J. Dairy Sci.

103:2006–2018
https://doi.org/10.3168/jds.2019-16439
© American Dairy Science Association®, 2020.

Association of pregnancy per artificial insemination with gonadotropin-

releasing hormone and human chorionic gonadotropin administered during
the luteal phase after artificial insemination in dairy cows: A meta-analysis
M. Besbaci,1,2* A. Abdelli,3 J. J. Minviel,4,5 I. Belabdi,1 R. Kaidi,1 and D. Raboisson4
1
Laboratory of Biotechnology in Animal Reproduction, Institute of Veterinary Sciences, University of Blida 1, 09,000, Blida, Algeria
2
High National School Veterinary El Harrach, Algers, 16,000, Algeria
3
Department of Agricultural Sciences, University of Bouira, 10,000, Bouira, Algeria
4
IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France
5
Université Clermont Auvergne, INRA, Vetagro Sup, UMR Herbivores, 63122, Saint-Genès-Champanelle, France

ABSTRACT Key words: human chorionic gonadotropin,

gonadotropin-releasing hormone, artificial insemination,
One strategy for improving fertility in cattle is cow
administration of GnRH or human chorionic gonado-
tropin (hCG) during the luteal phase, which increases
INTRODUCTION
progesterone (P4) secretion and delays luteolysis. To
provide an overview of how GnRH or hCG treatment A general belief across both the scientific and com-
between 4 and 15 d after artificial insemination (AI) mercial dairy communities is the antagonistic associa-
improves pregnancy per AI (P/AI) in cows, a meta- tion between milk production and reproductive perfor-
analysis was performed on 107 different trials from 52 mance of dairy cows (Bello et al., 2012). Low fertility
publications. Data from 18,082 treated cows and 18,385 of dairy cows is a big challenge for the industry (Lucy,
untreated controls were meta-analyzed. The meta-anal- 2001); however, the combination of improvements
ysis explained the relative risk for P/AI with GnRH in both reproductive management and genetics has
or hCG treatment under various circumstances. The greatly improved reproductive performance (Carvalho
results did not show any difference in P/AI between et al., 2018). Progesterone (P4) insufficiency has been
cows treated with hCG and cows treated with GnRH. reported to be associated with decreased pregnancy
Compared with no treatment, treatment with GnRH or per AI (P/AI), abnormally early embryo development,
hCG improved the chances of P/AI in cows with very and reduced signaling for the maternal recognition of
poor (<30%) and poor (30.1 to 45%) fertility, whereas pregnancy (Mann and Lamming, 2001; Spencer and
treatment did not benefit cows with very good fertility Hansen, 2015). Peripheral P4 concentrations are the
(>60.1%). Moreover, treatment with GnRH and hCG net result of secretion and metabolism. Increased P4
improved the chances of P/AI in primiparous cows. metabolism by the liver has been hypothesized to cause
The improvement was much better in primiparous the decreased P4 concentration in peripheral blood dur-
cows with very low fertility. Treatment with buserelin ing the estrous cycle in lactating dairy cows (Wiltbank
at a dose above 10 µg and with hCG at a dose above et al., 2006). Progesterone induces secretory changes in
2,500 IU was associated with increased chances of P/AI the endometrium and is essential for successful implan-
compared with lower doses. Treatment with GnRH 10 tation of the embryo. It also modulates the immune
d after AI was also associated with increased chances response of cows to prevent rejection of the embryo and
of P/AI compared with earlier treatment. The present inhibits uterine contractions. All of these changes fail to
meta-analysis showed that the use of GnRH and hCG occur if P4 is not produced at a sufficient concentration
after AI should be focused on cows expected to have (1 to 2 ng/mL on d 5 of estrous cycle; Starbuck et al.,
low or moderate fertility. Day and dose of treatment 2001), whereas a high P4 concentration favors embry-
have to be considered as well. onic development and higher P/AI (Lonergan, 2011).
Many studies tried to increase the P4 concentration
in the blood to improve P/AI in dairy cows (Nasci-
mento et al., 2013; Mendonça et al., 2017; Niles et al.,
2019). The administration of GnRH, GnRH agonists
Received February 6, 2019.
Accepted August 20, 2019. or hCG at specific times after AI stimulates corpus
*Corresponding author: besbaci@​univ​-blida​.dz luteum (CL) function by inducing accessory CL forma-

2006
 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2007

tion. Treatment with these hormones increases the P4 publications that were identified in the previous step
concentration and reduces estradiol production with were also included. Duplicate papers were excluded,
a consequent positive effect on embryonic survival and screening was conducted to determine whether the
(Thatcher et al., 2003). These studies failed to deliver final number of articles qualified for a meta-analysis
a clear consensus on the therapeutic benefits of such (Figure 1).
treatments. Some reported a beneficial treatment effect
of human chorionic gonadotropin (hCG) and GnRH Inclusion and Exclusion Criteria
after AI on P/AI, whereas others showed no benefit or
even a reduction in P/AI. Two studies (Peters et al., The papers retained for the analysis had (1) to record
2000; Nascimento et al., 2013) have attempted to sum the P/AI, (2) to follow a control-case protocol with
up factors that affect the efficacy of treatment with randomization to allocate treatment, (3) to include a
GnRH and hCG after AI. A first meta-analysis focused treatment with a single injection of GnRH or hCG dur-
on GnRH injected on d 11 to 14 after AI (Peters et ing the luteal phase (between 4 and 15 d after AI), and
al., 2000). A second meta-analysis was performed in (4) to be carried out on dairy cows. Based on these
the 2010s and focused on hCG administered to dairy criteria, 52 papers (107 trials) were included (Supple-
cows on d 5 after AI (Nascimento et al., 2013). The mental Table S1; https:​/​/​doi​.org/​10​.3168/​jds​.2019​
meta-analyses included 14 and 10 studies, respectively. -16439). The collective results represented 36,464 cows:
The first (Peters et al., 2000) confirmed that the ef- 18,082 cows treated with GnRH or hCG and 18,385
ficacy of GnRH administration between d 11 and 14 untreated control cows. Of these 52 publications, 33 (57
after AI was not consistent among the studies. Part of trials) were conducted with GnRH (21,825 cows, with
this variation may be explained by differences in study 10,845 treated cows and 10,980 untreated control cows).
protocol, particularly the method and timing of the Twenty-nine publications (50 trials) were conducted
pregnancy diagnosis. These results suggest a need for with hCG (14,642 cows, with 7,237 treated cows and
further research to identify factors at the cow or farm 7,405 untreated controls). Ten out of the 52 included
level for use of GnRH after AI to improve dairy cow both GnRH and hCG treatments (double counted in
fertility. The second meta-analysis confirmed that hCG the 33 and 29 publications). All of these papers were
administration at d 5 after AI considerably improved published between 1982 and 2019, and there was no
pregnancy in primiparous cows but not in multiparous restriction on the publication data. Similarly, there
cows. An update of these previous works, including a was no restriction on the country where the study was
broader approach, appeared necessary. The objective of conducted.
this study was to analyze the association of GnRH and
hCG treatments, administered 5 to 14 d after AI, with Data Extraction and Classification
P/AI in dairy cows and to focus on factors that influ- of Studies Analysis
ence this relationship. We hypothesized that a meta-
analysis can identify factors affecting the efficiency of Data extracted from publications included the num-
GnRH and hCG treatment after AI and consolidating ber of treated and untreated (control) cows, P/AI,
the estimate of the treatment response are possible repeat breeders, the nature of the treatment after AI
with a meta-analysis. (hCG or GnRH), the day of treatment (after AI), the
dose of treatment, the molecule of GnRH, the cow par-
MATERIALS AND METHODS ity, the use of a synchronization protocol, the number of
days after AI of the pregnancy diagnosis, the method of
Literature Search the pregnancy diagnosis, and the season. Details of the
extracted classes are reported in Table 1. In brief, the
Publications comparing hCG-treated or GnRH-treat- moderator (i.e., variable) day of treatment was trans-
ed cows to nontreated control cows were selected from formed into a categorical moderator with thresholds
the English-language literature through June 2019. The of 5 and 10 d for hCG and GnRH, respectively. The
literature search was conducted in PubMed (http:​/​/​ day of treatment was not specified in 30 trials, and the
www​.ncbi​.nlm​.nih​.gov/​pubmed), Science Direct (http:​ average was taken. The dose of hormone was also cat-
/​/​www​.sciencedirect​.com), and Google Scholar (http:​ egorized for hCG and GnRH (buserelin only; only one
/​/​scholar​.google​.com). The key words that were used dose was observed for the gonadorelin). The thresholds
separately or in different combinations for the search that were used for the moderator day of treatment and
were cow, cattle, hCG, GnRH, after, post, AI, embryo, dose of hormone were defined by the authors’ expertise
mortality, loss, fertility, pregnancy, and rate. All of the or based on previous publications for instance for the
publications that were referenced by at least one of the number of days after AI GnRH has been injected (Pe-
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Figure 1. Preferred reporting items for systematic reviews and meta-analysis (PRISMA) study flow diagram of the manuscript selection
process.

ters et al., 2000; Nascimento et al., 2013). The molecule statistical significance. Statistical heterogeneity was as-
of the hormone was also retained as a moderator for sessed using the Cochran Q statistic and the I2 statistic
GnRH (buserelin or gonadorelin). The thresholds used of the proportion of total variation due to heterogeneity
to classify the fertility (very good, good, poor, and very (Higgins and Thompson, 2002). For response variables
poor) were obtained (Table 1) from Yan et al. (2016). with high I2, uni- or multivariable meta-regressions
were then performed to explore the sources of hetero-
Statistical Analysis geneity and reduce them. Meta-analysis is a statistical
method used to aggregate, synthesize, and compare the
All meta-analyses were conducted using the metafor results from several related studies in a systematic way.
package (version 2.0–0) of R (version 3.5.1; R Foun- Briefly, the meta-analysis includes (1) a check for study
dation for Statistical Computing, Vienna, Austria). selection bias, (2) performance of a meta-regression—
Random-effects models were first used to estimate the a regression applied to metadata, each variable be-
log-effect size, its 95% confidence interval, and the ing called a moderator, and (3) a search for outlier
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contributors (a study included in the regression that outliers. Publication bias was assessed by performing
leads the results). The meta-regression is consequently Egger’s test (Egger et al., 1997), a regression test for
a step of the meta-analysis. The meta-regression was funnel plot asymmetry and inspection of the associated
conducted by regressing the observed effect sizes on one funnel plots. Outliers were also identified by conduct-
or multiple study characteristics (moderators). We used ing an influential case diagnostic [i.e., DFFITS value,
the following moderators: fertility, parity, repeat breed- Cook’s distances, covariance ratios, estimates of τ2, and
ers, nature of treatment after (hCG or GnRH), day of test statistics for (residual) heterogeneity].
treatment, dose of treatment, molecule of GnRH, use of
a synchronization protocol, number of days after AI for RESULTS
the pregnancy diagnosis, method of pregnancy diagno-
sis, and the season (Table 1). The variable publication The funnel plot did not suggest a bias of publica-
was kept as a random effect, and it was estimated as tion (Figure 2). Table 2 summarizes the univariable
combined relative risk (RR) and its 95% confidence in- meta-regressions. The meta-regressions performed by
terval. A sensitivity analysis was performed to identify subpopulations are provided in Supplemental Table S2

Table 1. Descriptive statistics of the pregnancy per artificial insemination (P/AI) for the moderators used in the meta-regression

P/AI

No. of Treatment1 Control2

Moderator Class trials [% (no./total)] [% (no./total)]
Treatment GnRH 57 45.85 (4,973/10,845) 43.81 (4,811/10,980)
hCG3 50 41.97 (3,038/7,237) 39.06 (2,893/7,405)
Parity Nulliparous 4 49.24 (195/396) 47.08 (178/378)
Primiparous 11 40.12 (634/1,580) 35.18 (551/1,566)
Multiparous 22 30.78 (1,183/3,843) 30.54 (1,191/3,899)
Use of synchronization No (spontaneous estrus) 12 58.17 (1,149/1,975) 55.67 (1,094/1,965)
GnRH/PGF2α 45 35.20 (2,745/7,797) 33.20 (2,598/7,823)
PGF2α only 9 48.70 (566/1,162) 40.48 (455/1,124)
Progesterone 9 38.68 (610/1,577) 39.96 (625/1,564)
Mix (different protocol) 9 51.17 (1,177/2,300) 50.70 (1,230/2,426)
Season No (all year long) 23 39.08 (1,542/3,945) 36.57 (1,480/4,046)
Cold (winter) 12 57.70 (1,172/2,031) 56.76 (1,246/2,195)
Hot (summer) 14 27.44 (614/2,237) 26.42 (564/2,134)
Mix (different season) 22 48.68 (2,050/4,211) 44.55 (1,869/4,195)
Method of pregnancy Rectal palpation 49 51.04 (3,379/6,620) 47.80 (3,290/6,882)
diagnosis Ultrasonography 31 41.18 (2,421/5,878) 39.60 (2,332/5,888)
Both 16 39.02 (1,600/4,100) 37.08 (1,510/4,072)
Week of pregnancy diagnosis ≤4 wk 15 36.84 (948/2,573) 34.19 (887/2,594)
4 to ≤6 wk 34 42.83 (2,816/6,574) 41.83 (2,773/6,629)
>6 wk 33 53.07 (2,765/5,210) 49.61 (2,682/5,406)
Repeat breeders Yes 17 40.25 (349/867) 37.15 (327/880)
No 90 44.50 (7,662/17,215) 42.14 (7,377/17,505)
Fertility Very poor (<30%) 31 27.14 (1,269/4,675) 23.79 (1,113/4,677)
Poor (30.1 to 45%) 39 40.34 (2,382/5,904) 36.87 (2,208/5,987)
Good (45.1 to 60%) 26 56.13 (2,725/4,854) 53.02 (2,663/5,022)
Very good (>60.1%) 11 61.72 (1,635/2,649) 63.72 (1,720/2,699)
Day of treatment with hCG ≤5 d 26 42.02 (1,543/3,672) 37.97 (1,439/3,789)
>5 d 24 41.93 (1,495/3,565) 40.21 (1,454/3,616)
Day of treatment with GnRH ≤10 d 29 41.14 (2,362/5,741) 40.45 (2,372/5,864)
>10 d 28 51.15 (2,611/5,104) 47.67 (2,439/5,116)
Dose of treatment with hCG ≤1,500 IU 17 51.55 (794/1,540) 45.81 (734/1,602)
>1,500 and ≤2,500 IU 12 40.62 (804/1,979) 42.54 (847/1,991)
>2,500 IU 20 39.43 (1,427/3,619) 34.59 (1,284/3,711)
Dose of treatment with GnRH <10 µg 4 60.54 (554/915) 63.41 (598/943)
(buserelin only) ≥10 and ≤10.5 µg 17 60.90 (2,572/4,223) 57.14 (2,454/4,294)
≥12 µg 5 59.02 (121/205) 48.94 (116/237)
Molecule of GnRH Buserelin 26 60.77 (3,247/5,343) 57.87 (3,168/5,474)
Gonadorelin 27 30.90 (1,655/5,355) 29.78 (1,596/5,359)
Lecirelin 3 46.71 (64/137) 32.59 (44/135)
1
Treatment percentage of cows pregnant to AI (P/AI).
2
Control percentage of cows pregnant to AI (P/AI).
3
hCG = human chorionic gonadotropin.

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Table 2. Results of the univariate meta-regressions

Model Estimate Risk

no. Moderator Class (SE) ratio 95% CI P-value I2 %
1 No moderator Intercept 0.07 (0.01) 1.07 1.03–1.11 0.0002 45
2 Intercept 0.06 (0.02) 1.06 1.01–1.11 0.01 45
Treatment GnRH Referent
hCG1 0.02 (0.03) 1.02 0.96–1.09 0.85
3 Intercept 0.16 (0.04) 1.17 1.07–1.28 0.0003 39
Fertility Very poor Referent
Poor −0.06 (0.05) 0.94 0.85–1.04 0.24
Good −0.09 (0.05) 0.91 0.82–1.01 0.10
Very good −0.24 (0.06) 0.78 0.69–0.88 <0.0001
4 Intercept 0.13 (0.04) 1.13 1.04–1.24 0.002 42
Parity Primiparous Referent
Nulliparous −0.09 (0.08) 0.91 0.77–1.06 0.23
Multiparous −0.09 (0.05) 0.91 0.81–1.01 0.09
5 Intercept 0.08 (0.02) 1.08 1.02–1.15 0.005 41
Dose of GnRH ≥10 and ≤10.5 µg Referent
(buserelin) <10 µg −0.13 (0.05) 0.87 0.79–0.97 0.009
≥12 µg 0.08 (0.09) 1.15 0.90–1.29 0.39
6 Intercept 0.01 (0.02) 1.01 0.97–1.06 0.52 31
Day of GnRH ≤10 d Referent
>10 d 0.08 (0.03) 1.08 1.02–1.15 0.007
7 Intercept 0.06 (0.02) 1.06 1.01–1.11 0.006 31
Molecule of GnRH Buserelin Referent
Gonadorelin −0.02 (0.04) 0.98 0.90–1.05 0.47
8 Intercept 0.14 (0.04) 1.15 1.05–1.25 0.002 46
Dose of hCG >2,500 IU Referent
≤1,500 IU −0.06 (0.07) 0.94 0.98–1.07 0.36
>1,500 and ≤2,500 IU −0.16 (0.06) 0.85 0.74–0.97 0.01
9 Intercept 0.09 (0.05) 1.09 0.99–1.20 0.06 53
Day of hCG ≤5 d Referent
>5 d −0.02 (0.06) 1.00 0.86–1.10 0.70
10 Intercept 0.14 (0.05) 1.15 1.03–1.28 0.01 39
Synchronization PGF2α Referent
GnRH/PGF2α −0.08 (0.06) 0.92 0.81–1.03 0.18
Progesterone −0.14 (0.07) 0.86 0.74–1.00 0.06
Spontaneous estrus −0.09 (0.07) 0.91 0.78–1.04 0.17
Mix −0.10 (0.07) 0.90 0.77–1.05 0.18
1
hCG = human chorionic gonadotropin.

(https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16439). The estimat-

ed pooled effect size obtained with the random-effects
model 1 (Table 2) with no moderator (RR = 1.07, P =
0.0002) showed that the treatment was associated with
7% higher chances of P/AI compared with no treat-
ment. The heterogeneity of the data set was moderate
(I2 = 45%; Q statistics: χ2 = 192.42, df = 106, P <
0.0001). Including the type of treatment (GnRH or
hCG) in model 2 did not decrease the heterogeneity,
and no significant difference between GnRH and hCG
treatment (P = 0.85) was observed.
Inclusion of the fertility status of cows in the meta-
regression allowed a 6% reduction in heterogeneity.
Treatment with GnRH and hCG resulted in 17%
higher chances of P/AI compared with no treatment
for cows with very poor, poor, or good fertility (P >
0.05) for poor and good groups compared with very
poor (model 3, Table 2, Supplemental Figure S1, https:​ Figure 2. Funnel plots for the meta-regression of model 1. Funnel
plots of the effective size of the pregnancy per AI (P/AI; displayed on
/​/​doi​.org/​10​.3168/​jds​.2019​-16439). Treated cows with the horizontal axis) for cows treated with GnRH and human chorionic
very good fertility had 22% lower chances of P/AI gonadotropin (hCG) after AI.

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 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2011

compared with treated cows with very poor fertility. trend toward lower P/AI was observed for other syn-
Moreover, treatment with GnRH and hCG resulted in chronization protocols.
5% (17–22) lower chances of P/AI compared with no The other moderators listed in Table 1 did not re-
treatment for cows with very good fertility. The related duce the residual heterogeneity of the models and were
submodel showed that the treatment given to cows not associated with statistically significant explanatory
with very good fertility was not significantly associated variables.
with P/AI (RR = 0.98, P = 0.15, Supplemental Table Table 3 reports the final bivariate meta-regressions.
S2, https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16439). Adding the moderators fertility and parity (model 2.1),
Inclusion of the parity of cows in the meta-regression fertility and GnRH molecule (model 2.2), and dose and
allowed a 3% reduction in heterogeneity. Treatment day of GnRH treatment (model 2.3) allowed for de-
with GnRH and hCG resulted in 13% higher chances creases in heterogeneity of 6, 25, and 13%, respectively,
of P/AI compared with no treatment in primiparous compared with the model with the moderator fertility
cows (used as the reference class in model 4, Table 2; (model 3). For nulliparous or primiparous cows with
Supplemental Figure S2, https:​/​/​doi​.org/​10​.3168/​jds​ very poor fertility, 44% higher chances of P/AI were
.2019​-16439). Treated primiparous cows tended to have observed in cases of treatment compared with no treat-
better treatment efficacy (P = 0.09) compared with ment (model 2.1, Figure 3). The treatment was a less
treated multiparous cows. effective for primiparous cows with poor fertility, with
Inclusion of the GnRH dose allowed a 4% reduction 25% (44–19) higher chances of P/AI were observed in
in heterogeneity of the data set (model 5, Table 2). The cases of treatment compared with no treatment. For
moderate dose (10–10.5 µg) of GnRH was associated multiparous cows with poor fertility, 16% (37–21) high-
with 8% higher chances of P/AI for treated cows com- er chances of P/AI were observed in cases of treatment
pared with nontreated cows. No difference between the compared with no treatment. For cows with very poor,
average and higher doses was observed. Lower chances poor, and even good fertility and treated with busere-
of P/AI were also observed for cows treated with a low- lin, 17% higher chances of P/AI in cases of treatment
er dose compared with cows treated with the moderate compared with no treatment were observed (model 2.2,
dose. This result shows a better P/AI for doses >10 µg, Figure 4). Cows treated with moderate and high doses
despite only 4 publications reporting low doses (<10 of buserelin more than 10 d after AI had 11% higher
µg). The moderator day of GnRH treatment allowed chances of P/AI in cases of treatment compared with
heterogeneity to be reduced by 10% (model 6, Table no treatment (model 2.3 Figure 5).
2; Supplemental Figure S3, https:​/​/​doi​.org/​10​.3168/​jds​ The models including parity and synchronization
.2019​-16439). For treated cows, the GnRH treatment protocols did not allow a reduction in heterogeneity.
administered more than 10 d after AI was associated Collinearity between the moderators synchronization
with 9% higher chances of P/AI compared with cows and fertility was observed, with less fertile cows be-
treated earlier (RR = 1.09, P < 0.001; Supplemental ing more likely to be synchronized. No other bivariable
Table S2, https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16439). models were statistically significant, and no interaction
Including the GnRH molecule in the model reduced was observed for either of the models.
heterogeneity but failed to produce a significant differ- The sensitivity analysis indicated 2, 3, and 1 outli-
ence between the 2 classes (Supplemental Figure S4, ers for the final models 2.1, 2.2, and 2.3, respectively
https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16439). (Supplemental Figure S5, https:​/​/​doi​.org/​10​.3168/​jds​
Inclusion of the hCG dose also failed to reduce the .2019​-16439), but exclusion did not change the signifi-
heterogeneity of the data set. The higher dose of hCG cant associations or the coefficient that was observed in
was associated with 15% higher chances of P/AI for the final meta-regressions, which have been retained as
treated cows compared with nontreated cows; more- final meta-regression models.
over, 15% lower chances of P/AI were observed for cows
given a moderate dose compared with cows treated with DISCUSSION
the highest dose (RR = 0.85, P = 0.01). The moderator
day of hCG was not significantly associated with the The present meta-analysis focuses on the association
chances of P/AI, and its inclusion in the model did not between hCG or GnRH treatment and P/AI in cows.
allow a reduction in heterogeneity (model 9, Table 2). Two types of coefficients were provided, and 3 kinds of
Inclusion of synchronization protocol type allowed a models were constructed. The first type of coefficient is
6% reduction in heterogeneity of the data set (model that of the intercept (effect size), which represents the
10, Table 2). Cows treated with PGF2α had 15% higher RR for P/AI in the case of treatment compared with no
chances of P/AI compared with nontreated cows. A treatment. When the regression was performed on the

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whole data set without a moderator, the mean effect shown) for the moderators of interest. In the present
size (RR = 1.07 Table 2, kind 1) represents the mean data set, synchronization with PGF2α or the combina-
efficacy of the treatment. When the regression was per- tion of GnRH/PGF2α (Ovsynch or Presynch) was more
formed to a subset of data without a moderator, the frequently used in cows with low fertility, suggesting
effect size represents the mean efficacy of the treatment that readers should focus on model 10 (Table 2).
for the subpopulation selected (Supplemental Table S2, Previous studies showed that administration of
kind 2, https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16439). When GnRH, a GnRH agonist, or hCG after AI is coincident
the regression includes a moderator(s), the effect size with the presence of the dominant follicle in the first
represents the mean efficacy of the treatment for the and second follicular waves, which may stimulate CL
subpopulation(s) sectioned as reference(s) (Tables 2 function, induce accessory CL formation, increase P4
and 3, kind 3). The second type of coefficients are those concentration, and reduce estrogen production with a
that were obtained for the moderators and associated consequent positive effect on embryonic survival (Ste-
risk; they represent the RR for P/AI for the selected venson et al., 2007). In addition to inducing an acces-
class compared with the reference class. The coefficient sory CL and increasing progesterone after AI, ovulation
(or RR) then compares 2 types of treatment or 2 condi- of a first-wave follicle can change follicular dynamics
tions of treatment used, with the reference being the at the time of luteolysis, thereby extending the time
reference class of the moderator. of luteolysis and allowing more time for an embryo to
The meta-analysis was conducted according to stan- elongate (Araujo et al., 2009). The present study pro-
dard recommendations (Viechtbauer, 2010; Schwarzer vides an update on the association between GnRH or
et al., 2015). The final choices for the models were hCG treatments administered between 4 and 15 d after
selected on the basis of decreased heterogeneity. More AI and P/AI.
than one model was reported for the same outcome The results show that treatments with GnRH and
because the authors judged all of these models to hCG improve P/AI in cows with low fertility (very poor
have biological significance and to be of interest to the <30% and poor 30.1 to 45%), whereas treatment of
scientific community. The bivariate models provided cows with very good fertility (>60.1%) did not result
in Table 3 show close coefficients compared with the in any benefit. The low fertility (poor and very poor)
univariate models that are provided in Table 2, and may result from poor ovulation, inappropriately timed
the addition of a new moderator allows reduced het- estrus or simply insufficient endogenous circulating P4
erogeneity. This observation suggests a low collinearity to support the survival of the early embryo. A lower P4
between the moderators that are included in the uni- concentration has been measured in low-fertility cows
variate models, in accordance with the collinearity tests after estrus (Shelton et al., 1990). Cows with good
that were performed at the start of the study (data not fertility do not need treatment to improve luteal func-

Table 3. Results of the bivariate meta-regressions

Model
no. Moderator Class Estimate (SE) Risk ratio 95% CI P-value I2 %
2.1 Intercept 0.37 (0.10) 1.44 1.18–1.78 0.002 37
Fertility Very poor Referent
Poor −0.21 (0.08) 0.81 0.67–0.96 0.01
Good −0.37 (0.11) 0.69 0.54–0.87 0.001
Very good −0.24 (0.19) 0.78 0.54–1.13 0.2
Parity Primiparous Referent
Nulliparous −0.11 (0.16) 0.89 0.65–1.22 0.48
Multiparous −0.21 (0.07) 0.81 0.69–0.94 0.004
2.2 Intercept 0.16 (0.07) 1.17 1.02–1.36 0.01 0
Fertility Very poor Referent
Poor 0.01 (0.06) 1.01 0.91–1.13 0.74
Good 0.008 (0.06) 1.04 0.88–1.15 0.90
Very good −0.17 (0.07) 0.63 0.72–0.98 0.01
Molecule Buserelin Referent
Gonadorelin −0.14 (0.05) 0.86 0.77–0.98 0.01
2.3 Intercept 0.11 (0.03) 1.11 1.04–1.19 0.001 34
Dose of GnRH ≥10 and ≤10.5 µg Referent
(buserelin only) ≥12 µg 0.07 (0.09) 1.27 0.89–1.30 0.44
<10 µg −0.10 (0.05) 0.90 0.81–1.00 0.05
Day of GnRH >10 d Referent
(buserelin only) ≤10 d −0.11 (0.04) 0.89 0.81–0.97 0.004

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 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2013

tion because they probably already have adequate P4 or spontaneously, was actually detrimental to embryo
for embryo development and the subsequent signaling survival and may have caused embryo loss by exces-
of pregnancy to the cow. In such situations, treatment sive growth stimulation, thus leading to asynchrony
with GnRH or hCG was not beneficial to P/AI. Supple- between the embryo and its environment.
mentation with P4 has previously been reported to in- The present work shows that treatment with GnRH
crease P/AI in herds with low fertility (Robinson et al., and hCG improved P/AI in primiparous cows. Primip-
1989; Van Cleeff et al., 1991; Yan et al., 2016), which is arous cows belong, generally, to a category of animals
consistent with the present study. Increased embryonic with very poor fertility. Therefore, the P/AI was greatly
survival was observed at P4 concentrations above 3 ng/ improved after their treatment. Primiparous cows have
mL and decreased embryonic survival at P4 concentra- many physiological and pathological differences from
tions higher than 9 ng/mL, indicating that low and multiparous cows, including lower milk production,
high P4 concentrations are both associated with a low fewer reproductive problems, increased anovulation,
embryo survival rate (Starbuck et al., 2001). The upper fewer disease problems, and reduced body and organ
limit of P4, whether occurring by P4 supplementation sizes compared with their counterparts (Bamber et al.,

Figure 3. Forest plot for model 2.1. Log risk ratio (RR) and 95% CI of studies on the effect of fertility and parity on pregnancy per AI (P/
AI). The gray diamonds represent the predicted effect sizes using the meta-regression model.

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 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2014

Figure 4. Forest plot for model 2.2. Log risk ratio (RR) and 95% CI of studies on the effect of fertility and GnRH molecule used after AI on
pregnancy per AI (P/AI). The gray diamonds represent the predicted effect sizes using the meta-regression model.

Journal of Dairy Science Vol. 103 No. 2, 2020

 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2015

2009). Any or all of these issues may underlie the parity phase or season were independent of the type of GnRH
differences that were observed between studies. (gonadorelin vs. buserelin) that was used in a study
Treatment with a medium or high dose (>10 µg) of (Armengol-Gelonch et al., 2017). Moreover, the 3
buserelin was associated with greater P/AI compared GnRH analogs (gonadorelin, lecirelin, and buserelin),
with low-dose buserelin treatments. A higher dose of at the doses indicated for induction of ovulation, are
GnRH may have enhanced LH secretion within 2 to almost equally effective for inducing the disappearance
4 h of administration in peripheral circulation, which of the dominant follicle at d 6 to 7 of the estrous cycle
in turn increased the ovulation rate (Chenault et al., (Picard-Hagen et al., 2015).
1990). In contrast, no effect was observed with low In the present work, GnRH treatment after d 10
(<10 µg) buserelin doses in another study (Nakao et post-AI was associated with higher P/AI compared
al., 1992). An intramuscular injection of buserelin at a with GnRH treatment before d 10 post-AI. This finding
dose of 10 µg or higher was reported to be as effective is not in accordance with previous results showing that
as intramuscular administration of 10,000 IU of hCG the ovulatory response to GnRH is not very good after
for the treatment of ovarian follicular cysts in cows. d 10 of the cycle due to the lack of a dominant follicle,
However, lower results for the treatment of ovarian fol- particularly from d 10 to 14 in 2-wave cows, and the
licular cysts were observed after treatment with a very inhibitory effect of high progesterone on the induction
small dose of buserelin (6 µg). of an LH surge (Giordano et al., 2012). The results
Treatment with buserelin was associated with higher observed in the present study are in accordance with
P/AI compared with treatment with gonadorelin in the fact that the administration of GnRH after 10 d co-
the final model 2.2. The characteristics of the GnRH- incides with the critical period of maternal recognition
induced LH surge profile due to the estrous cycle of pregnancy and before the initiation of the luteolytic

Figure 5. Forest plot for model 2.3. Log risk ratio (RR) and 95% CI of studies on the effective dose and day of treatment with buserelin on
pregnancy per AI (P/AI). The gray diamonds represent the predicted effect sizes using the meta-regression model.

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 Besbaci et al.: GONADOTROPIN-RELEASING HORMONE AND HUMAN CHORIONIC GONADOTROPIN 2016

mechanism characterized by the embryonic secretion Ataman, M. B., H. Erdem, B. Bülbül, S. Ümütlü, and M. Çolak.
2011. The effect of buserelin injection 12 days after insemination
of antiluteolytic factors (Mann and Lamming, 2001). on selected reproductive characteristics in cows. Acta Vet. Brno
Treatment with GnRH induces ovulation or luteiniza- 80:171–177. https:​/​/​doi​.org/​10​.2754/​avb201180020171.
tion of the dominant follicle and the formation of ac- Bamber, R. L., G. E. Shook, M. C. Wiltbank, J. E. P. Santos, and
P. M. Fricke. 2009. Genetic parameters for anovulation and preg-
cessory CL. Consequently, GnRH treatment increases nancy loss in dairy cattle. J. Dairy Sci. 92:5739–5753. https:​/​/​doi​
P4 and decreases the estradiol concentration. A lower .org/​10​.3168/​jds​.2009​-2226.
serum concentration of estradiol during d 14 to 17 after Bartolome, J. A., P. Melendez, D. Kelbert, K. Swift, J. McHale, J.
Hernandez, F. Silvestre, C. A. Risco, A. C. M. Arteche, W. W.
AI has been associated with higher P/AI in lactating Thatcher, and L. F. Archbald. 2005. Strategic use of gonadotro-
beef cows (Pritchard et al., 1994). phin-releasing hormone (GnRH) to increase pregnancy rate and
A high hCG dose (>2,500 IU) was associated with reduce pregnancy loss in lactating dairy cows subjected to syn-
chronization of ovulation and timed insemination. Theriogenol-
increased P/AI in the present work. A review of hCG ogy 63:1026–1037. https:​/​/​doi​.org/​10​.1016/​j​.theriogenology​.2004​
treatment after AI (De Rensis et al., 2010) indicated .05​.020.
that an hCG dose ranging from 1,000 to 10,000 IU did Bello, N. M., J. S. Stevenson, and R. J. Tempelman. 2012. Invited
review: Milk production and reproductive performance: Modern
not seem to affect the ovarian response. interdisciplinary insights into an enduring axiom. J. Dairy Sci.
No difference in the nature of the treatment (hCG or 95:5461–5475. https:​/​/​doi​.org/​10​.3168/​jds​.2012​-5564.
GnRH) was observed. The GnRH and hCG treatments Beltran, M. P., and J. L. M. Vasconcelos. 2008. Conception rate in
Holstein cows treated with GnRH or hCG on the fifth day post ar-
have similar effects on the ovary (Fricke et al., 1993), tificial insemination during summer. Arq. Bras. Med. Vet. Zootec.
induction of ovulation (Burns et al., 2008), and forma- 60:580–586. https:​/​/​doi​.org/​10​.1590/​S0102​-09352008000300009.
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of buserelin acetate on conception rate in repeat breeding dairy
cattle. Indian J. Anim. Reprod. 35:47–49.
CONCLUSIONS Burns, M. G., B. S. Buttrey, C. A. Dobbins, C. A. Martel, K. C. Olson,
G. C. Lamb, and J. S. Stevenson. 2008. Evaluation of human cho-
rionic gonadotropin as a replacement for gonadotropin-releasing
The present meta-analysis quantifies the change hormone in ovulation-synchronization protocols before fixed timed
in P/AI in cases of treatment with GnRH and hCG artificial insemination in beef cattle. J. Anim. Sci. 86:2539–2548.
https:​/​/​doi​.org/​10​.2527/​jas​.2008​-1122.
compared with no treatment. The meta-analysis sug- Carvalho, P. D., V. G. Santos, J. O. Giordano, M. C. Wiltbank, and
gests that in certain circumstances GnRH and hCG P. M. Fricke. 2018. Development of fertility programs to achieve
may produce significant benefits. It did not show any high 21-day pregnancy rates in high-producing dairy cows. The-
riogenology 114:165–172. https:​/​/​doi​.org/​10​.1016/​j​.theriogenology​
difference between GnRH and hCG. Treatment with .2018​.03​.037.
GnRH and hCG improved P/AI in primiparous cows Chagas e Silva, J., and L. Lopes da Costa. 2005. Luteotrophic influ-
with low fertility, whereas treatment of cows with good ence of early bovine embryos and the relationship between plasma
progesterone concentrations and embryo survival. Theriogenology
fertility did not gain any benefit. A buserelin treatment 64:49–60. https:​/​/​doi​.org/​10​.1016/​j​.theriogenology​.2004​.10​.019.
above 10 µg, with an hCG dose above 2,500 IU and a Chenault, J. R., D. D. Kratzer, R. A. Rzepkowski, and M. C. Good-
GnRH treatment after d 10 post-AI, were associated win. 1990. LH and FSH response of Holstein heifers to fertirelin ac-
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pu. 2010. Clinical use of human chorionic gonadotropin in dairy
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Journal of Dairy Science Vol. 103 No. 2, 2020