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Influence of Different Doses of Equine Chorionic Gonadotropin on Follicular Population and Plasma Estradiol Concentration in Chinese Holstein Dairy Cows.

Byline: Shu-Bin Fu, Hasan Riaz, Muhammad Kasib Khan, Hua-Lin Zhang, Jian-Guo Chen and Li-Guo Yang


The present study was designed to compare the effect of different doses of equine chorionic gonadotropin (eCG) on follicular population and plasma estradiol concentration in Chinese Holstein cows synchronized with prostaglandin F2? (PGF2?). One hundred cows were divided into five treatment groups. Each group (n=20) was administered with an injection of eCG at dose rate of 2, 2.5, 3, 3.5, or 4 IU/kg b.w., respectively, followed by administration of PGF2? 48 h later. Ovarian scanning was performed by transrectal ultrasonography around estrus, while plasma estradiol concentration at administration of eCG, PGF2?, estrus and 12 h after estrus was measured by Radioimmunoassay (RIA). The results showed that medium follicles (5-10 mm) varied (Pless than 0.05) among all treatment groups with an increasing trend. However, there was no significant difference (Pgreater than 0.05) in the number of large follicles (greater than 10 mm). Plasma estradiol concentration differed non-significantly (Pgreater than 0.05) in all groups before estrus. At estrus, the concentration was at peak in high dose treatment group (4 IU/kg, Pless than 0.05) than those of low dose treatment groups (2 and 2.5 IU/kg). The study concluded that eCG increases the recruitment and development of medium follicles in dose dependent manners however it has no subsequent effect on large follicles population. Copyright 2013 Friends Science Publishers

Keywords: eCG; Estrus; Medium follicles; Large follicles; EstradiolIntroduction

Equine chorionic gonadotrophin (eCG) has been used as synchronizing agent for promoting follicular growth in cows. Numerous reports have been shown that treatment with eCG resumed follicular activity (Baruselli et al., 2004; Rostami et al., 2011; Jabeen et al., 2013) and exhibited a significant linear relationship with number of follicular population (Monniaux et al., 1984) in heifers. In routine, eCG has been used to induce follicle maturation and to attain better oocyte and/or corpus letuem (CL) quality for obvious increase in pregnancy rate (Bo et al., 2003). In heifers, treatment with eCG doubled follicular growth rate (Sa Filho et al., 2010). Normally, an injection of eCG leads to an initial increase in the number of small follicles followed by their recruitment and selection into medium and large follicles (Driancourt et al., 1991;).High doses of eCG treatment may have a mixed response in regulating ovarian follicular population. In one way, it increases the developmental process of follicles and high doses of eCG may be helpful in recruiting more follicles into medium and large follicles, thus increasing the chances of multiple ovulation. Moreover, a relationship is already existed between eCG dose and multiple ovulations and/or births in dairy cattle (Andreu-Vazquez et al., 2012). On the other hand, treatments with eCG can affect ovarian endocrine function and lead to an obvious increase in estradiol productions (Liu et al., 2007). Some previous reports observed that eCG increased the amount of mRNA for cytochrome P45017a resulting in increased estradiol production (Soumano et al.,1996; 1998). Many studies have revealed that a significant amount of estradiol is produced by the dominant follicles (Ireland et al., 1984). The conception rates are most probably influenced by the overall estradiol concentration, mainly produced by the dominant follicles (Kiewisz et al.,2011; Enginler et al., 2012).Taken together, these observations imply that both number of follicles and estradiol concentrations might be affected by the treatment of different eCG doses and thus needed to be optimized in dairy animals. The present study was designed to investigate the effect of different doses of eCG on growing follicular populations and their association with estradiol concentrationMaterials and Methods


All the experimental procedures were approved by the Animal Care and Use Committee (Approval ID: SCXK (Hubei) 2008-0005), College of Animal Science and Technology, Huazhong Agricultural University, PR China. eCG injection commercially available as Serum Gonadotrophin (1000 IU) and PGF2a commercially sold as Cloprostenol Sodium Injection (0.2 mg) were purchased from Ningbo Sansheng Pharma chemicals, China. Briefly,100 non-pregnant and healthy Chinese Holstein dairy cows were selected during the period of March and April. The cows were medium sized (Body condition score rangesfrom 3 to 3.5), weighing 450 to 550 kg and were housed in Jingdu dairy farm, Hubei, China. Before starting the experiment, all the cows were confirmed to be free from anyreproductive problems. The animals were kept under samenutritional and management conditions while their cyclicity was monitored based on farm records. All the cows were randomly divided into five treatment groups of 20 animals each. Synchronization treatments were initiated on days8-10 of the estrous cycle with a single intramuscular (IM)injection of respective doses of eCG (2, 2.5, 3, 3.5 or 4IU/kg) in five different groups, followed by intramuscular administration of PGF2? (0.4 mg/cow) 48 h later. Cows were observed for estrus by a teaser bull thrice a day.

Ovarian Ultrasonography

Ovarian trans-rectal ultrasonography was undertaken at estrus period by a B-mode veterinary ultrasound scanner equipped with a 5.0 MHz linear array transducer (Zhang et al., 2010). The ultrasonographic images of ovaries were collected to analyze the number of follicles (Fig. 1). Photographs of the ovaries were transmitted to a computer workstation and were analyzed to assess the extent of ovarian follicular activity during the experiment. The location and size of each follicle (=5 mm in diameter) was recorded on an ovarian map.

Blood Collection and Estradiol Assay

Blood samples for estradiol concentrations were collected by tail vein puncture in heparinized, evacuated tubes at the time of administration of eCG, PGF2?, estrus and 12 h after estrus. All blood samples were kept at 4C and centrifuged within 6 h of collection; plasma was removed and frozen at-20C until assayed. Serum estradiol concentration was measured by RIA and was analyzed by a solid-phase radioimmunoassay kit (Beijing Chemclin Biotech Co. Ltd, Beijing, China). The intra- and inter assay coefficients of variation for estradiol calculated from the precision profileswere less than 10% and 15%, respectively. The sensitivitiesfor the least detectable amount of estradiol were less than 3 pg/mL. Statistical Analysis

All data were analyzed by ANOVA using SPSS (version13.0, SPSS Inc., Chicago, IL) and have been presented as mean S.E.M. The probability of Pless than 0.05 was considered statistically significant.


Follicle Population

In this experiment, more than 70% cows showed estrus signs, including restlessness, vulvar discharge and standing heat. Among different groups, the number of medium follicles (5 to 10 mm) at estrus increased in a dose dependent manner. However, there was no significant difference in the number of medium follicles between the treatment groups (P?0.05) received 3 and 3.5 IU/kg of eCG. While the animal group treated with 4 IU/kg of eCG had higher number of medium follicles (Pless than 0.05). Treatment with different eCG doses imparted a non-significant (Pgreater than 0.05) effect on large follicle population (greater than 10 mm). The ultrasonographic images of various medium and large follicles have been presented in Fig. 1.

Estradiol Concentration

Plasma estradiol concentration was similar among all groups at the time of eCG administration. After eCG treatment, serum estradiol concentrations started to increase (Pgreater than 0.05) in all groups. Following PGF2? administration, there was sharp increase (Pless than 0.01) in estradiol concentration (Fig. 2).Mean peak plasma estradiol concentration in all groups was attained at the time of estrus. Moreover, estradiol concentration was higher in the treatment group received high dose of eCG (4 IU/kg) (Pless than 0.05) as compared to those received low doses of eCG (2 and 2.5 IU/kg). After estrus, serum estradiol concentration started to decrease in all treatment groups. The estradiol concentration was also prolonged in high dose treatment group (4 IU/kg) 12 h after estrus (Pless than 0.01), while it was lowest in low dose treatment group (2 IU/kg).


The present study confirmed that exogenous eCG administration has a positive effect on follicular development in Chinese Holstein cows. A similar discovery has been previously reported in Holstein cows with improved ovulatory stimulus after eCG treatment (Souza et al., 2009). The eCG administration also appreciated the induction of new follicles in ruminants (Aba et al., 2005). However, several reports indicated that eCG significantly increased the number of normal preantral follicles but without altering number of normal antral follicles (Warriach and Ahmad, 2009).

Table 1: Follicular population among five different doses###

of eCG (mean S.E.M)###

eCG dose###Cows in###Number of Follicles (n)###

(IU/kg)###Estrus (n)###Medium Follicles###Large Follicles###






A positive influence of different doses of eCG on follicular population in Chinese Holstein cows was demonstrated in this study, which is an agreement with previous report in Pelibuey ewes (Gonzalez-Reyna et al.,1999). The findings that the number of medium folliclesincreased at the highest dose of eCG are in agreement with a previous report in Holstein cows (Gonzalez et al., 1994). However, the number of large follicles remained unaffectedamong all treatment groups. This conclusion may besupported by many previous results, which indicated that large follicles might inhibit the development of other growing follicles and reduced the superovulatory response to eCG (Guilbault et al., 1991; Huhtinen et al., 1992).Results from this study demonstrated that a sudden increase in estradiol concentrations was observed after administration of PGF2? up to estrus in all treatment groups. It is generally believed that plasma estradiol concentration increased significantly after follicles reach 8-9 mm diameter (Sheldon and Dobson, 2000). Similar increase is also reported in beef cows where estradiol began to rise about 48 h before estrus in all groups and reached highest at the time of estrus (Henricks et al., 1973). Moreover, the plasma estradiol concentration in the highest eCG dose group (4IU/kg) was higher than the other groups (2 and 2.5 IU/kg) at estrus. This finding might be due to the reason that estradiol is mainly produced by the dominant follicle (Evans et al.,1997; Yimer et al., 2012) and an increasing tendency of total number of large follicles was observed as the dose of eCG increased in this study.The plasma estradiol concentrations started to decline after estrus, and the estradiol concentration in the higherdose groups was prolonged than in the lower dose groups.High doses of eCG lead to excessive follicular development which often resulted in incomplete ovulations, and these un-ovulated follicles secretes abnormally high levels of estradiol, which might be a reason of high plasma estradiol concentration in the group administered with high dose of eCG in the present study. However, the high estradiol concentration may have adverse effects on embryo development (Ziecik et al., 2005). Some reports revealed that large eCG doses caused reproductive disorders during final follicular development and oocyte maturation which are known to proceed according to a chronologically fixed hormonal sequence in normally cyclic cows (Gonzalez et al.,1994) and lead to lower pregnancy rate (Fu et al., 2012).In conclusion, this study showed that the higher dose of eCG for synchronization increased the number of medium follicles and affected the longevity of estradiol concentrations.


This work was supported by Special Fund for Agro scientific Research in the Public Interest (201003060) and the earmarked fund for Modern Agro-industry Technology Research System (CARS-37-04B).


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Author:Fu, Shu-Bin; Riaz, Hasan; Khan, Muhammad Kasib; Zhang, Hua-Lin; Chen, Jian-Guo; Yang, Li-Guo
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Apr 30, 2014
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