• Reproductive and Developmental Biology
• /
• 제35권3호
• /
• pp.233-238
• /
• 2011

This study was performed to investigate the FSH levels for superovulation procedure in Korean Native Cattle (Hanwoo). The effectiveness of 200 mg and 400 mg of FSH to initiate superovulation was examined in Hanwoo. Donors, at random stages of the estrous cycle, received a CIDR 7 days later, 200 mg FSH group was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection fur 4 days. Also, 400 mg FSH group was treated with 80, 60, 40, 20 mg FSH levels. On the 3rd day administration of FSH, 25 mg $PGF_2$ ${\alpha}$ was administered and CIDR was withdrawn. Donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 ${\mu}g$ GnRH at time of 1st insemination and embryos were recovered 8 days after the 1st insemination. As a results, average number of CL treated with FSH 200 mg was higher as $20.9{\pm}1.20$ than $15.8{\pm}0.63$ for donors treated with FSH 400 mg, respectively(p<0.05). Treated group of 200 mg FSH level increased (p<0.05) the number of embryos recovered per procedure compared to 400 mg FSH level ($18.2{\pm}1.18$ vs. $12.38{\pm}0.52$, respectively). When treatment of 200 mg FSH was performed, average transferable embryos/ova increased (p<0.05) to $14.1{\pm}1.12$ from $6.8{\pm}0.33$ of treated of 400 mg FSH. Group of 200 mg FSH increased (p<0.05) to $8.3{\pm}0.76$ from $2.0{\pm}0.26$ in morula stage compare to 400 mg FSH group. Mean of total early blastocyst and expanded blastocyst stage embryos was similar (p<0.05) between the 200 mg and 400 mg FSH levels group ($4.7{\pm}1.19$ vs. $2.9{\pm}0.18$ and $1.2{\pm}0.40$ vs. $1.9{\pm}0.17$). These results suggest that 200 mg FSH level-based superovulation protocol with CIDR may be effectively used fur production of superior embryos in Hanwoo. In other words, the less level of FSH may be effectively applied for Hanwoo (Korean Native Cattle), because Hanwoo was smaller body size than beef or daily cow.

• 한국동물생명공학회지
• /
• 제34권2호
• /
• pp.117-122
• /
• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• Reproductive and Developmental Biology
• /
• 제34권1호
• /
• pp.47-53
• /
• 2010

Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권12호
• /
• pp.1566-1577
• /
• 2010

Nandrolone, 19-nortestosterone, is a synthetic androgenic-anabolic steroid promoting muscle growth. Nandrolone is also present in pig meat and sera at non-negligible levels. A number of scientific reports have suggested a positive relationship between incidence of infertility and increased meat consumption in humans. The present study was designed to determine out the effect of feeding nandrolone on the testis of the male reproductive tract. Mixtures of food and nandrolone at different concentrations (0.005 ppm and 0.5 ppm) were supplied to pubertal male rats for 6 weeks. Body weight was recorded every week during the entire experimental period. At the end of the treatment, the testis, epididymis, and epididymal fat were collected and weighted. Sperm numbers in the caudal epididymis were counted. Differential gene or protein expression of steroidogenic enzymes in the testes among experimental groups was determined by semi-quantitative real-time PCR or western blotting analysis, respectively. Histological changes of the testis induced by nandrolone treatment were examined by hematoxylin and eosin staining. Immunohistochemical analysis was employed to detect changes in the localization of steroidogenic enzymes in the testes among experimental animals. There were no significant changes on body, testis, epididymis, and epididymal fat weights among experimental groups. A significant increase of caudal sperm number was found in the 0.5 ppm nandrolone-treated group. Histological examination of the testes noted a high frequency of germ cell sloughing in seminiferous tubules of 0.5 ppm nandrolone-treated rats. Even though transcript levels of $3{\beta}$-hydroxysteroid dehydrogenase (HSD) I, $17{\beta}$-HSD4, and $17{\alpha}$-hydroxylase were influenced by nandrolone treatments, protein levels of all molecules examined in the present study were not significantly affected. Immunohistochemical analysis showed no visible changes in the localization of steroidogenic enzymes in the testes among experimental groups. The current study showed that oral intake of nandrolone in male rats for 6 weeks did not cause significant damage to the testis. It is considered that a feeding effect of nandrolone on male fertility would not be remarkable.

• Reproductive and Developmental Biology
• /
• 제33권4호
• /
• pp.257-262
• /
• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

• 한국수정란이식학회지
• /
• 제30권3호
• /
• pp.161-170
• /
• 2015

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade $1^{{+}{+}}$, $1^{+}$, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield $1^{{+}{+}}$, $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield $1^{{+}{+}}$, $1^{+}$, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield $^{{+}{+}}$ and $1^{+}$ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield $1^{{+}{+}}$and $1^{+}$ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, $1^{+}$ and $1^{{+}{+}}$, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield $1^{{+}{+}}$, $1^{+}$, 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

• 원예과학기술지
• /
• 제33권1호
• /
• pp.114-123
• /
• 2015

식물은 다양한 생화학적 및 생리적 과정에 속한 유전자들의 발현 수준을 조절함으로써 저온 스트레스에 반응 및 적응을 할 수 있다. 이러한 스트레스 환경은 막 기능 손실, 세포벽의 변화, 대사 속도 변화 등과 같이 부정적인 영향을 초래한다. 따라서 본 연구는 배추(Brassica rapa ssp. pekinensis)에서의 시간 변화에 따른 저온 스트레스 반응 기작 관련 유전자 상호발현 네트워크를 구축하였다. 배추의 저온 스트레스 네트워크는 2,030개 node, 20,235개 edge, 및 34개 connected component로 구성되었으며, 구축된 네트워크는 배추에서 저온에 관여하는 유전자가 생육도 조절한다는 것을 보여 주었다. 구축한 네트워크를 이용하여 배추에서 저온 스트레스($4^{\circ}C$) 처리가 미치는 영향을 분석한 결과 WRKY 전사인자와 살리실산 신호에 의해 chitinase 부동 단백질이 활성화되고, 전신적 획득저항성을 작동하기 위해 기공 개폐 및 탄수화물 대사과정이 조절됨을 확인하였다. 또한 저온 처리 후 48시간 후에 저온 스트레스가 영양생장에서 생식 생장 및 분열 조직 단계의 변화를 초래하는 것으로 나타났다. 본 연구에서 구축한 네트워크 모델은 배추에서 저온 저항성 관련 유전자들의 발현 패턴을 정확히 유추하는 데 이용될 수 있을 것이다.

• 한국동물생명공학회지
• /
• 제38권4호
• /
• pp.236-246
• /
• 2023

Background: This study helps to evaluate the Ovarian potential of Cameroonian Zebu cattle (Bos indicus) slaughtered in Etoudi - Yaoundé for implementing Assisted Reproductive Techniques (ARTs). The aim was to enhance the productivity of the cattle sub-sector in Cameroon while conserving local genetic resources. Methods: A total of 144 cows, including 94 cycled cows and 50 pregnant cows, were included in the study. Live weights were determined by measuring the thoracic perimeter of each cow using a Rondo measuring tape. Age was determined postmortem through examination of dentition and horns, while the uterus and ovaries were removed, weighed, and classified based on physiological status (pregnant or nonpregnant). Follicles were counted, and their diameters were measured and categorized into small (Ø < 3 mm), medium (Ø 3-8 mm), and large (Ø > 8 mm). Results: The results revealed an average follicular population of 32.02 ± 9.31 per cow, with 22.43 ± 8.45 small follicles, 8.42 ± 3.87 medium follicles, and 0.76 ± 0.34 large follicles. The weight of the right ovary was significantly higher than that of the left ovary (p < 0.05), and cows aged 6 to 9 years exhibited a higher number of follicles compared to other age groups. Cows with medium (BCS = 3) and large (BCS = 4-5) body reserves had the heaviest ovaries. Additionally, pregnant cows had heavier uteri compared to non-pregnant cows, and cows with a body condition score of 3 or higher had higher uterine weights than lean cows (BCS = 1-2). Conclusions: This study demonstrates that age, body condition score, and pregnancy status influence ovarian weight. Body Condition Score serves as a reliable indicator of cow nutritional status, and cows with BCS of 3-5 are excellent candidates for in vitro production of embryos.

• Asian-Australasian Journal of Animal Sciences
• /
• 제31권1호
• /
• pp.32-39
• /
• 2018

Objective: In this study, we investigated the adverse effects of dietary zearalenone (ZEA) (0.5 to 1.5 mg/kg diet) on the localization and expression of the growth hormone receptor (GHR) in the uteri of post-weaning gilts and explored alternative mechanism of the reproductive toxicity of ZEA on piglets. Methods: A total of forty healthy piglets (Duroc${\times}$Landrace${\times}$Large White) aged 28 d were selected for study. Piglets were transferred to single cages after 10 days' adaptation on an obstetric table. The animals were allocated to one of four treatments: a normal basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0), or 1.5 (ZEA1.5) mg/kg purified ZEA, and fed for 35 d after the 10-d adaptation. Analyzed ZEA concentrations in the diets were 0, $0.52{\pm}0.07$, $1.04{\pm}0.03$, and $1.51{\pm}0.13mg/kg$, respectively. At the end of the feeding trial, piglets were euthanized after being fasted for 12 h. Two samples of uterine tissue from each pig were rapidly collected, one of which was stored at $-80^{\circ}C$ for analysis of the relative mRNA and protein expression of GHR, and the second was promptly fixed in Bouin's solution for immunohistochemical analysis. Results: The relative weight of the uteri and thickness of the myometrium and endometrium increased linearly (p<0.001) and quadratically (p<0.001) with an increasing level of ZEA. The results of immunohistochemical analysis indicated that GHR immunoreactive substance was mainly localizated in the cytoplasm of uterine smooth muscle, glandular epithelial, luminal epithelial, stromal, and vascular endothelial cells. In contrast, nuclear staining was rarely observed. The immunoreactive integrated optic density of GHR in the myometrium, luminal epithelium, glandular epithelium, and whole uteri of weaning gilts increased linearly (p<0.001) and quadratically (p<0.05) with an increasing level of ZEA. The mRNA and protein expression of GHR in the uteri of weaning gilts increased linearly (p<0.001) and quadratically (p<0.05) with an increasing level of ZEA. Conclusion: In conclusion, ZEA at a concentration of 0.5 mg/kg was sufficient to significantly thicken the myometrium and endometrium, and at a concentration of 1.0 mg/kg induced a high level of GHR expression to promote growth and development of the uteri. This revealed an alternative molecular mechanism whereby ZEA induces growth and development of the uteri and provides a theoretical basis for the revision of Chinese feed hygiene standards.

• Journal of Animal Science and Technology
• /
• 제49권3호
• /
• pp.309-320
• /
• 2007

본 연구에서는 남성 생식 기관인 efferent ductules(ED)에서 monocarboxylate transporter (MCT) isoform과 Basigin(Bsg)의 mRNA 발현을 탐구하고, MCT1의 발현이 에스트로젠 수용체 α에 의해 조절되는지를 연구하였다. 생후 여러 연령대의 백서 ED에서 MCT isoform과 Bsg의 mRNA 발현 여부는 real-time PCR에 의해 조사 되었고, 정상 생쥐와 에스트로젠 수용체 α knockout 생쥐를 사용하여 에스트로젠 수용체 α에 의한 MCT1의 발현 조절 여부는 immunohistochemistry 방법에 의해 간접적으로 알아 보았다. 본 연구 결과는 백서의 ED에서 MCT1, 2, 3, 4와 8 그리고 Bsg 유전자의 발현은 연령에 따라 다르게 나타나며, MCT1의 발현이 ED의 ciliated 세포의 basolateral 지역에서 발현되고 어떤 측면에서 ERα에 의해 조절되어 질 수 있음을 보여 주었다. 따라서 본 연구 결과는 MCT가 monocarboxylate의 세포 내, 외부로 운반을 조절함으로써 남성 생식기의 일부인 ED의 역할을 조절하는데 관여함을 시사한다.