This study aimed to investigate the relationship between increases in the luteinizing hormone (LH) profiles in the serum and vaginal mucus of cows induced by
gonadotropin-releasing hormone (GnRH). Samples for LH
determination were collected from Japanese Black beef cows during estrus, which was induced
with a controlled internal progesterone-releasing device and the administration
of cloprostenol immediately before GnRH administration and every 30 min from
the start of GnRH administration until 6.5 h. The peak serum LH concentration
was clearly identified at 2.5 h post-GnRH administration, with serum
concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak vaginal mucus LH concentration was identified 4.5 h
after GnRH administration. These results indicate that the LH
secretion peak in vaginal mucus appeared about 2 h after peak LH secretion
in the serum.
Introduction
Technological progress has led to the development and application of
sensor-based monitoring systems that can continuously monitor and record
detailed information for estrus detection in cows
(Saint-Dizier and Chastant-Maillard, 2012). The
measurement of luteinizing hormone (LH) concentrations is laborious, costly,
time-consuming, and requires sophisticated equipment; thus, it is not
suitable for field conditions. Quantifying LH concentrations during estrus
improves the accuracy of predicting the optimal time for insemination to
ensure conception. If an electronic device could be developed to measure
LH concentrations, it could provide the necessary information to pinpoint the
optimum timing for more accurate and efficient insemination and embryo transfer. Intravaginal measurement is probably the most practical method
for LH detection, other than blood, because of the labour and animal handling
requirements. Therefore, the present study investigated the relationship
between increases in LH profiles in the serum and vaginal mucus of cows induced by gonadotropin-releasing hormone (GnRH).
Materials and methods
All animal handling and experimental procedures were performed in accordance
with the Guidelines for Animal Experiments of the Tokushima University. All of
the animals were housed and maintained in accordance with the guidelines of
the Institutional Animal Care and Use Committee. Seven cows (Japanese Black
beef cows between 10 and 15 years old) with regular estrous cycles were used for this
experiment, which was conducted from July to December 2020. The cows were
kept in groups of two in a concrete-floored paddock bedded with sawdust.
The cows were fed Italian ryegrass hay and concentrate with water ad libitum. The diet
was formulated to meet the nutritional requirements of non-lactating
Japanese beef cows (Japanese feeding standard for beef cattle, 2008).
The insertion of a controlled internal drug-releasing device (PRID-Delta;
Aska Animal Health Co., Tokyo, Japan) containing 1.55 g of progesterone and 10 mg of
estradiol benzoate was conducted on day 0 (start of the protocol) in cows
with unknown estrous cycles; intramuscular administration of 500 µg
of cloprostenol (Estrumate®; Intervet K.K, Tokyo, Japan) was
performed on day 9, along with PRID-Delta removal; and 100 µg of GnRH
(fertirelin acetate, Conceral®; MSD Animal Health, Tokyo,
Japan) was administered (intramuscularly) on day 11. Samples for LH
determination were collected immediately before GnRH administration and
every 30 min from the start of GnRH administration for 6.5 h. Blood was
collected from the jugular vein using an indwelling catheter. Briefly,
intravenous 18G catheters (NIPRO, Osaka, Japan) were connected to three-way
valves (3 W-RC type; NIPRO, Osaka, Japan) and inserted into the jugular
veins for blood sampling. Vaginal mucus was collected from the vagina using
a kitchen sponge. Briefly, a kitchen sponge made of polyurethane (3.5 cm
diameter, 7 cm length) with a handle (40 cm length) was inserted into the
vagina using a vaginal speculum. The sponge was glued to the mucosal surface
to absorb the mucus and was then placed in a 50 mL syringe; the mucus
(about 1–3 mL) was subsequently squeezed out into a 15 mL conical polypropylene tube.
Both samples in the conical tubes were immediately chilled in ice in
a styrofoam box and stored in a refrigerator until centrifugation. Serum and
vaginal mucus were separated by centrifugation at 3000 rpm for 15 min at 4 ∘C and stored at -80 ∘C until assayed for LH
concentrations. The LH concentrations of the serum and vaginal mucus were
determined using a double-antibody radioimmunoassay (RIA), according to the
method described by Naniwa et al. (2013), using a bovine LH RIA kit
provided by the National Hormone and Peptide Program (NHPP; Baltimore, MD,
USA). In addition, for measuring LH concentrations in the mucus, samples
were mixed well with a vortex mixer until the samples were homogenized and
deliquesced, and the supernatant was then applied to the RIA. The lowest
detectable concentration of LH in both samples was 0.049 ng mL-1 in a 100 µL sample, and the intra- and inter-assay coefficients of
variation were 10.4 % at 0.56 ng mL-1 and 8.0 % at 0.61 ng mL-1
respectively.
Results
Our study was not designed to validate the treatment effects on ovulatory
response, but ovulation occurrences in all cows treated by the synchronization program
were observed by transrectal ultrasonography. LH concentrations in the serum
and vaginal mucus versus time after intramuscular administration of GnRH are
shown in Fig. 1. The mean peak in serum LH concentrations (28.89 ± 8.47 ng mL-1) observed after GnRH administration was approximately 40-fold
higher than the vaginal mucus LH concentrations (0.69 ± 0.32 ng mL-1). The serum
LH concentration (range of 8.45–74.28 ng mL-1) peaked at 2.5 h post-GnRH
administration, with serum concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak in the LH concentration
(range of 0.18–2.42 ng mL-1) in vaginal mucus was observed 4.5 h after GnRH
administration. The mean time interval between serum and mucus peaks
averaged 1.92 ± 0.23 h (range of 1.0–3.0 h). Moreover, surge-like
LH secretion was clearly identified in the serum of all cows, while the LH
peak concentration in the vaginal mucus of some cows was not as distinct as
in the serum.
Mean (± standard error of the mean) concentrations of luteinizing hormone (LH) in
serum and vaginal mucus from the start of gonadotropin-releasing hormone
(GnRH) injection to 6.5 h. All cows received a PRID-Delta for 9 d, which
was removed at the time of cloprostenol treatment. A total of 2 d after the
cloprostenol treatment, cows received GnRH. Samples were collected
immediately before GnRH administration and every 30 min up to 6.5 h after
GnRH treatment. The peak LH concentration was observed at 2.5 and 4.5 h after GnRH injection in
serum and vaginal mucus respectively.
Discussion
Previous studies have reported that cervical mucus has a strong relationship with the
progesterone concentration and ovulation time (Layek et al.,
2013), and the estrus-specific odour in mucus has been suggested to
influence LH surges in cows in the same barn (Nordeus et al., 2012).
Moreover, the vaginal electrical resistance is known to fluctuate with the
stages of the estrous cycle and is closely related to the timing of
ovulation (Canfield and Butler, 1989). These reports indicate that cervical
mucus may be used as a tool to determine the proper time of insemination.
Moreover, the preovulatory LH surge has also been suggested as a more
precise indicator of ovulation time (Larsson, 1987; Rajamahendran et al.,
1989). In the present study, we observed that the LH profile in the
vaginal mucus of all cows showed surge-like LH secretion after GnRH injection, similar
to the serum LH profile. Moreover, the surge-like LH secretion appeared
2.5 and 4.5 h after GnRH injection in serum and vaginal mucus respectively; thus, the surge-like
LH secretion in vaginal mucus appeared about 2 h later than the surge-like
LH secretion in serum. In previous studies, a GnRH-induced LH increase has been
reported to appear 1.5 to 2 h after GnRH administration in dairy and beef
cows (Colazo et al., 2008; Armengol-Gelonch et al., 2017; Wijma et al.,
2017), which was similar to the time of surge-like LH increase in the serum
observed in the present study. Furthermore, the LH peak concentrations in the
vaginal mucus were approximately 1/40 of those in the serum. The peak in the
LH concentrations in the vaginal mucus was not distinct in some cows. In the
present study, the sponge sampling method might have had a cleaning effect,
resulting in a lower LH concentration. On the other hand, if the LH
measuring device was left in the vagina for an extended period, the increase
in the LH concentration may have been delayed and, consequently, obscured, as
secreted LH is likely to be added to the mucus present in the vagina.
Therefore, these results indicate that precise electronic equipment for
measuring LH concentrations needs to be developed to obtain the necessary
information from vaginal mucus regarding the optimal timing for insemination. In
conclusion, the results from the current trial confirmed the peak in LH
secretion in vaginal mucus, which appeared about 2 h later than the peak in
LH secretion in the serum. However, measuring the dynamics of the peak LH
secretion from vaginal mucus requires an accurate measurement device due to the extremely low LH concentration in vaginal mucus.
Data availability
The data sets used in this paper are available from the corresponding author upon request.
Author contributions
All authors made substantial contributions to each step of the experimental
procedure and paper preparation. NY and TO conceived the study and wrote the
paper. MY, NY, and TK performed the majority of experiments. TO designed the
study, coordinated all of the experiments, and reviewed the paper. CH, KT,
YH, and MN contributed to the laboratory work and statistical analysis. YM
and FT revised the paper. All of the authors read and accepted the paper.
Competing interests
The contact author has declared that none of the authors has any competing interests.
Ethical statement
This study was approved by the Ethical Committee for the Care and Use of
Experimental Animals of the Tokushima University (approval no. T2019-10), Japan, and was performed in accordance with the Guidelines for Animal
Experiments of the Tokushima University.
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
The authors thank Yuri Kitagawa and Rei Ichikawa (Graduate School of
Bioagricultural Sciences, Nagoya University) for technical support with respect to
measuring the LH concentrations. We also thank Satoshi Ohkura (Nagoya
University) for critical suggestions on LH concentration measurements during
preparation of the manuscript.
Review statement
This paper was edited by Joachim Weitzel and reviewed by two anonymous referees.
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