• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 한국동물위생학회지
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• 제31권4호
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• pp.547-554
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• 2008

Bacteriospermia is a frequent finding in fresh boar semen and can result in detrimental effects on semen quality and longevity. The objectives of this study was to evaluate types of bacterial contaminants in porcine fresh semen and the reducing effect of antibiotic and density gradient with percoll on the bacterial contaminants. Fresh semen was collected by gloved-hand method into a pre-warmed($37^{\circ}C$) thermostable bottle, and was inoculated onto blood agar and MacConkey agar, respectively. After incubated for 48 hour, 7.5% $CO_2$ at $37^{\circ}C$, bacterial colonies were selected and identified by Gram staining, oxidase test, catalase test and finally identified using API kits and Vitek system. Aerobic culture yielded a variety of bacteria from different genera. The most prevalent contaminant of fresh semen were Leclecia adecarboxylata, Acineobacter banmanni, Staphylococcus epidermidis, Staphylococcus cohni spp urealyticus, Proteus mirabilis. Most of identified bacteria were Gram(-) and non-pathogenic bacteria. It seems that bacterial contaminants in fresh semen were seem originated from multiple sources at the stud/farm, and were from animal and non-animal origins. Gentamicin treatment did not eliminate the bacterial contaminants completely but 3 step-density gradient with percoll completely removed the bacterial contaminants in fresh semen. Therefore, future study is necessary to prove that density gradient method with percoll can eliminate bacteria in fresh semen without significantly affecting sperm viability or function.

• 한국수정란이식학회지
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• 제15권3호
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• 한국수정란이식학회지
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• 제22권3호
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• pp.173-178
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• 2007

돼지 정자의 성 선별에는 일반적으로 유속 세포 분석기를 이용한다. 유속 세포 분석은 DNA량의 차이에 기초하여 정자를 분리하는 기술로써 X 정자와 Y 정자를 90% 정확도로 분리할 수 있다. 그러나 이러한 유속 세포분석 기술은 정자의 손상을 야기해 정자의 기능과 수정능에 영향을 미치므로, 본 연구에서는 특정한 핵산 서열을 탐지할 수 있는 Chromogenic in situ hybridization(CISH)을 그와 비교하여 평가하였다. 유속 세포 분석을 수행하기 위해 정자를 SYBR 14와 PI로 염색하였고, histogram, dotplot, density, contour를 측정하였다. Y 염색체 특이적인 primer를 이용한 PCR로 유속 세포 분석의 정확도를 검사하였다. HRP/DAB 시스템에 기초한 CISH 분석에는 X 또는 Y 염색체에 상보적으로 결합하는 probe가 사용되었다. CISH 분석은 기존의 방법들보다 빠르고 쉬우며 비용이 적게 든다는 장점이 있다. 또한, CISH는 보다 정화한 정자의 선별을 가능하게 하는 것으로 나타났다. 본 연구에 따르면 CISH가 기존의 선별 방법들을 평가하는 기술로서만이 아니라 특정한 성별을 가진 포유동물의 생산에도 사용될 수 있을 것이다.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.67-81
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• 1997

To determine the concentration and the physiologic role of metal components in blood plasma and seminal plasma in relation to male infertility, the concentrations of twelve metal components in blood plasma and seminal plasma including Na, Mg, K, Ca, Cr, Mn, Fe, Cu, Zn, Se, Cd and Pb were measured by atomic absorbance spectrophotometery or ion selective electrode analysis. Semen and blood samples were obtained from a total of 110 men including 70 male infertility patients, 20 vasectomized persons and 20 fertility proven volunteers visited to the Male Infertility Clinic of Pusan National University Hospital. The concentrations of Ca, Zn, Mg, Cr and Cd in control group were higher in seminal plasma than in blood plasma, and additionally Pb were higher in infertility group. The concentrations of all metal components revealed no significant difference according to patients' age, resident, occupation, sperm density, motility and hormone level in blood plasma, but some metal components including Ca, Mg, Cu, Mn, Cd and Pb revealed a significant difference according to each these parameters except patient's age in seminal plasma. The concentrations of Mn, Cd and Pb in the vasectomy persons were higher than in the infertility group III including testicular and epididymal factors, but not in blood plasma. We conclude that the quantitative changes of metal components in the seminal plasma may have effects on not only spermatogenesis and sperm function, but also contribute to diagnostic parameter according to organ specificity of the metal in the male reproduction.

• Clinical and Experimental Reproductive Medicine
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• 제51권1호
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• pp.28-41
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• 2024

Objective: Chronic scrotal hyperthermia (SHT) can lead to serious disorders of the male reproductive system, with oxidative stress playing a key role in the onset of these dysfunctions. Thus, we evaluated the impact of caffeine, a potent antioxidant, on cellular and tissue disorders in mice with chronic SHT. Methods: In this experimental study, 56 adult male NMRI mice were allocated into seven equal groups. Apart from the non-treated control group, all were exposed to heat stress. Two groups, termed "preventive" and "curative," were orally administered caffeine. The preventive mice began receiving caffeine immediately prior to heat exposure, while for the curative group, a caffeine regimen was initiated 15 consecutive days following cessation of heat exposure. Each treated group was subdivided based on pairing with a positive control (Pre/ curative [Cur]+PC) or a vehicle (Pre/Cur+vehicle). Upon conclusion of the study, we assessed sperm characteristics, testosterone levels, stereological parameters, apoptosis, antioxidant and oxidant levels, and molecular markers. Results: Sperm parameters, testosterone levels, stereological parameters, biochemical factors (excluding malondialdehyde [MDA]), and c-kit gene expression were significantly elevated in the preventive and curative groups, especially the former, relative to the other groups. Conversely, expression levels of the heat shock protein 72 (HSP72) and nuclear factor kappa beta (NF-κβ) genes, MDA levels, and apoptotic cell density were markedly lower in both caffeine-treated groups relative to the other groups, with more pronounced differences observed in the preventive group. Conclusion: Overall, caffeine attenuated cellular and molecular abnormalities induced by heat stress in the testis, particularly in the mice treated under the preventive condition.

• 한국수의병리학회지
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• 제2권1호
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• pp.47-52
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• 1998

The incidence of spontaneous testicular atrophy and its morphological changes during stage-specific spermatogenesis were investigated in male Sprague-Dawley rats at 10, 19, and 32 weeks of age. The incidence of testicular atrophy was 0.2%(2/90) 7.9%(9/114) and 10%(4/40) in 4, 13 and 26 weeks respectively. The epididymis with testicular atrophy had low sperm density. In the minimally affected tests scattered tubules showed complete depletion of germ cells without stage specificity. Testes with moderate to severe testicular atrophy showed seminiferous tubules lined with dense Sertoli cell population. While Leydig cells in the interstitium appeared hypertrophy they were immunohistochemically negative for proliferating cell nuclear antigen a marker of cell proliferation.

• Applied Microscopy
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• 제41권1호
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• pp.13-20
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• 2011

바다빙어목(Osmeriformes), 바다빙어과(Osmeridae)에 속하는 빙어(Hypomesus nipponensis)의 성숙란 난막구조를 광학현미경과 전자현미경을 이용하여 관찰하였다. 빙어의 성숙란은 두 개의 난막으로 둘러싸여 있었으며 바깥쪽 난막의 외층은 한 층의 여포세포가 분포하고 있었고 안쪽의 난막 바깥쪽은 호염기성을 나타내는 구조물들이 배열하고 있었다. 난막의 단면을 투과전자현미경으로 관찰한 결과 두 개의 난막 중 바깥쪽 막은 전자밀도가 균일한 한 층으로 되어 있었고 안쪽막은 전자밀도가 높은 6층이 전자밀도가 낮은 5층에 의해서 분리되어 층상구조를 하고 있었다. 주사전자현미경으로 관찰한 결과 바깥쪽 막은 홈들이 관찰되었고 안쪽막 표면은 무정형의 구조물들이 산재하고 있었다. 또한 동물극 쪽에 한 개의 난문이 관찰되었다. 이상과 같이 빙어의 성숙란은 지금까지 보고된 경골어류의 경우 한 개의 난막으로 둘러싸여 있는데 비해서 두 개의 난막으로 둘러싸여 있다는 것은 매우 중요한 빙어의 종특이성을 나타내며 난막의 미세구조적 특징들은 종을 분류하는 데 이용될 수 있다.

• Applied Microscopy
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• 제31권4호
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• pp.333-345
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• 2001

두족류 3종(Octopus minor, Octopus ocellatus, Todarodes pacificus)의 성숙정자를 전자현미경을 통해 관찰한 결과 다음과 같았다. 팔완류인 서해박지와 주꾸미의 성숙정자 전체 길이는 각각 $390{\mu}m$와 $125\sim130{\mu}m$정도였고, 십완류인 살오징어의 길이는 $35{\mu}m$정도로 매우 짧았다. 서해낙지는 나선형의 첨체와 약간 굽은 바나나 모양의 머리를 소지하고 있었으며, 주꾸미는 꼬인 첨체와 막대 모양의 긴 머리를 가지고 있었다. 이들 침체 내강에는 규칙적 구조(periodic structure)인 많은 가로무늬가 관찰되었고, 머리의 내강에는 치밀전 (dense plug)을 형성하였다. 그러나 살오징어의 첨체는 전자밀도가 낮은 둥근 모자 모양이 였으며, 머리는 길쪽한 장타원형을 나타내었다. 특히 첨체 하단부에는 2개의 작은 강(cavity)이 관찰되었고, 이들 내강에는 전자밀도가 높은 물질들(juxtanuclear acrosomal materials)로 채워져 있었다. 3종의 성숙정자 중편은 서해낙지와 주꾸미에서 미토콘드리아가 mitochondrial sleeve를 형성하였지만, 두 종간 미토콘드리아의 수는 각각 $11\sim12$개와 $8\sim9$개로 관찰되어 수의 차이를 보였다. 반면, 살오징어는 미토콘드리아가 축사와 분리되어 mitochondrial spur를 형성하였고, 이들 spur내에는 $10\sim13$개의 미토콘드리아와 전자밀도가 높은 물질들이 밀집되어 있었다. 서해낙지, 주꾸미, 그리고 살오징어의 축사는 공히 9+2구조에 9개의 금은섬유가 둘러싸고 있었고, 살오징어의 축삭내에서만 작은 입자형의 글리코겐이 관찰되었다. 금은섬유들은 서해낙지와 살오징어에서는 꼬리의 주편까지만 관찰되었으나, 주꾸미에서는 단편에서도 관찰되었다.

• Applied Microscopy
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• 제27권4호
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• pp.417-432
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• 1997

The structures of the fertilized egg envelope in three species of cichlidae, golden severum (Cichlasoma severum var.), convic cichlid (Cichlasoma nigrofasciatum) and discus (Symphysodon aequifasciatus) were investigated by routine light and electron microscopies. The fertilized eggs of all three species were of the non-transparent, ellipsoidal, adhesive and non-floted type. The egg envelopes have a single micropyle, which is thought to the pathway of sperm in the area of the animal pole. In golden severum, an outer surface of egg envelope was covered by a reticular layer, that of convic cichlid was covered by an amorphous reticular layer and that of discus was covered by a branched reticular layer. The fertilized egg envelopes consisted of two distinct layers, an adhesive outer layer and an inner layer, consisted of lamellae alternating with interlamellae of lower electron density, in all three species; an inner layer of golden severum was $15\sim17$ layers, that of convic cichlid was $14\sim16$ layers, and that of discus was $18\sim19$ layers.