Development of Superovulation Program and Heterologous in vitro Fertilization Test Assessment in Hamsters
Rosnina H. Yusoff,
Soe W. Naing
Superovulation has become a common assisted reproductive technology in the field of animal reproduction. In addition, zona-free hamster oocytes have been used in heterologous in vitro fertilization research to evaluate sperm function. A study was conducted to compare eight different superovulation protocols for golden hamsters using two concentrations of human Chorionic Gonadotropin (hCG) given at two time intervals post-pregnant mares serum gonadotrophin (PMSG) injection and two time intervals of oocyte harvesting. Fifty-six female golden hamsters were randomly and equally assigned into eight superovulation groups. Hamsters were superovulated initially with PMSG followed by human Chorionic Gonadotrophin (hCG). All the groups received 40 IU PMSG, either 40 or 45 IU hCG given at either 48-50 or 55-57 h post PMSG injection and the oocytes recovered at either 12-15 or 16-18 h after hCG injection. Higher number of recovered oocytes (51.57±0.83) and maturation rates (94.20%) (p<0.05) were detected in hamsters which received 45 IU hCG at 55-57 h after PMSG injection when the oocytes were recovered later at 16-18 h compared with hamsters in the other groups. Mean fertilization rate of hamsters given 45 IU hCG at 55-57 h post PMSG injection ranged from 77.89-78.84% and were significantly higher (p<0.05) than those that received hCG at 48-50 h post PMSG injection. In conclusion administration of 40 IU PMSG followed by 45 IU hCG injection at 55 and 57 h post PMSG injection followed by oocyte recovery after 16-18 h gave the highest response in oocyte recovery and maturation in golden hamsters.
to cite this article:
Kazhal Sarsaifi, Mohamed-Ariff Omar, Rosnina H. Yusoff, Abd-Wahid Haron, Homayoun Hani, Nurhusien Yimer, Jaya Vejayan, Soe W. Naing and Abas-Mazni Othman, 2013. Development of Superovulation Program and Heterologous in vitro Fertilization Test Assessment in Hamsters. Asian Journal of Animal and Veterinary Advances, 8: 796-805.
Received: March 03, 2013;
Accepted: August 22, 2013;
Published: September 25, 2013
Superovulation has become a common technology in the reproductive management
of farm and laboratory animals. This technique involves the induction of multiple
ovulations during one oestrous cycle by administering exogenous hormones. Superovulation
was first described by Gates and Runner (1957) in mice
and was later applied to other species, such as rat (Mukumoto
et al., 1995; Popova et al., 2005;
Kito et al., 2010), mouse (Nagy
et al., 2003; Martin-Coello et al., 2008;
Luo et al., 2011), rabbit (Treloar
et al., 1997; Kauffman et al., 1998;
Satheshkumar, 2006), hamster (Fleming
and Yanagimachi, 1980; Roldan et al., 1987;
Bavister, 1989; Lee et al.,
2005), cattle (Kanitz et al., 2002; Baruselli
et al., 2006), sheep (Ryan et al., 1991;
Grazul-Bilska et al., 2007), pig (Hunter,
1964; Sommer et al., 2007) and man (Market-Velker
et al., 2010).
Various gonadotropins have been used for superovulation. Equine chorionic gonadotropin
(eCG) formerly known as Pregnant Mare Serum Gonadotropin (PMSG) has been used
in laboratory animals to induce multiple ovulations before it was applied to
livestock, namely cattle, buffalo and goats, to increase oocyte production.
PMSG is a glycoprotein having both FSH and LH activities. Also, human chorionic
gonadotropin (hCG), a glycoprotein hormone and similar in structure to Luteinizing
Hormone (LH) which simulates the physiologic effects of LH, has been used to
trigger the final follicular maturation before oocyte recovery in an ART program
(Griesinger et al., 2007).
The hamster has been used widely as a model for studying various diseases (Roberts
et al., 1978; Jorquera and Tanguay, 1997;
Nonaka, 1998; Milazzo et al.,
2002). However, only a few protocols (Barnett et
al., 1997; Ludwig et al., 2001; Lee
et al., 2005; Mckiernan et al., 2005)
have been developed for Assisted Reproduction Technologies (ART) in hamsters.
Yanagimachi et al. (1976) reported the first
successful heterologous in vitro fertilization in golden hamsters. Since
then, heterologous in vitro fertilization using hamster oocytes has
been effectively pursued to predict male fertility in numerous species, including
humans (Rogers et al., 1979), cattle and horses
(Brackett et al., 1982). In several important
aspects, the hamster in vitro fertilization system is one of the most
useful models that had been used for the study of mammalian gamete interaction
(Bavister, 1980). For the purpose of predicting male
fertility, superovulation minimizes the number of females utilized to produce
the required number of oocytes for gamete studies or any advanced embryological
studies. Access to a reliable source of high quality harvested oocytes, with
capability for fertilization and development, is an important requisite both
for basic reproductive studies and applied research (Martin-Coello
et al., 2008).
In most hamster studies, it is tacitly assumed that collected and cultured
oocytes and embryos from PMSG-stimulated females are normal. However,
there is increasing evidence that the quality of oocytes and embryos of these
stimulated animals may be different from oocytes of non-stimulated females (McKiernan
and Bavister, 1998). Therefore, the present study was conducted to establish
an effective superovulatory program for golden hamsters in order to obtain a
large quantity of high quality oocytes by manipulating the dose of hCG, time
interval between PMSG and hCG injection and time interval between oocyte recovery
and hCG injection.
MATERIALS AND METHODS
Experimental animals and design: Fifty-six pubertal female golden hamsters
(Mesocricetus auratus) with body weight ranging from 120-150 g and age
between 12 and 15 weeks old were purchased from Institute for Medical Research
(IMR) in Kuala Lumpur, Malaysia. Prior to the start of the study the hamsters
were adapted and maintained in a special room for two weeks under controlled
lighting 12:12 h light-dark cycle, with the light switched on between 07:00
and 19:00 h. The room temperature was maintained at 22°C with approximately
60-70% humidity. These hamsters had exhibited at least two consecutive normal
4 day oestrous cycles, which were checked based on the vaginal discharge. The
56 golden hamsters were assigned randomly and divided equally into eight groups:
G1 to G8 (Table 1).
|| Treatment groups of superovulation protocols with oocyte
recovery time after hCG injection in the golden hamsters
|1n = 7 hamsters per group
Superovulation was induced by intraperitoneal (i.p.) injection of 40 IU PMSG
(Folligon® Intervet International, B.V. UK) in all groups regardless
of the stage of estrus of the hamsters. Then, ovulation was induced by administration
of either 40 or 45 IU hCG (Chorulon®; Intervet International,
B.V. UK) injected intraperitoneally at 48-50 or 55-57 h after PMSG injection.
Oocytes were then collected from the hamsters either 12-15 or 16-18 h after
Recovery of oocytes: Prior to oocyte recovery, the females were anesthetized
with a combination of ketamine (80 mg kg-1 b.wt., Troy Laboratories
PTY Limited, Australia) and xylazine (10 mg kg-1 b.wt., Troy Laboratories
PTY Limited, Australia) following the recommendation of Heffner
and Harrington (2002) intraperitoneally and then later sacrificed with an
intracardiac injection of pentobarbital sodium 250 m kg-1 b.wt. (AKORN;
Nembutal® Sodium Solution CII, USA). The oviducts were subsequently
flushed with modified Tyrodes solution (TALP) for oocyte retrieval following
the method described by Bavister and Yanagimachi (1977).
After collection, the maturation status of oocytes was assessed by degree of
cumulus expansion (De Loos et al., 1992), which
was also used to assess oocyte quality. Expansion was characterized by the extremely
sticky nature and enlargement of the cumulus mass to at least 0.5 mm in diameter
(≥500 μm) away from the zona-pellucida. Lack of expansion which is an
indication of poor-quality oocytes, was characterized by tight adherence of
cumulus cells to the zona-pellucida. Nuclear maturation of oocytes was evaluated
and confirmed by the formation of a first polar body extrusion examined under
a stereoscopic microscope at 40xmagnifications. This experimental procedure
has been reviewed and approved by the animal care and use committee of Faculty
Veterinary Medicine, Universiti Putra Malaysia (UPM/FPV/PS22.214.171.1241/AUP-R106).
Removal of cumulus oophorus and zona pellucida: Oocytes collected from
superovulated hamsters were treated with 0.1% (w/v) hyaluronidase (Type, I,
H-3506; Sigma-Aldrich St. Louis, Missouri, USA) to remove the cumulus oophorus.
The oocytes were then washed three times with wash-TALP medium. After washing
the oocytes were transferred into a droplet containing 0.1% (w/v) trypsin (Type
III, T-8003; Sigma-Aldrich St. Louis, Missouri, USA) placed in a petri dish
to dissolve the zona pellucida. Digestion and dissolution of the zona pellucida
were performed within 1 to 2 min and monitored under the dissecting microscope
to avoid under or over treatment. Then, zona-free oocytes were washed three
times with wash-TALP medium to remove any remnants of trypsin solution.
In vitro sperm capacitation: Straws (0.5 mL) which contained
frozen bull semen were thawed in a water bath at 38°C for 30 sec, pooled
in a 15 mL Falcon tube (three straws per tube; each straw contained approximately
25-30x106 live sperm at the time of freezing) and diluted with 5
mL of Sperm-TALP solution. Then, the diluted bull semen was centrifuged at 350xg
for 5 min motile sperm cells were isolated by the swim-up procedure as described
by Lu et al. (1987). The final pellet obtained
after the swim up was diluted in a capacitation medium (Sperm-TALP with 50 μg
mL-1 heparin; Sigma No. H-5765) and acrosome reaction induced by
adding 10 μM calcium ionophore A 23187 for 1 min (C-7522; Sigma, Aldrich
Chemie GmbH, Germany) (Tardif et al., 1999).
The capacitated sperm pellet recovered after discarding the supernatant was
diluted with the capacitation medium to give a sperm concentration of 2-4x106
sperm mL-1. The sperm droplets (100 μL each) were prepared in
a 35x10 mm petri dish covered with mineral oil and kept in an air incubator
Sperm-ocyte co-incubation, staining and penetration scoring: Between
10 and 15 washed zona-free hamster oocytes (ZFHOs) were placed in each sperm
droplet (Fert-TALP) in a medium described by Bavister and
Yanagimachi (1977) and incubated at 38°C for 3 h. After co-incubation,
the ZFHOs were removed, washed and fixed on glass slides and stained with 1%
Aceto-orcein, to examine sperm penetration. The presence of a distended sperm
head, with a tail and male pronucleus was taken as an indication of successful
sperm penetration, as described by Yanagimachi et al.
(1976). Fertilization Percentage (FP) was calculated using the following
Statistical analysis: Comparison between superovulation protocols on
the number of oocyte recovered, maturation rate and fertilization percentage
was done using one-way ANOVA, followed by a Duncan multiple range test. Differences
between superovulation protocols were considered significant at p<0.05. The
analysis was performed by SPSS (SPSS Inc. Version 20).
Oocyte recovery and maturation: The total number of oocytes recovered
and maturation rate from hamsters in the eight different superovulation groups
are shown in Table 2.
|| No. of oocytes harvested and maturation rate in golden hamsters
following different superovulation protocols
|1Data were expressed as total No. (Mean±SEM)
from seven replicates (hamsters) per group, abcValues with different
superscripts in the same column are significantly different at p<0.05
|| Fertilization rate of oocytes fertilized with capacitated
|1 ZFHOs: Zona-free hamster oocytes, 2
Data were expressed as mean±SEM from seven replicates abValues
with different superscripts in the same column are significantly different
In general, groups which received higher doses of hCG (45 IU) showed higher
number of oocytes recovered and subsequently, higher number of mature oocytes.
Group 8 gave a significantly higher number of oocytes recovered and matured
oocytes. In fact, among the eight groups, the highest number of oocytes recovered
was obtained from hamsters given 45 IU hCG at 55-57 h after PMSG injection and
when the oocytes were recovered 16-18 h later (mean of 51.57±0.83). Whilst
the lowest number of oocytes was recovered from G1 with a mean of 34.57±0.84.
Thus, the total number of oocytes recovered and the maturation rate of G8 was
significantly highest (p<0.05) among the 8 superovulation groups. When comparison
was made between 1-G6 groups which received equal doses of PMSG and hCG (40
IU each), there were no significant (p>0.05) differences observed in the
mean number of oocytes recovered and maturation rate. These results indicated
that when the time for oocytes recovery was delayed as in G2 and G6, the mean
number of oocytes recovered slightly increased but not significantly different
from G1 and G5 when the oocytes were recovered earlier at 12-15 h post hCG (G1;
34.57±0.84 vs. G2; 36±0.61 and G5: 36.42±0.84 vs. G6: 37.28±0.74).
Heterologous in vitro fertilization: Table 3
shows the results on heterologous in vitro fertilization of hamster oocytes
with capacitated bull spermatozoa. The fertilization rate of G7 and G8 were
significantly higher (p<0.05) than G1-G6 but not with G3 and G5 (Table
3). However, there was no significant difference (p<0.05) in fertilization
rate between G7 and G8. The superovulation protocols for G7 and G8 resulted
in similar fertilization rate achieved, 77.23 and 79.01%, respectively (Table
The association of ovulation rate and days of oestrous cycle was inversely
related with low ovulation rate found on days 3 and 4 of oestrous cycle in hamsters
as reported by Fleming and Yanagimachi (1980). The results
of the present study revealed that oocyte recovery and fertilization rates were
independent on the stage of the hamsters oestrous cycle. In the present
study the optimum superovulation protocol which resulted in the highest mean
number of oocytes recovered per female (51.57±0.83) and maturation rate
(94.20) was achieved by the administration of 40 IU PMSG and 45 IU hCG at 55-57
h interval followed by oocyte recovery 16-18 h after hCG injection.
A previous study by Wilson and Zarrow (1962) showed
dose of hCG, interval between PMSG and hCG injections and interval between hCG
and recovery of the maximum number of ova influenced the superovulatory response
in immature mice and rats. The optimum concentration of PMSG for superovulation
in hamsters was in the range of 30 to 45 IU as indicated by Greenwald
(1974, 1976), Lee et al.
(2005) and Kathiravan et al. (2008).
In the present study, the different groups of animals treated with equal doses
of PMSG and hCG irrespective of the differences in the interval between PMSG
and hCG injections and time of oocyte recovery generally showed insignificant
differences in both the mean number of oocytes recovered as well as the maturation
rate. This indicated that the time interval between PMSG and hCG injections
and time of oocyte recovery was not a significant factor in influencing the
variation in ovulatory rates. However, the optimum time interval between follicle
stimulating substance (FSH) injection and the ovulatory dose of chorionic gonadotropin
was reported to be 56 h in rats (Munalulu et al.,
1987), 40 h in mice (Fowler and Edwards, 1957; Gates
and Runner, 1957) and 54-56 h in hamsters (Bodemer et
al., 1959). In the groups that were administered with 40 IU hCG, the
number of recovered oocytes and mature oocytes was significantly lower than
the 45 IU hCG groups. This indicates that the dose of hCG plays an important
factor in influencing ovulatory rate. This finding is consistent with the earlier
studies of Erickson and Shimasaki (2001), Grimmett
and Perkins (2001) and Mehaisen et al. (2005).
In addition, when comparisons were made among groups which were given the same
dose of hCG, the number of oocytes recovered and the number of mature oocytes
increased. These results are consistent with Wang et
al. (2011) who reported that the percentage of mature oocytes can be
improved by extending the interval between hCG and oocyte recovery.
The dose dependent superovulatory response of hamsters observed in this study
is in agreement with Roldan et al. (1987) but
the dosages that were used in the present study were about 3 times higher. Similar
reports of using a combination of PMSG and hCG gave good response in other laboratory
animals such as rats (Corbin and McCabe, 2002; Cornejo-Cortes
et al., 2006) and mice, (Martin-Coello et
al., 2008; Luo et al., 2011). Corbin
and McCabe (2002) reported that higher doses of PMSG (30 IU) and hCG (25
IU) increased significantly the number of oocytes recovered in female rats.
Furthermore, according to Cornejo-Cortes et al. (2006),
equal doses of PMSG (50 IU) and hCG (50 IU) produced the highest mean number
of rat embryos when given 50 h apart. This was the highest PMSG: hCG dose combination
attempted compared with other doses (30:30, 30:50, 50:30) in rats.
The effectiveness of PMSG injection in producing ovulated oocytes was extremely
influenced by the stage of the oestrous cycle at which it was given (Lee
et al., 2005). Recovery rate was higher on days 3 and 4 of the oestrous
cycle. Given 45 IU hCG, the hamsters in the current study assumed to be in late
estrus showed significantly higher number of oocytes recovered compared with
the other groups. Fleming and Yanagimachi (1980) reported
that ovulation rate in golden hamsters was associated with the days of oestrous
cycle, whereby ovulation rate decreased on days 3 and 4 of the oestrous cycle
but increased on day 1. Thus in the present study it can be assumed that hamsters
that had low ovulation rates were in days 3 and 4 of the ooestrous cycle.
In agreement with Fleming and Yanagimachi (1980) the
present study indicated that, ovulation rate decreased on days 3 and 4 of the
oestrous cycle compared with day 1 in golden hamsters following PMSG injection.
The reduction in ovulation rate might be attributed to a deviation in gonadotropic
properties among endogenous FSH and exogenous PMSG around day 4. Fleming
and Yanagimachi (1980) concluded that extreme gonadotropin levels when PMSG
was added on the normal FSH level subsequently might have an inhibitory influence
on the development of follicles. The current optimum protocol comprising of
the administration of 40 IU PMSG followed by 45 IU hCG injection at 55-57 h
later and subsequent oocyte recovery in 16-18 h was shown in the present study
to result in highest number of oocytes recovered, leading to high maturation
Although the superovulation protocol (40 IU PMSG, 45 IU hCG) in the present
study produced the highest mean number of oocytes per female, maturation rate
and fertilization rate, it doesnt guarantee in producing subsequent higher
quality embryos compared with other protocols tested if the fertilized oocytes
were subjected to culture or embryo-transfer. Hence further subcellular studies
to evaluate the developmental competence of fertilized oocytes obtained by the
different protocols compared with the optimum protocol should be able to confirm
and identify the best protocol. According to Lee et al.
(2005) although hamsters can be effectively superovulated by injecting equal
doses of PMSG and hCG, a dose-dependent adverse effect on fertilized oocytes
(embryos) development and sub-cellular microfilament distribution can occur.
In the present study, the ability to induce superovulation by the PMSG administration
concurred with Roldan et al. (1987) which is
in agreement with previous studies that reported the ability to retrieve good
number of oocytes in Chinese hamsters and golden hamsters (Roldan
et al., 1987) as well as in other laboratory animals including rats
(Hiroe et al., 2005) and mice (Martin-Coello
et al., 2008). This has the advantage of minimizing the workload
as it avoids the need to program superovulation by identifying animals on a
particular stage (day 1) of the oestrous cycle to inject PMSG.
Heterologous in vitro fertilization test evaluated in the present study
indicated higher fertilization rates from the golden hamsters given 45 IU of
hCG. The extra follicles induced to ovulation by 45 IU hCG were physiologically
normal as attested by the high percentage of fertilized oocytes. These findings
implied that none of the doses of hCG and time interval tested inhibited growth
and nuclear maturation of oocytes.
Generally, the present study was able to optimize the superovulation protocol
in golden hamsters to obtain higher number of good quality oocytes. Though,
the time interval between injection of PMSG and hCG and recovery interval of
oocytes might have an impact on oocyte recovery, the dose of hCG appeared to
be the most important factor in determining the superovulatory response of hamsters
to the different superovulation protocols. The different interventions in the
superovulation protocols on developmental competence of fertilized oocytes needs
to be addressed in future studies.
In general, the present study succeeded to make effective the superovulation
procedure for golden hamsters in order to obtain a lofty number of high-quality
oocytes. Although the period between PMSG and hCG injections as well as recovery
of oocytes might contribute to the rate of oocyte recovery, the hCG dose appeared
to be the influential factor in determining the superovulatory response of hamsters
to the different superovulation protocols. In conclusion, among the three factors
examined in establishing the superovulation protocol for hamsters, hCG dosage
is the most prominent factor in achieving the highest oocyte recovery rate.
Higher dosage of hCG (45 IU) and longer interval between PMSG-hCG applications
(55-57 h) and oocyte recovery (16-18 h) resulted in higher number of oocytes
recovered, leading to higher maturation rate of the oocytes. Nevertheless, the
different interventions in the superovulation protocols on developmental competence
of fertilized oocytes needs to be addressed in future studies.
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