• 한국수정란이식학회지
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• 제33권4호
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• pp.321-326
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• 2018

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.235-243
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• 2000

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.143-148
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• 2013

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-$34^{TM}$ cell culture media at $37^{\circ}C$. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin ${\alpha}6$ and ${\beta}1$, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

• 한국식품과학회지
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• 제2권2호
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• pp.8-16
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• 1970

69종(種)의 각종 식물체(植物體)를 대상으로 하여 이것을 마쇄(磨碎)한 slurry로부터 총질소(總窒素)의 추출상황(抽？狀況)을 조사(調査)하고, 추출액(抽出液)으로 부터의 LPC의 제조방법(製造方法)을 검토(？討)하여 다음과 같은 결과(結果)를 얻었다. 1. 원료(原料)의 총질소(總窒素)의 함량(含量)이 클수록 총질소(總窒素)의 추출비(抽出比)는 커지며, 원료(原料)로부터의 추출(抽出)은 적어도 2회추출(回抽出)까지 필요(必要)하다. 원료엽체(原料葉體)로부터의 1차(次) 추출액(抽出液) $(E_1;\;%)$, 2次 抽出液$(E_2;\;%)$와 (T; %)사이에는 $E_1=0.8168T,\;E_2=0.1830T$의 1차(次) 관계식(關係式)이 성립(成立)한다. 2. pH 처리(處理)에 의한 LPC 생성(生成)에 있어서 pH 3 이하(以下)에서는 생성(生成)된 LPC의 색택(色擇)이 갈변(褐變)되고 제품(製品)의 탄력성(彈力性)이 커지며, 그 정도는 pH가 낮아질 수록 현저(顯著)해진다. pH 처리(處理)에 의한 LPC 생성(生成)의 지적(至適) pH 는 $3.5{\sim}4.5$ 라고 할 수 있다. 3 추출액(抽出液)으로 부터의 LPC의 생성(生成)은 TCA 처리(處理)> pH 4> pH 3> 열처리(熱處理)의 순서(順序)가 되며, TCA 처리(處理)에 비(比)하여 pH 4 처리(處理)는 10%감(減), pH 3 처리(處理)는 11.4%감(減), 열처리(熱處理)는 14.8%감(減)이었다. 4. pH 처리법(處理法)은 열처리법(熱處理法) 보다 LPC의 생성량(生成量)이 다소 높으나 대량처리시(大量處理時)의 pH 조절(調節)의 복잡성(複雜性)과 생성제품(生成製品)의 품질(品質)로 보아 열처리(熱處理)에 의한 LPC의 생성방법(生成方法)이 효과적(效果的)이고 간편(簡便)하다.