ITALIAN JOURNAL OF ANIMAL SCIENCE

2021, VOL. 20, NO. 1, 1882–1888

https://doi.org/10.1080/1828051X.2021.1925169

PAPER

The effect of various administration routs of D-cloprostenol at the time of

artificial insemination on conception rate in dairy cattle
Mohammad Javad Behzadi-Shahrbabaka, Naser Shams-Esfandabadib,c, Abolfazl Shirazib,c,d and
Taghi Taktaz-Hafshejanie
a
Depertment of Clinical Sciences, Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran; bDepartment of Clinical Sciences,
Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran; cResearch Institute of Animal Embryo Technology,
Shahrekord University, Shahrekord, Iran; dReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran,
Iran; eDepartment of Animal Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord Azad University, Shahrekord, Iran

ABSTRACT ARTICLE HISTORY

The aim of the present study was to evaluate the effect of various administration routes of Received 17 June 2020
PGF2a analogue, D-cloprostenol, at the time of artificial insemination (AI) on conception rate in a Revised 9 April 2021
large dairy herd. A total number of 659 lactating dairy cows (101 heifers and 558 cows) were Accepted 15 April 2021
enrolled in the study. Following heat detection, the cows were artificially inseminated according
KEYWORDS
to the AM-PM rule. The animals were divided into a control (29 heifers and178 cows) and three Prostaglandin F2a;
experimental groups; IM (53 heifers and 110 cows), IV (155 cows) and IU (19 heifers and115 D-cloprostenol; artificial
cows) groups that received intramuscular 150 mg, intravenous 150 mg and intrauterine 5 mg of D- insemination; conception
cloprostenol, respectively, at the time of AI. The control group did not receive any treatment at rate; dairy cattle
the time of AI. To measure serum progesterone concentration, blood samples were obtained
from 15 cows in each group at the time of insemination and on days 4 and 15 post-insemin-
ation. Pregnancy was diagnosed by rectal ultrasound examination between days 35-40 post-
insemination. The effects of treatments, parity, milk production, and days in milk (DIM) on con-
ception rate of adult cows were evaluated using the multivariate logistic regression model.
Intramuscular administration of D-cloprostenol at the time of AI significantly increased concep-
tion rate in dairy cows compared to the control group (60.1% vs. 40.6%; p ¼ .005). However,
intravenous and intrauterine administration of D-cloprostenol did not alter the conception rate
(32.3%; p ¼ .29 and 50%; p ¼ .11, respectively). D-cloprostenol treatment at AI could not signifi-
cantly increase the conception rate of heifers followingboth (IM & IU) administration routes. In
conclusion, this study showed that intramuscular administration of D-cloprostenol at the time of
insemination might improve the conception rate in adult cows, while its intravenous and intra-
uterine administration did not induce any positive effects on reproductive efficiency of
dairy cows.

HIGHLIGHTS
 Intramuscular administration of D-cloprostenol at the time of insemination might improve
the conception rate in adult cows.
 Intravenous administration of D-cloprostenol did not induce any positive effects on repro-
ductive efficiency of dairy cows.
 Intrauterine administration of D-cloprostenol did not induce any positive effects on concep-
tion rate of dairy cows.

Introduction
The decline in reproductive performance and fertility Rajala-Schultz and Frazer 2003). Moreover, reduction
in dairy cows has significantly influenced the eco- in conception rate is more serious during the hot sea-
nomic efficiency of dairy industry in recent decades sons worldwide (De Rensis and Scaramuzzi 2003;
(Lucy 2001). Conception rates to artificial insemination Torres-Ju
nior et al. 2008; Bernabucci et al. 2014;
have dramatically dropped since 1950s (Butler 1998; Schu€ller et al. 2014).

CONTACT Dr. Naser Shams-Esfandabadi drn_shams@yahoo.com Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord
University, Shahrekord, Iran
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 ITALIAN JOURNAL OF ANIMAL SCIENCE 1883

In clinical trials, various reproductive hormones Material and methods

have been prescribed before, during and after AI
Farm and reproductive management
to increase the conception rate after
insemination(Friedman et al. 2012; Friedman et al. This study was conducted in a well-managed herd of
2014). Hormonal treatments at the time of AI are 4000 dairy cattle in Shahrekord of Chaharmahal and
mainly consisted of GnRH and hcG administration, Bakhtiari province in central region of Iran (32 190 3200
which are mostly used to increase synchronisation N, 50 510 5200 E; height above sea level: 2060 m).The
between AI and ovulation time. Administration of average of daily minimum, maximum and mean air
these two hormones have been reported to result in temperature during the study period was 10.5, 32.9
different outcome and has been sometimes used as a and 21.9  C, respectively, and the average of daily min-
common protocol in dairy farms (Kharche and imum, maximum and mean relative humidity was
Srivastava 2007; De Rensis F et al. 2008; Paksoy and %10.41 and % 50.8 and 26, respectively.All dairy cows
Kalkan 2010; Gu €men et al. 2011). were of the Holstein breed. Animals were fed with a
Another hormone whose effects at the time of AI Total Mixed Ration (TMR) based on their milk yield lev-
has been studied is prostaglandin F2a, which has been els. The cows were milked three times a day. The aver-
used as a luteolytic agent in dairy cattle for oestrus age daily milk production was 40 kg per cow. The
synchronisation, regression of corpus luteum, induc- cows were kept in a free stall barn on concrete
tion of abortion, parturition and treatment of uterine flooring.To reduce the effects of high temperature on
infections (Wenzel 1991; Risco et al. 1994). There is cow’s performance in summer season, misting fans
evidence that PGF2a plays an important role in ovula- were used in the barns. Also, the lack of side walls in
tion, in addition to its luteolytic properties. The results the barns led to better air flow.
of previous studies conducted on ovarian follicles of A veterinarian examined ovarian and uterine health
different mammals have indicated that PGF2a concen- of the post-parturient cows through rectal palpation
trations increase immediately prior to ovulation at the and ultrasonography, 30-35 days after parturition. The
follicular level (Hedin et al. 1987; Dozier et al. 2008). cows were assigned to a voluntary waiting period
Moreover, it has been reported that administration of (VWP) of 50 days. The herd average days open was
prostaglandin F2a inhibitors prevents ovulation 120 over the period study.
(Edelman et al. 2013). As well as, it has been found
that prostaglandins play a critical role in follicular rup-
Animals and treatments
ture (Sirois et al. 2004). On the other hand, several
clinical trials have reported that, administration of A total of 558 cows and 101 heifers that became artifi-
PGF2a during AI, induces a positive effect on the cially inseminated over a 30-day period from 1 August
induction and synchronisation of ovulation in cows, 2014 to 6 September 2014 were included in this study.
which could consequently enhance conception rate All the animals were clinically healthy at the time of
(Lopez-Gatius et al. 2004; Leonardi et al. 2012; Pfeifer artificial insemination. The cows were in different par-
L et al. 2014). Gallo et al. (Gallo et al. 1992) also ity, milk yield level and days in milk (DIM).Therefore,
reported synchronisation of ovulation improves in they were different in the number of insemination.The
super-ovulated heifers treated with cloprostenol mean days in milk at the time of AI was 143 and 121
immediately before the onset of ovulation. A few stud- for primiparous and multiparous, respectively. The
ies have examined the effect of PGF2a administration cows were artificially inseminated after observation of
at the time of AI on conception rate of dairy and beef heat synch protocol- induced oestrus (day 0: 10 lg
cattle, that have reported varying results in different GnRH analogue, Gonadoreline, Aboureihan company,
conditions, including season of the year (warm or Iran; day 7: 150 lg D-cloprostenol, Rooyan Darou, Iran;
cold) (Lopez-Gatius et al. 2004), route of administra- day 8: 1 mg oestradiol benzoate, Aboureihan, Iran)
tion (Gabriel et al. 2011), parity (Lopez-Gatius et al. based on AM/PM rule by 4 skilled technicians while
2004), and dose of administered hormone (Ambrose artificial insemination of heifers was done following
et al. 2015). The aim of this study was to evaluate the spontaneous oestrus in the same way from 13 months
effect of administration of PGF2a analogue, D-clopros- onwards. No significant difference was detected in
tenol, at the time of AI on the conception rate in dairy conception rates obtained by each technician.At the
cattle in different conditions, including parity, milk time of AI, the heifers and Cows were randomly div-
yield, days in milk (DIM) and route of administration, ided into one control and three experimental groups;
in an industrial dairy farm in central regions of Iran. namely IM group(n ¼ 163: 53 heifers, 29 primiparous
1884 M. J. BEHZADI-SHAHRBABAK ET AL.

Table 1. Odds ratios of the treatment in the univariate logis- Statistical analyses
tic regression models for conception rate of heifers.
Total Conception rate% (n) Odds ratio p Value
The effects of D-Cloprostenol treatment, parity, milk yield
IM 53 73.6 (39) 1.702 .281 level, days in milk (DIM) and their interactions on con-
IU 19 84.2 (16) 3.259 .109 ception rate and double ovulation rate were investigated
Control 29 62.1 (18) – –
using the multivariate logistic regression model in SPSS
IM: intramuscular; IU: intrauterine.
software. The effects of treatments on conception rate of
heifers were analysed separately using univariate logistic
and 81 multiparous) that received 150 lg D-cloproste- regression. An overall analysis of serum progesterone
nol intramuscularly, IV group (n ¼ 155: 45 primiparous concentrations was repeatedly measured by ANOVA
and 110 multiparous) that received 150 lg D-clopros- using generalised linear procedure (GLM) in SAS.
tenol intravenously through milk vein, and IU group
(n ¼ 134: 19 heifers, 29 primiparous and 86 multipar- Results
ous) that received intrauterine infusion of 5 lg D-clo-
prostenol. The control group (n ¼ 207: 29 heifers, 56 The results of logistic model for the effect of treat-
primiparous and 122 multiparous) did not receive any ment on conception rate of heifers are shown in
Table.1. Neither intramuscular nor intrauterine admin-
treatment at the time of AI. Due to the invisibility of
istration of D-cloprostenolcould significantly increases
the milk vein in heifers they were not enrolled in the
the conception rate of heifers at the time of AI.
IV group. In the IU group, 5 lg of D-Cloprostenol was
According to the results of multivariate logistic
diluted in 0.5 mL normal saline followed by being
regression model with backward variable selection,
packaged inside a straw for intrauterine infusion by an
the effect of milk production level on conception rate
insemination pipette and similar to AI process infused
of adult cows was not significant and eliminated.
into uterine body immediately after insemination.
Therefore, the final model for the conception rate
Based on the herd reproductive management program
included parity, DIM and treatment variables.
in summer season, a Controlled Internal Drug Release Interactions of all the above variables with each other
CIDR (Cue-Mate, Bioniche Animal Health, Bellevile, were excluded from the logistic model. As shown in
New Zealand) was placed inside all the cow between Table 2, intramuscular administration of D-cloproste-
days 4 to 11 post-insemination. nolatthe time of AI in primiparous and multiparous
Pregnancy was diagnosed 35-40 days after AI by significantly increased the conception rate compared
transrectal ultrasonography (CTS-3300V, SIUI, to the control group(p ¼ .005). However, no significant
Guangdong, China). Moreover, ovaries of the pregnant differences were detected in conception rate in the
cows were simultaneously scanned for existence of groups treated with intravenous and intrauterine
two corpus luteums which are indicator of dou- administration of D-cloprostenol (p > 0.05).
ble ovulation. The mean serum progesterone concentrations of
various therapeutic groups on days0, 4, and 15 post-
insemination are presented in Table 3. No significant
Progesterone measurement differences in serum progesterone level were detected
between different treatment groups. Progesterone
Blood samples were collected from 15 cows of each
concentration on day 4 post-insemination was not sig-
experimental group by coccygeal venipuncture on
nificantly enhanced compared to that on day 0, how-
days 1, 4 and 15 post-insemination to measure the
ever, a significant increase in progesterone
serum progesterone level. All the samples were centri- concentration was detected on day 15compared to
fuged and serum was separated and stored at 20  C. those on days 0 and 4 post-insemination.
Progesterone concentration was measured by None of the treatments, parity, milk production
Electrochemiluminescence immunoassay (Elecssys level and DIM variables induced a significant effect on
2010, joint product from Hitachi of Japan and Roche double ovulation; therefore, they were removed from
of Germany, special kit made by Roche Company- the model.
Germany). Measurement interval in this method was
from 0.03 to 60 ng/mL of progesterone. And the val-
ues obtained from the samples of the present study
Discussion
were between 0.03 (only one sample) to 17.6 ng/mL of The effects of administration of PGF2a at the time of
progesterone. AI on conception rate in beef and dairy cattle have
 ITALIAN JOURNAL OF ANIMAL SCIENCE 1885

Table 2. Odds ratios of the included variables in the final multivariate logistic regression models (treat-
ments, parity and DIM) for conception rate of adult cows.
Total Conception rate % Odds ratio 95% confidence interval p Value
Control 207 40.6 1 –
IM 163 60.1 1.911 1.21  3.01 .005
IV 155 32.3 0.722 0.48  1.08 .294
IU 134 50 1.265 0.82  1.96 .110
Primiparous 159 44.3 1 – –
Multiparous 399 39.1 0.688 0.51  0.92 .014
DIM  100 265 43.02 1 – –
DIM > 100 293 38.23 0.689 0.51  0.93 .015
IM: intramuscular; IV: intravenous; IU: intrauterine; DIM: days in milk.

Table 3. Mean serum progesterone level (s) (ng/mL) of differ- (Archbald et al. 1992; Kauffold et al. 2009; Gabriel et
ent groups on days 0, 4, and 15post-insemination. al. 2011; Sauls et al. 2018).
Insemination day After 4 day After 15 day Administration of D-cloprostenol at the time of AI
Control 0.77 ± 0.6a 1.64 ± 0.62a 6.5 ± 0.66b in the two tested routes could not significantly
IM 0.37 ± 0.7a 1.02 ± 0.72a 8.94 ± 0.7b improve the conception rate of heifers. In the heifers,
IV 0.46 ± 0.62a 0.77 ± 0.66a 8.23 ± 0.66b
IU 0.68 ± 0.72a 1.03 ± 0.85a 8.72 ± 0.91b administration of D-cloprostenol was inconclusive that
Total 0.57 ± 0.33a 1.12±.036a 8.09 ± 0.37b may be due to small sample size of the heifers group.
Different lowercase letters indicate a significant difference between differ- PGF2a-triggered ovulation induction, which has
ent treatment groups on different days.
IM: intramuscular; IV: intravenous; IU: intrauterine. been demonstrated by numerous studies, might be
the underlying mechanism involved in the enhanced
conception rate. The results of a clinical trial have indi-
cated that PGF2a can induce ovulation in prepubertal
been one of the interesting research subjects in recent heifers through a luteolysis-independent mechanism
years. Although several studies have repeatedly (Leonardi et al. 2012). Some studies have reported
reported that fertility is enhanced following PGF2a that PGF2a administration at the time of insemination
administration at the time of insemination, mainly due can accelerate ovulation or increase ovulation rate in
to an increased ovulation rate (Lopez-Gatius et al. cattle (Pfeifer L et al. 2014; Pfeifer LFM et al. 2016), or
2004; Leonardi et al. 2012; Pfeifer L et al. 2014; Pfeifer it can increase ovulation rate in dairy cattle insemi-
LFM et al. 2016), to the best of our knowledge, only nated in warm season (Lopez-Gatius et al. 2004).
four studies have reported significant statistical Furthermore, some studies have explained the effect
increases in conception rates of dairy cattle, namely in of PGF2a administration at the time AI through post-
repeat breeders (Lopez-Gatius et al. 2004), normal insemination increased serum progesterone concentra-
cows (Prinzen et al. 1991; Ambrose et al. 2015) and tion (Neglia et al. 2008), which could not be confirmed
buffalos (Neglia et al. 2008). by the present study. It has been hypothesised that
In the present study, the probability of conception administration of PGF2a during AI may induce its
was 1.911 times (p ¼ .005) higher in the cows that effect via stimulating uterine myometrial contractions
received intramuscular administration of D-cloproste- and transferring spermatozoids to the uterine tube
nol at the time of AI. This increased probability in the (Rodriguez-Martinez et al. 1987; Willenburg et al.
studied herd, which received proper reproductive 2003). Some studies have proved this hypothesis,
management and presented a conception rate of while it has been rejected by others (Morrison et al.
40.6% in the control group is noteworthy. This result 1988).The significant positive results obtained from
is supported by the findings of Ambrose et al. the IM group of this study may be due to the fact
(Ambrose et al. 2015), who have reported an increase that the experiment was conducted during the sum-
in conception rate in lactating dairy cows following mer months. In a study conducted by lopez-Gatius et
intramuscular administration of 10 mg dinoprost tro- al. (Lopez-Gatius et al. 2004), administration of clopros-
methamine at the time of AI. Moreover, in a study per- tenol at the time of AI led to an increase in ovulation
formed by Neglia et al. (Neglia et al. 2008) to rate in cows inseminated during the warm season,
investigated the effect of intramuscular administration while it could not affect ovulation during the cold sea-
of cloprostenol on buffaloes, similar results have been son. On the farm where the study was conducted, the
obtained. On the other hand, some studies have adverse effects of hot season on reproduction were
reported that intramuscular administration of PGF2a evident so that, the difference between reproductive
failed to affect conception rate in dairy cattle performance during the cold and hot seasons of the
1886 M. J. BEHZADI-SHAHRBABAK ET AL.

year was significant. Therefore, administration of D-clo- In this study, no difference was observed between
prostenol during pre-ovulatory period might compen- the treatment and control groups in terms of serum
sate (eliminate) the adverse effects of hot season progesterone concentration on days 4 and 15 post-
on ovulation. insemination. Therefore, the hypothesis that D-clopros-
Despite the expectation that, compared to intra- tenol can increase conception rate by increasing pro-
muscular administration, intravenous administration of gesterone concentration during the post-insemination
D-cloprostenol could affect the ovary or uterus rapidly days could not be confirmed by the present study.
and to a greater extent, it could not induce a signifi- This finding is in agreement with those of Ambrose et
cant difference in conception rate under the condi- al. (Ambrose et al. 2015) and Sauls et al. (Sauls et al.
tions of our study. 2018) who have reported that PGF2a treatment at the
Considering the results of this study, the question time of AI failed to induce greater plasma progester-
definitely arises "why intravenous administration of D- one level 7 and 13 days following PGF2a treatment at
cloprostenol failed to increase conception rate, unlike the time of AI. However, it has been reported that in
its intramuscular administration?". Italian Mediterranean buffaloes higher progesterone
It is likely that intravenous injection, due to the level and corpus luteum diameter were detected on
short half-life of D-cloprostenol (Martin and Liptrap day 11 post PGF2a administered at the time of AI
1981), may not provide effective concentration of the (Neglia et al. 2008).
hormone on the verge of ovulation compared to intra- Our findings indicated no significant difference in
muscular administration.This finding contradicts the serum progesterone levels between day 0 and 4post-
Prinzen et al. (Prinzen et al. 1991) report that intraven- insemination, while it was significantly increased on
ous cloprostenol administration at the time of AI day 15 in all the treatment and control groups com-
resulted in a 15.2% increase in pregnancy rates. pared to that on day 4 post-insemination, as expected
To date, to the best of our knowledge, the differen- according to the previous studies (Willard et al. 2003;
ces between the effects of intramuscular and intraven- Stronge et al. 2005; Vadhanakul et al. 2008).
ous administration of PGF2a or its analogues have not There was no significant difference in double ovula-
been investigated in cattle. These differences have tion rate in any of the treatment groups, which is
been studied in buffalos (Neglia et al. 2008), indicating contradictory with the findings of Lopez-Gatius et
that both administration routes equally increased the al.(Lopez-Gatius et al. 2004)who have reported an
conception rate .These findings are in contrast with increase in double ovulation in dairy cows following
the results of the present study. However, it should be cloprostenol treatment at the time of insemination.
considered that, besides being more practical com- The results of this study indicated that parity, DIM,
pared to intravenous administration, intramuscular and milk production variables did not affect double
injection is also more effective. ovulation rates, while the results of a study by Lopez-
The effects of intrauterine infusion of PGF2a or its Gatius et al. (Lo
pez-Gatius et al. 2005) demonstrated
addition to semen have been evaluated in several
that parity and DIM were correlated with dou-
past studies that have reported different results.
ble ovulation.
Moreover, some older studies on the effect of intra-
uterine administration of PGF2a in rabbits (Sorgen and
Glass 1972; Spilman et al. 1973), sheep (Gustafsson et Conclusion
al. 1975) and horses (Bader et al. 1999) have achieved The results of the present study indicated that under
contradictory results. However, the effect of intrauter- the conditions of this study, Prostaglandin F2a ana-
ine administration of PGF2a in cattle has only been logue, D-cloprostenol, could effectively increase con-
evaluated by Gabriel et al. (Gabriel et al. 2011) who ception rate in dairy cows when administered
have reported an increase in conception rate (p ¼ .12). intramuscularly at the time of AI, however, intravenous
In the present study intrauterine administration of D- and intrauterine administration of D-cloprostenol
cloprostenol at the time of AI led to a 1.26 fold showed no favourable effect on conception rate in
increase in the conception rate for cows (p ¼ .11) dairy cattle.
which was consistent with those of reported by
Gabriel et al., (2011). Perhaps further studies on a
larger scale can prove the beneficial effects of intra- Ethical approval
uterine administration of PGF2a or its analogues at the The ethical Committee of Shahrekord University approved
time of AI on reproductive performance of cattle. the study.
 ITALIAN JOURNAL OF ANIMAL SCIENCE 1887

Disclosure statement insemination in lactating dairy cows. Czech J Anim Sci.

56(No. 6):279–283.
No potential conflict of interest was reported by Gustafsson B, Edqvist S, Einarsson S, Linge F. 1975. The fertil-
the author(s). ity of deep-frozen ram semen supplemented with PGF 2
alpha. Acta Vet Scand. 16(3):468–470.
Hedin L, Gaddy-Kurten D, Kurten R, Dewitt DL, Smith WL,
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