• Reproductive and Developmental Biology
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• 제36권3호
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• pp.163-166
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• 2012

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.479-483
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• 2011

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, $Seminark^{Pro}$ (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at $17^{\circ}C$ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/$Dead^{(R)}$ stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at $17^{\circ}C$) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90~6.86) and T4 (6.86~6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0~7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 ($20{\times}10^6$ sperm cells/ml : $5{\times}10^5$ cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

• 한국동물생명공학회지
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• 제34권1호
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• pp.57-63
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• 2019

This study was conducted to find out the effect that ${\kappa}-Carrageenan$ has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% ${\kappa}-carrageenan$ ($64.26{\pm}0.49$) was significantly higher than control ($40.24{\pm}8.27$) (p < 0.05). RPMs of extender with 0.1%, 0.2% ${\kappa}-carrageenan$ ($57.64{\pm}6.34$, $56.47{\pm}1.35$) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% ${\kappa}-carrageenan$ ($61{\pm}8.03$) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of ${\kappa}-carrageenan$ of 0.1% ($0.81{\pm}0.05$), 0.2% ($0.85{\pm}0.05$) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

• 한국가축번식학회지
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• 제23권3호
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• pp.191-203
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• 1999

본 연구는 rBST, Vito E 및 Se 투여가 한우 종모우의 정액성상에 미치는 효과를 검토하였고 정액의 동결보존 시 정액희석액에 taurine의 첨가가 융해 후 정액성상에 미치는 영향을 검토하였다. 1. rBST, Vit. E, Se 및 Vit. E와 Se 혼합 투여가 한우 종모우의 정액량, 정자농도 및 수소이온농도 (pH)에 미치는 영향을 조사한 결과, 정액량과 수소이온농도는 처리구간에 커다란 차이가 없었으며(P＜0.05), 정자의 평균농도는 각각 13.50$\pm$2.32, 16.26$\pm$2.65, 17.07$\pm$2.61, 19.23$\pm$2.07 및 19.46$\pm$2.06$\times$$10^{8}$ 정자/$m\ell$로서 Se 투여구와 혼합투여구가 대조구, rBST 투여구 및 Vit. E 투여구보다 통계적으로 유의하게 높은 정자농도를 나타냈다 (P＜0.05). 2. rBST. Vit. E 및 Se 투여가 한우 종모우의 정액의 동결융해시 정액성상에 미치는 영향을 조사한 결과, 정자의 운동율과 기형율은 투여구간에 통계적 유의차는 없었으나, 정자의 생존율은 혼합투여구가 48.24%로서 대조구 (44.48%)보다 통계적으로 유의하게 높은 생존율을 나타냈으며 (P＜0.05), rBST 투여구, Vit. E 투여구 및 Se 투여구는 각각 46.48%, 47.28% 및 46.34%로서 대조구와 통계적 유의차는 인정되지 않았다. 3. 정액의 동결보존시 정액회석액에 taurine의 첨가가 정액의 융해 후 정액성상에 미치는 영향을 조사한 결과는 총정자수, 운동율, 생존율 및 기형율에서 taurine 첨가구가 taurine 무첨가구보다 다소 좋은 결과를 얻었지만 커다란 차이가 없었다. 4. rBST, Vit. E 및 Se 투여가 정자의 수정능획득과 첨체반응에 미치는 효과를 조사한 결과, 수정능 획득과 침체반응이 모두 일어나지 않는 비율(F율)과 모두 일어난 비율 (AR율) 은 투여구간에 커다란 차이가 없었다 (P＜0.05). 5. 정액의 동결보존 시 taurine 의 첨가가 융해후 정자의 수정능획득과 첨체반응에 미치는 영향을 조사한 결과는 수정능 획득이 일어났지만 첨체 반응이 일어나지 않은 비율 (B율)과 AR 율에서는 투여구간에 커다란 차이가 없었지만, F율은 taurine 첨가구가 taurine 무첨가구보다 낮은 비율을 나타났다 (P＜0.05).

• 한국가금학회지
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• 제44권1호
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• pp.59-65
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• 2017

본 연구에서는 닭 정액의 운동성 분석에 미치는 요인을 분석하기 위하여 희석배율에 따른 생존성을 조사하고, 신선정액의 희석 시에 필요한 조건을 확보하기 위하여 희석제에 단백질원으로서 BSA와 FAF-BSA의 첨가가 닭정자의 생존성에 미치는 영향을 관찰하였다. 최종 희석율이 1/4이 되도록 닭 정액을 희석하여 생존성을 조사하면 신선정액에서 희석율이 1/8, 1/16 및 1/32로 낮아질수록 89.9%, 69.9% 및 53.2%로 생존율이 낮아지는 것을 관찰하였으며, 단백질원으로 BSA나 FAF-BSA를 이용하면 생존율을 유의적으로 증진시킬 수 있음을 증명하였다. 또한 이러한 현상은 동결정액의 희석에도 중요한 요인으로 관찰되었는데, FAF-BSA를 이용하여 희석할 때 단백질이 없는 경우보다 양호한 생존율이 나타난다는 것을 관찰하였다(17.6% 대 34.0%). 그러나 단백질원을 활용하더라도 닭정액의 희석율이 낮아질수록 생존율이 저하하는 현상은 지속적으로 나타나고 있으며, 신선정액에 비하여 동결정액은 장수성이 낮은 것으로 추정되었다. 그러므로 본 연구에서 사용된 정자의 분석방법은 닭 정액동결을 실시하여 운동성을 분석할 때 필요한 기본 정보를 제공할 수 있을 것으로 판단되며, 닭 희석배율을 낮추어 동결정액의 효율성을 높이는 연구에 기여할 것으로 추정된다.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.147-154
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• 2012

본 연구에서는 제주흑우의 유전자원 보존과 증식에 있어 성공적인 인공수정을 위한 안정적인 동결정액 제조법을 수립하고 동결 융해 후 정자의 품질을 개선하기 위하여 제주흑우의 동결 정액 제조시 LDL, taurine, hypotaurine 그리고 trehalose를 첨가하여 이들이 동결 융해 후 정자의 성상에 미치는 영향에 대하여 알아보고자 수행하였다. 제주흑우의 정액 동결 시 LDL, LDL-taurine, LDL-hypotaurine 그리고 LDL-trehalose의 첨가는 ($63.40%{\pm}7.39$, $69.70%{\pm}6.12$, $67.25%{\pm}3.21$, $64.55%{\pm}2.43$) 대조구에($56.25%{\pm}6.42$) 비하여 모두 유의적인 생존율의 증가를 나타냈다 (p<0.05). 정자막 온전성 검사를 통한 꼬리막 팽창 정자의 비율은 LDL-taurine을 첨가 실험구에서 $70.55%{\pm}5.16$, LDL-hypotaurine 첨가 실험구에서 $64.45%{\pm}5.85$, LDL-trehalose 실험구에서 $64.50%{\pm}2.78$, LDL 단독 첨가 실험구에서 $61.65%{\pm}5.18$로 대조구 $51.90%{\pm}9.99$보다 모두 유의적으로 높은 결과를 나타냈다 (p<0.05). 동결 융해 후 정자의 첨체막 변화 양상에 있어서는 B pattern의 비율은 대조구와 실험구 모두 유의적 차이가 없었으며, F pattern은 LDL-taurine의 조합만이 대조구와 유의적인 차이를 나타내며 증가하였다 (p<0.05). 그러나 AR pattern의 비율은 LDL-taurine, LDL-trehalose, LDL-hypotaurine 그리고 LDL 처리의 순으로 대조구에 비하여 모두 유의적으로 낮은 수준의 AR pattern의 비율을 나타냈다(p<0.05). 정자의 수정능력 평가에 있어 LDL-taurine, LDL-hypotaurine 그리고 LDL-trehalose 모두 대조구에 대하여 유의적으로 높은 웅성전핵 형성율과 SFI를 나타냈으며 (p<0.05), 특히 LDL-taurine의 첨가는 LDL의 단독 처리에 비하여도 유의적으로 높은 수준의 웅성전핵 형성율과 SFI를 나타냈다 (p<0.05). 또한, decondenced sperm 비율에 있어 LDL, LDL-taurine, LDL-hypotaurine 그리고 LDL-trehalose 실험구 모두 대조구에 비하여 유의적으로 높은 결과를 나타냈다 (p<0.05). 본 연구의 결과는 제주흑우 정액의 동결정액 제조에 있어 안정적인 제조법을 공급할 수 있으며, 동결 융해 후 정자의 기능 개선 방법에 보다 많은 정보를 제공할 수 있을 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.61-65
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• 2011

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in $37^{\circ}C$ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities ($300{\times}g$ and $700{\times}g$) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at $700{\times}g$ for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.241-246
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• 2010

The objective of this study was to investigate the effect of storage time on fresh boar semen in Androhep and Beltsville Thawing Solution (BTS). Boar semen samples extended in each extender were stored at $17^{\circ}C$ up to 4 days. Sperm motility kinematics was evaluated by computer assisted sperm analyzer (CASA) and capacitation status by chlortetracycline (CTC)/Hoechst 33258 staining. Sperm motility (%) was not decreased during storage in BTS and Androhep. No significant difference between extenders was observed. Only significant differences in kinematic parameters on linearity during storage were found. The percentage of dead sperm significantly decreased during storage (p<0.05). Also the percentage of noncapacitated, capacitated, and acrosome-reacted sperm significantly modified during storage (p<0.05). However, there was no significant difference between extenders except proportion of capacitated sperm. This finding supported that modification in these parameters was not significantly different between extenders during this short-term storage. Our finding strongly indicated that both Androhep and BIS maintained favorable conditions for motility, motility kinematics, and capacitation status during short-term storage. Despite modifications in some parameters were apparent during sperm storage in extenders, these may not affect the fertilizing capacity of boar semen.

• 농업과학연구
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• 제47권3호
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• pp.657-665
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• 2020

The objective of this study was to investigate whether supplementation of pentoxifylline (PTX; phosphodiesterase inhibitor) to thawed boar semen improves the post-thaw motility of sperm and affects the efficiency of artificial insemination (AI) and further development. To determine the concentration of PTX for AI, frozen-thawed semen was incubated with 0, 5, 10, and 20 mM PTX in an extender freezing medium, respectively, after thawing. Kinematic properties of sperm were examined with a computer-assisted semen analysis (CASA) system. In addition, viability and mitochondrial activity were also tested by LIVE/DEAD and a MitoTracker kit. There were no significant differences in the kinetic parameters of thawed sperm between control and treatment groups, but overall assessment parameters such as motility and rapid progressive were higher in the 10 mM PTX group. In the viability and mitochondrial assay, there were no significant differences observed in the PTX treatment, compared to the control. For further analysis, artificial inseminations were performed using frozen semen and 10 mM PTX treated cryopreserved semen, respectively. There were no differences in pregnancy rates and fetus weights among the groups until 30 and 40 days, but litter size was reduced and relatively low-birth weight was observed in the PTX group. In summary, our findings suggest that enhancement of in vitro sperm quality or non-toxicity supplemented by PTX may have detrimental effects on fetus development.