• Asian-Australasian Journal of Animal Sciences
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• 제25권5호
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• pp.629-634
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• 2012

This study was conducted to improve the yield of mature oocytes from in vitro-culture of ovarian primary follicles by optimizing follicle retrieval from neonatal mice of different ages. Primary follicles of 75 to $99{\mu}m$ in diameter were collected daily from 7- to 14-day-old neonatal mice, and subsequently cultured in ${\alpha}$-MEM medium. Number of primary follicles isolated, growth of the follicle during in vitro-culture and maturation of intrafollicular oocytes were monitored. Overall, mean number of preantral follicles per animal was improved from 10.7 to 88.7 as the age of follicle donors was increased from 7 to 14-day-old. Number of primary follicles was increased gradually up to 11-day-old (35.7 follicle per an animal), then reduced to 29 in 14-day-old (p = 0.0013). More follicles retrieved from 10-day-old or 11-day-old females maintained their morphological normality at the end of primary culture than the follicles retrieved from 9-day-old. Of those cultured, primary follicles retrieved from 11-day-old mice yielded largest larger number of early secondary follicles than the follicles retrieved from in the other ages (39 vs. 13 to 29%). More than 3.3-times increase (0.86 to 2.86; p<0.05) in an average number of mature oocytes per animal was observed in the group of 11-day-old, compared with 9-day-old. However, no difference was found in the percentage of primary follicles developing into the pseudoantral stage (21 to 30%; p = 0.5222) and in the percentage of oocytes mucified (32 to 39%; p = 0.5792). In conclusion, a positive correlation between retrieval time and follicle growth was detected, which influences the efficiency to derive mature oocytes by follicle culture.

• Clinical and Experimental Reproductive Medicine
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• 제12권2호
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• pp.71-79
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• 1985

Systemic extrogen therapy promotes multiple preantral follicular development in immature mice. Estrogen pretreated ovaries might therefore be a useful source of cells for in vitro studies of oocytes maturation. Silastic capsules (5.0 mm length; 3.18 mm outer diameter, 1.57 mm inner diameter) filled with diethylstilbesterol were implanted subcutaneously in experimental mice (ICR) for up to 6 days. Ovarian weight and histology in diethylstilbesterol pretreated and control animal were assessed before and after pregnant mare serum gonadotrophin treatment and after human chorionic gonadotrophin. The following results were obtained; 1. Ovarian weight was significantly increased by 6 days of diethylstilbesterol pretreatment. Subsequent ovarian weight gain in response to pregnant mare serum gonadotrophin and human chorionic gonadotrophin was increased. 2. Diethylstilnbesterol pretreatment stimulated the developed healthy preantral follicles. 3. Forty eight hours after pregnant mare serum gonadotrophin treatment, a larger number of the antral follicles which developed in diethylstilbesterol pretreated animals showed signs of atresia, whereas in the control ovaries there was a higher incidence of premature luteinization. 4. Forty eight hours after human chorionic gonadotrophin, numerous corpora lutea and occasional luteinized unruptured follicles were present in both control and diethylstilbesterol ovaries. 5. Ovulation rate, fertilization rate and subsequent preimplantation development in vitro were not adversely affected by diethylstilbesterol pretreatment. However, there was considerable variation in the ovulation rate the number of animals with more than 60 ovulations was greater in the diethylstilbesterol gorup (52.4%) as compared to the control (33.3%).

• Asian-Australasian Journal of Animal Sciences
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• 제20권9호
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• pp.1354-1360
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• 2007

The aim of this study was to evaluate if oocytes, aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration, could be efficiently used as ooplasm recipients for somatic cell nuclear transfer (SCNT). Within a common SCNT protocol, a comparison between oocytes recovered by direct aspiration (aspirated) from available ovarian follicles and oocytes flushed out from oviducts (flushed) was carried out. The results showed that maturation and enucleation rates of aspirated oocytes were 70.7% and 69.2%, significantly lower than 95.3% (p<0.01) and 83.6% (p<0.05), respectively, from flushed oocytes. However, following enucleation of matured oocytes as ooplasm recipients for SCNT, no difference was recorded in fusion and cleavage rates, as well as blastocyst development from cleaved embryos or hatching of blastocysts between aspirated and flushed groups. Additionally, some matured aspirated and flushed oocytes were also used for immediate parthenogenetic activation and the resulting embryo development was not significantly different. Results from this study show the following: i) the majority of oocytes aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration are matured and can be used directly as ooplasm recipients for SCNT; ii) the reconstructed embryos derived from these oocytes have similar in vitro developmental ability to those flushed from the oviducts.

• Asian-Australasian Journal of Animal Sciences
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• 제15권12호
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• pp.1695-1701
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• 2002

The purposes of this study were to optimize the in vitro maturation (IVM) and culture (IVC) systems of rabbit oocytes. Cytoskeletal structures in the germinal vesicle stage (GV) and during IVM are also investigated. Ovaries were transported from local slaughterhouses and the cumulus-oocyte complexes (COCs) were collected from ovarian follicles (${\geq}1mm$). COCs were randomly allocated to TCM199-based medium ($T_1$, TCM-199) supplemented with $NaHCO_3$, glucose, sodium pyruvate and FSH ($T_2$), $T_2+E_2+LH$ ($T_3$), $T_3+FBS$ ($T_4$), or $T_1+E_2+LH+FSH+FBS$ ($T_5$), for IVM. In Experiment 1, COCs were retrieved from the follicles and 51 GV oocytes were fixed in the fixative (MTSB-XF) for nuclear and cytoplasmic examinations. In Experiment 2, progressive changes of both the nucleus and the cytoskeleton were examined at 0, 6, 16, and 20 h after IVM. Maturation (MR) and developmental rates were assessed in Experiment 3. Cytoplasmic microtubules (MT) were clearly observed in rabbit GV oocytes. To our knowledge, this is the first report that describes the appearance of MT structures in the GV stage ooplasm. Tremendous variations in cytoskeletal alterations were observed among treatments with the exception of the vitelline ring (VR), which is constantly visible and unchanged during maturation. Germinal vesicle breakdown (GVBD) does not occur at 6 h after onset of maturation culture. When the oocytes for IVM were collected within 2 h, results from Experiment 3 showed that rates of nuclear maturation were 42, 8, 42, 37 and 65% at 16 h of IVM for $T_1$ through $T_5$, respectively, in which $T_1$, $T_4$ and $T_5$ had significantly greater MR than those in other groups (p<0.05). Morula/blastocyst development after parthenogenetic activation ranged from 20 to 63% with significantly greater rates in $T_3$, $T_4$ and $T_5$ (p<0.05). These results suggested that oocytes recovered from slaughterhouse ovaries can be matured and parthenogenetically activated in vitro, but the MR remained low in this study. Addition of $E_2$ and LH in the medium may be beneficial for cytoplasmic maturation, but FBS exerts a nega- tive role in the subsequent development of parthenogenetic embryos when energy substrates are provided in the IVC media. More studies are required for improving the MR and further development of the GV stage rabbit oocytes.

• 한국수정란이식학회지
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• 제15권3호
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• 한국양식학회지
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• 제17권2호
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• pp.128-132
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• 2004

이 연구에서는 자연산 참가자미의 주 어획지역인 강원도 주문진 인근해역에서 1998년 9월부터 1999년 10월까지 연간 생식소중량지수의 변화를 조사하였으며, 조직학적 조사에 의한 생식소 발달과정과 생식주기를 밝혔다. 정소는 정소소엽 형태이며, 각각의 정소엽은 여러 개의 소낭구조를 가진다. 각 소낭내의 생식세포들은 같은 단계의 발달상태를 보인다. 난소는 원추형의 낭상으로 체강벽에서 연결되는 난소간막에 의해 부착되어 있으며, 내부는 결체성 조직인 다수의 난소박판으로 구성되며, 이곳에서 난원세포가 유래한다. 수컷의 GSI는 1월에 가장 높았으며, 암컷의 GSI는 3월에 가장 높은 값을 보였다. 생식주기는 성장기(6∼9월), 성숙기(10∼12월), 완숙 및 산란기(1∼3월)그리고 회복 및 휴지기(4∼5월)로 나눌 수 있었다. 난모세포의 발달양식은 난군동기발달형에 속하였다.

• Fisheries and Aquatic Sciences
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• 제3권3_4호
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• pp.180-187
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• 2000

Reproductive effort and spawning frequency of the two palaemonid prawns, Exopalaemon modestus and Palaemon gravieri, were investigated. In both embryos of the two species, egg size was larger in E. modestus than in P. gravien but for a given size, number of eggs (EN) was fewer in E. modestus. The statistical results revealed that there were significant differences in egg size and EN between the two species. E. modestus living in freshwater environments had larger and fewer offspring than P. gravieri inhabiting marine environments. These findings are consistent with predictions from r- and K-selections models. Reproductive effort (RE) also was higher in E. modestus, suggesting the possibility for E. modestus to invest larger amount of energy per individual offspring. In the two prawns the ovarian dry weight of females with eyed eggs was significantly higher than those with non-eyed eggs. This indicates that the ovarian maturation occurs during the period between the two embryonic stages, suggesting females being potentially of continuous breeding within a single reproductive period. In the both species brood loss did not occur during the incubation period.

• 한국수산과학회지
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• 제33권6호
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• pp.554-558
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• 2000

참서대 암컷의 생식소중량지수는 6월에 가장 높았다. 난모세포의 발달양식은 난군동시발달형이며, 초기 성장기 난모세포에서 난병과 난황핵이 관찰되었다. 생식주기는 성장기 (2${\~}$5월), 성숙기 (5${\~}$6월), 완숙 및 산란기 ($6{\~}8$월) 그리고 회복 및 휴지기 ($8{\~}2$월)로 구분된다 전장 $TL 28.1{\~}30.8 cm$의 개체당 절대포란수는 2,197 개였으며, 체중 g당 상대포란수는 10.0개였다.

• 한국패류학회지
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• 제19권2호
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• pp.153-153
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• 2003

2001년 1월부터 2001년 12월까지 전라북도 김제시 심포 앞바다에서 각장 18.6-52.7 mm 의 가무락조개 (Cyclina sinensis) 를 대상으로 자원증식 및 적정 관리를 위해 조직학적 방법에 의해 생식소발달단계에 따른 생식주기와 군성숙도를 조사하였고 인공산란 유도에 의해 산란량과 산란빈도를 조사하였다. 생식소 발달단계에 따른 생식주기를 조직학적으로 조사한 결과 가무락조개의 생식주기는 초기활성기 (2-4월), 후기활성기 (3-6월), 완숙기 (5-8월), 부분산란기 (7-9월), 퇴화 및 비활성기 (9-2월) 의 연속적인 5단계로 구분할 수 있었다. 7월부터 산란하기 시작하여 9월 중순까지 일어났고 산란성기는 7-8월이었다. 각장 25.1-30.0 mm인 암컷 개체들의 군성숙도는 64.3%이었고, 각장 40.1 mm이상인 개체들의 군성숙도는 100%이었다. 인공산란 유도에 의해 각 개체들로부터 방란된 난수는 개체들의 각장이 증가됨에 따라 증가되었다. 2차 산란유도에 의해 방란된 평균 난수는 1차 산란수의 평균 76.87%이었다. 가무락조개의 각 산란 간격은 대략 15-17일 (평균 16.5) 이었고, 한 산란기 중 2회 이상의 산란이 일어날 것으로 추정되었다.

• 한국동물생명공학회지
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• 제34권1호
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• pp.35-39
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• 2019

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.