• Asian-Australasian Journal of Animal Sciences
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• v.26 no.3
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• pp.334-342
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• 2013

Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs) pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3), as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO), LH-supplemented FBO (LFBO), or Lutalyse-supplemented FBO (LuFBO)). TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

• Korean Journal of Veterinary Research
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• v.38 no.3
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• pp.652-658
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• 1998

The efficiency of nuclear transfer using donor embryos originated from ovum pickup(OPU) and activated recipient cytoplasts were examined for induction of twinning in Korean native cattle(KNC). After aspiration of follicle by OPU, regardless of the vacuum applied, we obtained same result in proportions of recovered cumulus-oocyte complex (COCs) with compact cumulus. Under electric stimulation(1.0kV/cm DC for $40{\mu}s$), most of activated oocytes proceed to anaphase II/telophase II within 3h(84.7%). In the treatment of oocyte activation, the preactivation which was performed before fusion had significant effect on the developmental rates to morula/blastocyst stage(9.4 vs 4.0%). In embryo transfer of nuclear transferred embryos, we obtained 2 twins from KNC recipients and 1 twin from a Holstein recipient. Our results showed that it is possible to obtain twins using nuclear transfer technique in KNC.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.51-69
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• 1996

본 실험은 촉진 및 호르몬 측정으로는 알기 힘든 난소의 정상 혹은 병리학적 형태 및 변화를 알아보기 위하여 실시하였다. 실험에서 도살장으로부터 채취된 난소 100개를 수침법으로 초음파 사진을 찍고 그 실제 단면과 비교하였다. 그리고, 홀스타인 50두에서 직장 벽을 통해 난소를 초음파로 관찰하여 난소의 정상 상태 및 병적 상태를 조사하였다. 이 기초 실험을 바탕으로 하여 홀스타인 소 1두를 과배란 처리하여 42일간 난소의 형태 및 변화를 직장 벽을 통해 초음파로 관찰하고 난포의 크기를 측정하였다. 수침법을 통한 난소의 관찰로 난포와 황체의 정상적 . 비정상적인 구조를 관찰할 수 있었고, 과배란 처리한 소 난소의 관찰로 난포와 황체의 능동적 상호작용을 관찰할 수 있었다.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.147-156
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• 1997

Ultrasound-guided follicular aspiration was performed in Holstein heifers once weekly with or without pretreatment of single or multiple decreasing doses using a total of 400 mg FSH. Oocytes were aspirated with a 6.5 MHz convex-array ultrasound trasducer designed for intravaginal use. All the visible follicles larger than 4 mm in diameter were punctured with a 17 gauge, 55 cm needle at each aspiration session and the follicular fluids containing oocytes were obtained by vacuum suction. The results obtained were as follows: As a preliminary experiment, the recovery rates of folicular oocytes by ultrasound-guided aspiration from the isolated ovaries of Korean native cows were compared between suction methods using manual syringe or vacuum pump. The recovery rate of oocytes using vacuum pump (80.7%) was significantly (P<0.05) higher than that using manual syringe (47.1%). The follicles were counted by their size in diameter with ultrasound image, and recovery rates and grades of follicular oocytes collected by ultrasound-guided aspiration were investigated in Holstein heifers pretreated with or without FSH. A group of heifiers were injected with multiple decreasing doses (twice a day for 3 days) of a total of 400 mg FSH. The other 2 groups were injected with a single dose of 400 mg FSH mixed with 25% PVP. Ultrasound observation of follicle population and/or ultrasound-guided transvaginal oocyte aspiration were performed 12 hrs following the last FSH injection in the multiple dose group, and 48 or 60 hrs after FSH injection in the single dose groups. Most of the visible follicles had small size of less than 3 mm in diameter in unstimulated heifers (71.0%), but medium size in all the heifers treated with FSH. (70.5 to 92.8%). The number of OPU follicles per session (4.6$\pm$1.9) were much less, compared to the vilsible follicle counts (9.7$\pm$2.2), in the nustimulated heifers due to the small dominant follicles. Among 4 goups of heifers the most visible as well as OPU follicles were observed in the heifers at 60 hrs following treatment of a single dose of 400 mg FSH (21.2$\pm$2.3 and 21.0$\pm$2.0), and the differences in both the follicle counts between the groups was found significant (P<0.05) The rates of oocyte recovery from the follicles by ultrasound-guilded aspiration were varied 46.3 to 75.0% in the heifers unstimulated and treated with a single dose of 400 mg FSH, but the group difference was not significant. The number of recovered oocytes per session a, pp.ared to be highest at aspiration at 60 hrs following single FSH (10.6$\pm$2.2) than at aspiration at 48 hrs after single FSH (7.8$\pm$2.7) or in the unstimulated heifers (3.4$\pm$3.0). The proportion of grade I and II oocytes to all oocytes collected was varied 31.8 to 64.0% between the groups. However, there was found no significant difference in both the number of oocytes recovered per session and the percentage and the percentage of grade I and II oocytes. From the above results it was concluded that the more oocytes of superior quality might be recovered economically by ultrasound-guided aspiration at 60 hrs following the pretreatment of a single dose of 400 mg FSH and by suction using a vacuum pump system of about negative pressure of 75 to 85 mmHg.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.237-244
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• 1990

These studies were carried out to investigate the effects of the follicles size, hormone supplementation, semen types and capacitation methods on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean Native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18~20hrs. with motile capacitated sperm in the TCF(Tyroide calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1%, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in dimeter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% FCS and hCG, HCG, $\beta$-estradiol were 76.0%~82.3% and 26.2%~70.0%, and those values were higher the supplementation of the hormone than the non-supplementation. 4. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymis cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8%, 38.3, respectively. 5. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%, 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin than other methods.r methods.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.189-195
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• 2008

The objective of this study is to investigate the changes in concentrations of leptin and insulin in serum of Korean cattle (Hanwoo) with reproductive disorders and to examine the relationship among leptin, insulin, and body condition score (BCS). The concentration of leptin in serum of pregnant Hanwoo showed insignificant difference from that in serum of Hanwoo with reproductive disorder, such as repeat breeding, follicular cyst, corpus luteum cyst, ovarian atrophy, and feeble estrus (p>0.05). However, the concentrations of leptin and insulin in serum were changed with different BCS value. In emaciated Hanwoo (BCS $2.0\sim2.9$), they were significantly decreased compared to BCS $3.0\sim3.4$ (p<0.05). The leptin showed different genotypes with different BCS value. In BCS $2.0\sim2.9$, C/T genotype was expressed (83.3%) more than C/C (16.7%) or T/T (0%) genotype, whereas C/C genotype was expressed (62.5%) more than C/T (25.0%) or T/T (12.5%) genotype in BCS $3.5\sim4.0$. The insulin concentration in follicular fluid obtained from ovary with follicular cyst which has follicles having diameter of $25\sim40 mm$ was significantly higher (p<0.05) than those in normal follicle fluid which has follicles having diameter of $3\sim10 mm$. These results showed that concentration of leptin and insulin in serum were related to BCS value and follicular size and suggest that the changes in concentration of leptin and/or insulin in serum could be a potent biomarker for diagnosis of bovine reproductive disorder.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.4
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• pp.455-461
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• 2001

The effect of exogenous gonadotrophins on superovulation in rabbits was examined. One hundred and sixteen sexually mature California, Chinchilla and New Zealand White rabbits were randomly allocated to control (100 IU hCG), PMSG-treated (100 IU HCG following 150 IU PMSG) and FSH-treated groups (0.3 mg/head /12 h for 3 days followed by 100 IU hCG). All does were mated after hCG injection and were sacrificed or laparotomized within 1 to 4 days postcoitus for counting the number of ovulation points. The number of ovulations was higher in FSH-treated animals than in the control and PMSG-treated groups (37.2 vs. 10.4 and 14.5, p<0.05). Follicle haemorrhagicum was observed in many cases in the PMSG-treated group. No significant difference in ovulation number was observed between left and right ovaries regardless of gonadotropin treatment. In another experiment, 2-cell stage embryos were collected at 26 h postmating and blastomeres were separated by mechanical pipetting or gentle pressure with a fine glass needle. Aggregated or chimeric embryos were produced from two single blastomeres from two breeds, New Zealand White and Chinchlla, with different coat colors. All the embryos were cultured in Ham's F-10 medium supplemented with 1.5% BSA (bovine serum albumin fraction V) and 10% PRS (pregnant rabbit serum), and incubated in a humidified atmosphere with 5% $CO_2$ at $38^{\circ}C$. After development to morula or early blastocyst, the embryos were transferred into the oviducts of recipient does. Results showed that 7 out of 10 does (70%) receiving intact embryos (control) became pregnant and 41 kits were delivered. However, no pregnancy was obtained from the recipient of either denuded demi- or aggregated embryos. It is suggested that embryos without zona pellucida could not develop to term in rabbits.

• The Journal of the Korean Society for Microbiology
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• v.10 no.1
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• pp.1-8
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• 1975

Despite a number of recent studies on appendix its function appears to remain unknown. The present studies were undertaken in order to extend and confirm the previous studies concerning the role of appendix in immune response. An early hemagglutinin response of mercaptoethanol sensitive antibody(IgM antibody) in rabbit injected intravenously(i.v.) with 200mcg of bovine gamma globulin(BGG) was abolished by lethal whole body irradiation(900 r), but preserved in animals whose appendix and bone marrow were shielded during irradiation. Late formation of mercaptoethanol resistant antibody(IgG antibody) and the development of memory in bone marrow shielded animals were not affected by irradiation of the appendix. Formation of either IgM or IgG antibody to sheep red blood cells(SRBC) injected i.v. as determined by direct plaque forming cell(DPFC) technique in spleen were effectively abolished by appendectomy, thymectomy, or both followed by irradiation. When bone marrow was shielded in combination with autologous appendix reconstitution, DPFC response was about 5 times greater than the sum of two. Lysed appendix cells failed to restore the response. Lethally irradiated rabbits restored with combination of autologous appendix and thymus cells showed DPFC responses which were essentially normal. Three pools of appendix were obtained by manual separation technique and were stimulated with soluble concanavalin A(Con A), phytohemagglutinin-P(PHA) and pokeweed mitogen(PWM). Rabbit appendix cells responded to Con A, PHA and PWM. Cells of thymus dependent area(TDA) of the appendix were relatively enriched in their response to T cell mitogens compared to dome and follicle cells. The PHA/Con A responsive ratio of appenix TDA subpopulation was high, indicating that Con A responsive cells have a wider distribution among appendix. This finding showed that interfollicular area of the appendix is thymus-dependent. The present studies confirmed other evidence that the rabbit appendix cells itself are unable to form antibody and T lymphocytes in appendix TDA may be heterogenous, and that the appendix cells are synergistic with either bone marrow or thymus cells in the early hemagglutinin on splenic antibody response to BGG or SRBC.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.84-88
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• 2023

Because multiple ovulation embryo transfer (MOET) in cattle includes several benefits such as wide spreading of genetically superior offspring for long distance, this biotechnological method has been widely applied to Hanwoo. When the recipients are not stayed close after embryo recovery from donor, the embryos are moved to other farms via several vehicles (car, train, and airplane). However, air travel induces lesser oxygen level, increased vibration, lower air pressure, higher noise, and increased exposure of cosmic radiation to living things than ground level. It was still unknown that fresh embryos obtained from multiple ovulation of Hanwoo could maintain their fertility after being transported via air plane, the present case report introduced a clinical case of MOET in Hanwoo after shipping fresh embryos via air transportation. The donor was multi-ovulated via follicle-stimulating hormone series of injection, which was followed by a gonadotrophin-releasing hormone injection and artificial insemination twice. The embryos were recovered by the uterine flushing, packed in ministraws, transported to recipients for 6 h including 1 h air flight, and then transferred to the synchronized recipients. During pregnancy diagnosis of early gestation period, 5 of 7 recipients (71.4%) presented no heat signs and showed fetal sacs with fluid under transrectal ultrasonography. After normal gestation period, all recipients naturally delivered healthy calves (male n = 2 and female n = 3) without abortion, stillbirth, and premature birth. The present case report indicated that transportation of fresh embryos for MOET via domestic flight in Korea did not affect to their fertility.