• 한국수정란이식학회지
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• 제27권1호
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• pp.21-28
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• 2012

A study was conducted on four crossbred bulls, used as artificial insemination (AI) sires, to correlate their semen quality with their non return rate (NRR). Semen was collected once a week via an artificial vagina, diluted in egg yolk-citrate and maintained at $+7^{\circ}C$ for three days. It was evaluated for sperm motility, viability, morphology immediately after collection and was examined daily for sperm motility, viability and morphology of acrosome, mid piece and tail for a total of three days. A total of 2016 cows were inseminated by two AI technicians. The proportions of sperm with normal heads were 83.4% (63.7~91.7%), the proportion of spermatozoa exhibiting normal morphology (acrosome, mid piece and tail), motility and viability were 89.2% (82.3~92.0%), 71.3% (61.7~75.0%) and 76.7% (65.7~85.0%), respectively in fresh ejaculates. Sperm motility and sperm viability was significantly ($p$ <0.05) lower in Holstein-Friesian ${\times}$ Local bull than in other bulls during all three days of storage. The overall NRR for four bulls was 82.7% (72.9-87.5%). Bulls with higher sperm motility, viability and normal morphology of spermatozoa of individual bull had significantly (each $p$ <0.05) higher NRR. The highest ($p$ <0.01) NRR (87.5%) was observed in a Red Chittagong bull whose semen qualities were significantly ($p$ <0.05) higher than Holstein-Friesian ${\times}$ Local bull (NNR 72.9%). The results of the present study concluded that NRR at 56 days post AI is related to parameters of semen quality. Therefore, semen evaluation may allow the discarding of bulls with poor fertility in an AI program.

• Clinical and Experimental Reproductive Medicine
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• 제46권2호
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• pp.60-66
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• 2019

Objective: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment. Methods: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean${\pm}$standard deviation) was compared. Results: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p= 0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p= 0.974). A strong correlation (r = 0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r = 0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads. Conclusion: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples.

• 한국발생생물학회지:발생과생식
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• 제17권4호
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• pp.345-351
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• 2013

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: $99.5{\pm}0.8%$, DMSO 7.5%: $99.5{\pm}0.7%$, and DMSO 10.0%: $99.6{\pm}0.6%$). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: $96.2{\pm}2.3%$, DMSO 7.5%: $95.3{\pm}3.6%$, 10.0%: $96.6{\pm}1.8%$) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm ($98.4%{\pm}0.5$) and DMSO concentration level of 5.0% ($97.8{\pm}0.1%$) were not significantly different. However, with 7.5% ($97.2{\pm}0.6%$) and 10.0% DMSO concentrations ($95.9{\pm}0.2%$) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; $66.8{\pm}1.8%$, HR: $82.0{\pm}12.9%$) and 2 years (FR; $78.5{\pm}14.8%$, HR: $79.3{\pm}0.6%$) cryopreserved sperm were lower than control (FR; 100%, HR: $91.1{\pm}3.6%$) and 2 days cryopreserved sperm (FR; $99.6{\pm}0.6%$, HR: $96.6{\pm}1.8%$). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

• 한국임상수의학회지
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• 제28권1호
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• pp.13-19
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• 2011

요 약: 정액 동결 과정은 활성산소종의 생성을 유발하며, 생성된 활성산소종은 정자의 손상을 일으키는 것으로 알려져 있다. 따라서 본 연구의 목적은 동결 과정 중 항산화 효소 중 하나인 catalase (CAT)를 첨가함으로써 융해 후 정자의 기능과 활성산소종의 수준에 미치는 효과를 알아보고자 하였다. 5마리 돼지에서 채취한 정액은 0 (대조군), 200, 400 U/mL CAT가 첨가되어 있는 동결 희석액으로 각각 동결하였다. 융해 후, 정자 운동성, 생존성, 정상 형태율, 형질막 온전성, 미토콘드리아 기능, 세포내 ROS를 평가하였다. CAT는 400 U/mL의 농도에서 전체 정자 운동성을 향상시켰지만 (P < 0.05), 전진 운동성, 생존성, 기형율, 형질막 온전성, 미토콘드리아 기능의 향상을 나타내지 않았다. 활성산소종의 평가에서, CAT는 융해된 생존 정자의 ${\cdot}O_2$의 감소에는 효과를 나타내지 않은 반면 $H_2O_2$를 감소시켰다(P < 0.05). 결론으로 CAT는 동결 및 융해된 정자의 질을 향상시키는 데 큰 효과를 나타내진 않았지만 생존 정자에서 $H_2O_2$을 제거함로써 생존정자의 산화적 손상을 감소시킬 수 있으리라 판단된다.

• 한국수정란이식학회지
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• 제25권2호
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• pp.111-116
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• 2010

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

• 한국수정란이식학회지
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• 제25권4호
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• pp.237-245
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• 2010

Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were $21.5{\pm}0.7\;cm$, $43.5 {\pm}5.4%$, $83.5{\pm}6.7$ million and $88.3{\pm}4.1%$, respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ($2.7{\pm}1.1$ and $1.4{\pm}1.3$, respectively), whereas higher percentages of abnormalities ($7.0{\pm}1.8$) were observed in mid piece and tail portion. The proportion of live spermatozoa was $28.5{\pm}5.4$. It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.

• 한국수정란이식학회지
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• 제27권1호
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• pp.37-43
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• 2012

This study evaluated a method of sorting X and Y chromosomes based on size using the forward angle light scatter related refractive index (FSC) of a flow cytometer. Hanwoo bulls sperm were separated to X and Y chromosomes by the parameters of FSC or Hoechst 33342 intensity. As a result, using monitor program linked flow cytometry during sorting processing, the purities were $97{\pm}0.57$ or $96{\pm}0.67%$ for the X-fraction and $96{\pm}0.33$ or $97{\pm}1.33%$ for the Y-fraction in the two sperm sorting methods. There were no differences in the X and Y ratios (X and Y %) between the sperm sorting methods based on FSC or DNA content. The proportions of female and male embryos used for in vitro fertilization and development were $66.03{\pm}3.31$ or $69.37{\pm}1.41%$, and $70.56{\pm}2.42$ or $56.11{\pm}3.09%$ when sperm were processed using the sex sorting method by FSC or Hoechst 33342. In conclusion, further study is needed to determine the optimum procedure and improve the nozzle to enhancing sorting accuracy or efficiency. Also, the findings of this study do not negate the possibility that the difference method of sperm sorting cannot use a UV laser beam.

• Clinical and Experimental Reproductive Medicine
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• 제50권3호
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• pp.160-169
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• 2023

Objective: Cryptorchidism is one of the main causes of infertility and can result in testicular cancer. This study aimed to present quantitative data on the damage caused by cryptorchidism using stereological analysis. Methods: Thirty newborn rats were randomly divided into control and experimental groups. The experimental group underwent surgery to induce unilateral cryptorchidism in the left testis, whereas the control group underwent a sham surgical procedure 18 days after birth. The testes were removed at designated time points (40, 63, and 90 days after birth) for stereological evaluation and sperm analysis. Total testicular volume, interstitial tissue volume, seminiferous tubule volume and length, and seminiferous epithelium volume and surface area were measured. Other parameters, such as sperm count, sperm morphology, and sperm tail length, were also examined. Results: Statistically significant differences (p<0.05) were observed between the experimental and the control groups at different ages regarding the volumes of various parameters, including the surface area of the germinal layer, the length of the seminiferous tubules, sperm count, and sperm morphology. However, no significant differences were observed in the epithelial volume and the sperm tail length of the groups. Conclusion: Given the substantial effect of cryptorchidism on different testicular parameters, as well as the irreversible damage it causes in the testes, it is important to take this abnormality seriously to prevent these consequences.

• 생명과학회지
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• 제26권8호
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• pp.943-947
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• 2016

본 연구는 간단히 활용할 수 있는 나노 크기의 마그네틱 비드를 이용하여 정자의 품질을 향상시킬 수 있는지 여부를 규명하기 위하여 실시하였다. 돼지 정자 시료는 인공수정 센터에서 공급받아 실험실로 2시간 이내로 이송한 후 CASA 측정을 통하여 4개의 활력을 나타내는 그룹으로(1, > 90%; 2. 80~90%; 3. 70~80%; 4. < 70%) 분류하였다. 정액은 BTS 희석제를 사용하여 보존하였고, 총 정자수와 동일한 농도의 마그네틱 비드를 정액에 20분간 처리한 후, 5분간 실온에서 마그네틱 비드에 반응한 정자를 자석을 이용하여 분리하였다. 마그네틱 비드 처리 전 과 후 정자의 생존율 및 활력은 CASA를 이용하여 측정하였고, 기형율과 정자응집의 정도는 현미경으로 검사하였다. 처리 후의 정자 활력은 4개 그룹 모두에서 유의하게(p<0.0.5) 높은 차이를 보였으며 처리 전에 비하여 평균 7.11% 향상되었다. 정자 활력의 변화는 처리 전 낮은 활력을 보인 그룹에서 보다 현저한 차이를 보였다(< 70% and 70~80%; 19.12±1.08% and 5.67±0.71%, p<0.0.5). 정자 활력을 VCL, VSL, VAP 및 LIN (%)로 구분한 성적에서도 유사한 패턴을 나타냈고, 이러한 현상은 활력 70% 이하를 나타낸 그룹에서 개선 효과가 더욱 뚜렷하였다. 마그네틱 비드 처리 후 정자 생존율은 처리 전에 비하여 평균 4%가 향상 되었고(p<0.0.5), 정자 기형율 또한 3.7~4.5%(p<0.0.5) 정도 감소하였다. 정자 응집의 정도 또한 마그네틱 비드 처리를 통하여 처리 전 낮은 활력을 나타낸 그룹에서 감소됨을 알 수 있었다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2005년도 춘계 국제심포지엄 및 학술대회
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• pp.158-158
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• 2005