concentrations
was significantly lower in pregnant cows (P < 0.05) (Table 3).
Mean (±SE) of milk production, number of services, body condition scores (BCS), parity and milk fat progesterone concentrations between four groups before initiation of
GroupsMilk production (kg)Number of services BCS Parity Milk fat progesterone (ng/ml)
GnRH (group 1)
hCG (group 2)
CIDR (group 3)
Control (group 4)
B. Khoramian et al./Research in Veterinary Science 90 (2011) 312–315
Poor luteal activity has been associated with pregnancy failure
in cattle (Wiebold, 1988). Luteal cells from CL of subfertile cows
had decreased progesterone secretion per unit of luteal tissue
when compared with cells from cyclic and pregnant cows (Shelton
et al., 1990). The results of the present study demonstrated that
progesterone supplementation via once-used CIDR early in the lu-
teal phase (days 5–6) or administration of hCG in the same time in-
creased P/AI in repeat-breeder cows.
Administration of progesterone has been shown to increase
endometrial protein secretion and growth factors involved in
the control of early embryo development (Garrett et al. 1988)
and can enhance both secretion of anti-luteolytic interferon-s
and embryo survival (Mann et al., 2006; Stronge et al., 2005; Cha-
gas e Silva et al., 2002). Mann and Lamming (2001) indicated that
a delay in post-ovulatory rise in progesterone compromises
conceptus development which results in reduced conception
rates. However in few studies progesterone had been applied in
repeat-breeder or subfertile cows (Villarroel et al., 2004; Walton
et al., 1990; Wiltbank et al., 1956; Henrick, 1953). We used a
relatively low level of progesterone supplementation with a
once-used CIDR to minimize any down regulatory effects on
endogenous progesterone secretion. In few studies, previously
used or modified CIDR were applied to improve pregnancy rates
(Larson et al., 2007; Mann et al., 2001; Mann et al., 2006; Van
Cleeff et al., 1991) and most of them reported positive effects
on embryonic growth and conception rates. Larson et al. (2007)
detected an increase in conception rates when a previously used
CIDR was inserted between days 3 and 10 after AI. Results of our
study also showed the benefits of low dose progesterone (once-
used CIDR) supplemented in repeat-breeder cows.
Conflicting results on conception rates of cows receiving hCG or
GnRH were achieved by many authors. While some studies have
shown increase conception rates as a result of the administration
of hCG on day 4 (Breuel et al., 1989), day 5 (Santos et al., 2001)
and day 7 (Rajamahendran and Sianangama, 1992) after AI, others
have reported no effects or reduced conception rates when hCG
was given on day 4 (Tefera et al., 2001), day 5 (Walton et al.,
1990; Schmitt et al., 1996; Hanlon et al., 2005) or day 6 (Funston
et al., 2005) after insemination. Also, some studies reported no sig-
nificant increase on conception rates after administration of GnRH
agonists between 1 and 9 days after AI (Macmillan et al., 1986; Les-
lie et al., 1986; Schmitt et al., 1996; Bartolome et al., 2005; Howard
et al., 2006; Sterry et al., 2006; Stevenson et al., 2007).
Administration of hCG or GnRH after insemination at specific
times coincident with the presence of the dominant follicle/s of
the first and second follicular waves may stimulate CL function, in-
duce accessory CL formation, increase progesterone and reduce
estrogen production with a consequent positive effect on embryo
survival (Thatcher et al., 2003). It seems that timing of administra-
tion, potency of drugs, or both, may determine ability of GnRH to
Schmitt et al. (1996) reported that heifers treated with hCG con-
sistently had a greater luteinization of the preovulatory follicle and
greater concentrations of progesterone when compared with heif-
ers treated with buserelin. This may be due to differences in
dynamics of LH-like exposure induced by these two pharmacolog-
ical agents that produce an induced CL with different steroidogenic
capabilities. The majority of LH receptors are expressed in the
small luteal cells (Fitz et al., 1982), and small luteal cells are trans-
formed into large luteal cells during CL development (Alila and
Hansel, 1984). Human chorionic gonadotropin also has a longer
half life than GnRH; therefore it can persist longer in the
We were not able to improve P/AI with higher dose of GnRH.
The lack of effect of GnRH in this study is in agreement with other
studies. Leslie et al. (1986) administrated 250 lg Cystorelin on the
fourth day after AI and reported that pregnancy rates were not sig-
nificantly different between GnRH-treated and control cows. In
other study pregnancy rate was not increased in lactating dairy
cows treated with 100 lg Cystorelin, either on day 5 or 15 after
timed insemination (Bartolome et al., 2005). Perhaps hCG induces
a greater luteinization of the preovulatory follicle and subsequent
differentiation into an active CL than does buserelin. Administra-
tion of hCG or buserelin also may have differential effects on the
original CL that could account for differences in plasma progester-
one (Schmitt et al., 1996).
In present study, milk fat progesterone concentrations in preg-
nant cows on day 5–6 after insemination in hCG group were lower
than non-pregnant cows (P < 0.05). This indicated that hCG admin-
istration in cows with lower progesterone have more benefits than
normal cows. Starbuck et al. (1999) reported that highest preg-
nancy rates occur when day 5 milk progesterone concentrations
are between 3 and 9 ng/ml and pregnancy rates significantly de-
crease both below and above this range.
In conclusion this study demonstrated that post insemination
early stages of pregnancy improved P/AI in repeat-breeder cows.
subsequent concentrations of
Conflict of interest statement
This work was supported by research fund no. 1/645 of Fer-
dowsi University of Mashhad, Iran. Authors would like to thanks
Salehi-Saffari (Chaltasion) dairy farm manager, Mr. Shahryar Saf-
fari for his assistance and collaboration.
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Pregnancy per AI among four groups in 45 days after AI.
a,bGroup with different superscripts are significantly different at P < 0.05.
Milk fat progesterone concentrations (ng/ml) on day 5–6 in pregnant and non-
Groups Pregnancy statusMedian Range
(1) GnRH Positive
a,bGroup with different superscripts are significantly different at P < 0.05.
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