Abstracts for the KSAR and JSAR Joint Symposium

Fertility control in female domestic animals:
From basic understanding to application

Current Research Orientation in Livestock Reproduction in Korea
Choong-Saeng PARK and Seong-Keun CHO
Laboratory of Animal Reproduction, College of Agriculture and Life Sciences,Gyeongsang National University, Jinju, Korea
The genetic improvement of farm animals has been increased greatly byartificial insemination and multiple ovulation-embryo transfer. The nuclear transfer(NT) technology has been developed to increase the frequency of genes of genetically
outstanding animals in a population. The introduction of new genetic materials into apopulation and the production of knock-in and knock-out animals forxenotransplantation have also been attempted by transgenic and somatic cell nuclear
transfer (SCNT) technologies. Furthermore the human somatic cell nuclear
transfer-embryonic stem (SCNT-ES) cell lines will be contribute to develop newtechnologies in cell therapy. During the period of 15 years since 1988, the researchworks in the field of embryo cloning and SCNT have also been performed withsuccessful results in Korea. Some of the interesting results related to embryo cloning,
SCNT, transgenic SCNT, and human SCNT-ES cell lines from Korean laboratories arediscussed by animal species in my talk.

2The Basis of the Oocyte Production
Yuji Hirao
National Agricultural Research Center for Tohoku Region, Morioka, Iwate, JapanThe ovary is a multi-functional organ and is in particular responsible forproducing fertile ova. Each one of ova is derived from a follicle, a specialized structurethat encapsulates the oocyte and supports its development up to ovulation. In the meantime, however, many more of other oocytes are to be lost by atresia as a result ofovarian regulatory actions. Therefore, the overall mechanisms for the oocyte (ovum)
production in the ovary involve complicated processes, many of which are more likely
to permit a limited number of follicles to survive than simply nurture the oocyte. In thisreview, the focus is on a simple model of an intrafollicular environment that supports
oocyte development. In vitro culture of growing oocytes with companion ovarian cells
provides us with a way to know the necessary and sufficient conditions for continued
oocyte growth. Such culture systems have been developed to understand the regulatory
mechanisms of oocyte development in mice, but are also potentially powerful tools forstudies of oogenesis in larger animals. From application point of view, establishment of
the oocyte culture technology would lead to expand the population of livestock andother valuable animals. In a typical mouse system, oocyte-granulosa cell complexes cangrow on the surface of the matrix. Only recently, such a substratum-adherent system has
been developed for bovine oocytes, in which the oocytes, starting from about 75% of
the maximum diameter, approached the full-size during a two-week culture period. In
this case, no cell types other than granulosa cells were necessary to support oocyte
growth. A proportion of the oocytes were capable of undergoing in vitro fertilizationand embryo development. It should be emphasized that a high concentration of
polyvinylpyrrolidone in culture medium, a novel modification, influences theorganization of complexes, resulting in a firm association between the oocyte andsurrounding granulosa cells. Studies with mice have revealed that the oocyte has key
roles in regulation of its own microenvironment through establishing the bidirectional
communication with granulosa cells. These findings taken together would imply that acontinuous and evolving regulatory loop between the oocyte and surrounding granulosa
cells is essential as a basis for the growth and development of animal oocytes.

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In Vitro Production of Cloned Porcine Embryos
Deog-Bon Koo, Kyung-Kwang Lee and Yong-Mahn Han
Laboratory of Development and Differentiation, Korea Research Institute of Bioscienceand Biotechnology, Daejeon, Korea
Although cloned piglets have been successfully generated from embryos producedin vitro after somatic cell nuclear transfer (SCNT), many problems such as a low
developmental rate and a poor quality of SCNT-derived embryos remain to be solved.
The acquisition of developmental competence of SCNT-derived embryos during in vitro
culture is a prerequisite step to produce cloned piglets. Despite recent improvements in
the in vitro culture system for porcine embryos, the efficiency of the production ofpiglets after transfer of SCNT- or IVF-derived embryos is still inferior to other speciessuch as sheep and cattle. Therefore, our researches are focused on improving in vitro
developmental ability and structural integrity of porcine embryos. Firstly, we found thatelectrical stimulation was more effective for parthenogenetic activation of in vitro
matured porcine oocytes than chemical reagents. Then, effects of different activation
methods on developmental ability of SCNT embryos were also assessed. Electricalpulse made significant increases in both cleavage rate and development rate to theblastocyst stage (P<0.05) of SCNT embryos compared to A23187 only or combined
A23187/6-DMAP treatment. And then to investigate the optimal conditions forelectrical method, the field strength and activation timing after fusion on thedevelopment of porcine SCNT oocytes were examined. The results demonstrate thatfield strength and activation timing after electrofusion could affect developmental
ability and quality of porcine SCNT embryos. SCNT-derived embryos had a lower
developmental competence to the blastocyst stage than IVF-derived embryos (P<0.05).
Total cell number of SCNT-derived blastocysts was also inferior to that of IVF-derivedembryos (P<0.05), however no difference was detected between the two groups in theratio of ICM to total cells. To investigate what proportions of in vitro-produced porcine
embryos represent normal structural integrity, differentially-stained blastocysts wereindividually classified into three presumptive groups according to the ratio of ICM tototal cells (I; <20%, II; 20 to 40%, and III; >40%). Low proportions of NT- andIVF-derived blastocysts were assigned to Group II, whereas almost all of invivo-derived embryos were allocated to Group II. From our observations, it isconcluded that limited structural integrity may lead to poor survival to the term ofSCNT- or IVF-derived porcine embryos produced in vitro.

4Hormonal Control of Follicular Maturation, and Luteal Development
and Maintenance in Domestic Ruminants
Noritoshi Kawate
Laboratory of Theriogenology, Graduate School of Agriculture and Biological Sciences,
Osaka Prefecture University, Osaka, Japan
Ovarian activity in domestic animals is regulated intrinsically and can be, at least
partially, controlled extrinsically by hormones. In domestic ruminants, luteinizing
hormone (LH) has a pivotal role in the regulation of ovarian functions, such as follicularmaturation, ovulation, and luteal development and maintenance. In this joint symposium,
I will describe our observations including artificial controls using hormones of ovarianfollicular maturation for fertility improvement, and gene expressions of a few
luteotropic factors during luteal development and their regulation by LH in ruminants.
In Shiba goats, which are originated from a Japanese native dwarf breed and are
non-seasonal breeder, estrus is not observed usually during the suckled early postpartum
period. Effects of a low dose of human chorionic gonadotropin (hCG) on the inductionof estrus were examined in suckled early postpartum goats pretreated intravaginallywith progesterone (Controlled Internal Drug Release dispenser; CIDR). Treatment withthree times of a low dose of hCG at 12h intervals after the inclusion of CIDR for 7 days
promoted follicular maturation and fertile estrus induction in the goats (Kawate et al, JReprod Dev, 2002).
In suckled postpartum Japanese Black beef cows, inclusion of a CIDR for 7 days
from the start of Ovsynch protocol (GnRH on Day 0, PGF2 on Day 7, GnRH on Day9) improved conception rates to timed-AI (Kawate et al, Theriogenology, 2004). To
clarify one of mechanisms of the improved conception by the combination of Ovsynch
and CIDR, timings of follicular maturation and ovulation of the beef cows wereexamined when hormonal treatments were started a few days before initiation of corpus
luteum regression. These studies showed that dominant follicles matured before thesecond GnRH treatment and ovulated before AI in the Ovsynch protocol alone. The
addition of a CIDR to the Ovsynch prevented such earlier occurrences of follicularmaturation and ovulation, and made those phenomena occur at an appropriate time.
To examine a part of mechanisms of corpus luteum (CL) development andmaintenance in domestic ruminants, gene expressions of LH receptors, vascularendothelial growth factor (VEGF) and its receptors and prostacyclin (PGI2) synthase,
which catalyzes the conversion of PGH2 into PGI2, in the CL, and their regulations byLH were examined using the Shiba goats. The PGI2 synthesis was enhanced at thebeginning of CL formation, followed by increases of LH receptors and its mRNA,VEGF165 and KDR/Flk-1 (one of VEGF receptors) mRNAs during the CL
development, and then VEGF165 and Flt-1 (another VEGF receptor) mRNAs increased
during the CL maintenance (Kawate et al, Mol Reprod Dev, 2002, 2003, 2004). The
increase of LH receptor during the CL development was inhibited by5gonadotropin-releasing hormone antagonist, but other factors were not affected. Theseresults suggest that the PGI2 may play a role in initiating caprine CL development, andthat the LH receptor, VEGF and KDR/Flk-1 may participate in the CL development.
Besides, the LH receptor may be, at least partly, up-regulated by LH itself during theCL development (Kawate, J Reprod Dev, 2004).

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Synchronization of estrus and ovulation in dairy cattle
Ill Hwa Kim
College of Veterinary Medicine, Chungbuk National University, Chongju, Chungbuk,
Korea
The failure to accurately detect heat is one of main reasons for increasing thecalving interval in dairy herds. However, the estrus periods in nearly half of normalcycling dairy cows may not be detected. In addition to poor heat detection, delayed
ovulation and failure to ovulate is one of the most frequent causes of infertility in highyielding dairy herds. Typical protocols for estrus synchronization have focused on
extending or restricting the luteal phase by the use of exogenous progestins and/ orPGF2α. These protocols allow dairy producers to easily detect cows in estrus within
limited periods (2 to 7 days). Previously developed protocols for estrus synchronization
using PGF2α show large variations in the time to estrus due to the stage of developmentof follicular waves at the time of treatment. In fact, the synchrony of estrus andovulation not only depends on the control of luteal life span but also on the phase offollicular development. Thus, protocols for manipulation of follicular development toinduce precise synchronization of ovulation have been developed in cattle. The Ovsynch
protocol, which combines GnRH (Day 0), PGF2α (Day 7) and GnRH (Day 9)
treatments, may be used to regulate follicular development and subsequent ovulation.
This protocol induces synchronized ovulation within 24 to 32 h after a second injectionof GnRH, thereby enabling AI without the need to detect estrus. It has been widely usedin many beef and dairy herds. However, ovulation failure in response to the initial
GnRH injection in the Ovsynch protocol may result in reduced pregnancy rates
following timed AI due to asynchronous ovulation. For example, premature estrus
between the first GnRH and the injection of PGF2α has been reported in 5 to 11.8% ofcows. An alternative is to utilize a progestin-based regimen. However, in order toprevent the development of a persistent follicle that can result in reduced fertility
following AI, the induction of a new follicular wave is important in progestin-based
synchronization regimens. Recently, researches showed that treatment with GnRH in a
CIDR-based protocol improved pregnancy rates to timed AI in beef and dairy cattle.
The protocol resulted in the emergence of synchronous follicular waves andsynchronous ovulation, which gave acceptable pregnancy rates to timed AI. At the sametime, the interval from treatment to the emergence of a new follicular wave appears toinfluence pregnancy rates, possibly because of the growth and size of the newpreovulatory follicle. Further studies on the delicate regulation of follicular dynamics
and luteal function may lead to enhanced fertility in dairy cattle.

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Improvement of Pregnancy Rates by Efficient Selection of Recipientsin Bovine Embryo Transfer
Masahiko Nishigai
Nasu ET Institute, 7-10, Shimakata, Kuroiso, Tochigi, JapanTo improve pregnancy rates in bovine embryo transfer, the correlation of thepregnancy rate with the luteal function in recipients was investigated. Blood concentrationsof progesterone (P) and estradiol-17β (E2), which closely related the luteal function weredetermined in parous Japanese Black beef cattle on the previous day of frozen embryotransfer i.e., on day 6 (the day of appearance of estrus designated day 0) and the day of thetransfer i.e., on day 7. Both on the previous day and on the day of embryo transfer, thepregnancy rate tended to be increased with the increase in blood P concentration ofrecipients; the pregnancy rate (60.7%) for recipients, which showed a blood P
concentration of ≧2.5 ng/ml on the previous day of embryo transfer, was significantly(P<0.05) higher than that (38.6%) of recipients showing a blood P concentration of ＜2.5ng/ml on the previous day of embryo transfer. The pregnancy rate tended to be increased
according to the decrease of blood E2 concentration on the day of embryo transfer. To
investigate the mechanism involved in the improvement of pregnancy rate being positively
correlated with the high luteal function in recipients, we injected 1,500 IU of humanchorionic gonadotropin (hCG) in Holstein dairy cattle on the day of ovulation or 5 days
after ovulation, and then reaction of the ovaries to the administration was investigated. The
cattle administered with hCG 5 days after ovulation showed the formation of induced
corpus luteum resulting in a significantly (p <0.05) higher blood P concentration than thatof control cattle. They showed a rapid decrease of blood E2 concentration after hCGadministration and maintained it at a low level. Based on these results, the following
treatments were conducted prior to embryo transfer and the pregnancy rate was compared;
1,500 IU of hCG was injected in parous Japanese Black beef cattle recipients on days 1(Day 1-hCG group) and 6 (Day 6-hCG group), and 5 ml of saline was injected in thecontrol cattle (control group) on day 6. The pregnancy rate (67.5%) in the Day 6-hCGgroup was significantly (p <0.05) higher than that (45.0%) in the control group and that(42.5%) in the Day 1-hCG group.
These results revealed that the administration of hCG 1,500 IU to recipients on day 6
improve the pregnancy rate in the non-surgical frozen embryo transfer carried out on day 7
by enhancing luteal function and depressing E2 secretion in recipients.

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