• Reproductive and Developmental Biology
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• 제29권3호
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• pp.141-144
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• 2005

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5 ${\mu}g/mL$ R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; $53.29{\~}72.94\%$, 6 h; $21.40{\~}58.90\%$, 12 h; $8.26{\~}25.93\%$, 24 h; $1.00{\~}13.78\%$, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging $70.8{\~}77.8\%$ and $52.1{\~}84.5\%$, respectively, were not significantly different. However, in vitro development rates of the same groups ranging $25.7{\~}40.0\%$ and $12.9{\~}1.8\%$, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

• Animal Bioscience
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• 제34권2호
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• pp.198-204
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• 2021

Objective: The aim of this study was to ascertain the effects of adding green tea extract (GTE) to skim milk-egg yolk (SM-EY) extender on both the quality of post-thawed bull semen and the pregnancy rates of the recipient cows. Methods: Twelve ejaculates from four Simmental bulls, aged 3 to 5 years and weighing 900 to 950 kg, were diluted SM-EY extender, added with 0, 0.05, 0.1, and 0.15 mg GTE/100 mL extender and then frozen. After four weeks storage in liquid nitrogen, the sperm were thawed and evaluated for viability, motility, intact plasma membrane (IPM), and DNA fragmentation. Meanwhile, the estrus cycles of 48 recipient cows were synchronized by intramuscular administration of a single injection of 5 mg prostaglandin F2α. Estrus cows were divided into four equal groups and inseminated artificially 18 to 20 h after the onset of estrus by using semen from each extender group. Pregnancy was diagnosed by measuring serum progesterone levels at 21 days, followed by transrectal palpation 90 days after insemination. Results: The findings revealed that adding 0.1 mg of GTE/100 mL extender produced the highest percentages of sperm viability (70.67%±1.75%), motility (69.17%±1.47%), and IPM (69.23%±1.21%) and the lowest percentage of DNA fragmentation (3.00%±0.50%). The pregnancy diagnosis revealed that all cows (36/36) inseminated using frozen semen in GTE addition extender were pregnant (pregnancy rate 100%), whereas the pregnancy rate of the control group was 83.33% (10/12). Conclusion: It may be concluded that 0.1 mg GTE/100 mL extender yields the best quality of spermatozoa and that all variants doses of GTE in extender produce a higher pregnancy rate among recipient cows.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.155-162
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• 2005

본 연구는 돼지 정자 동결시 taurine과 $\alpha$-tocopherol의 첨가가 융해후 정자 성상과 정자 기능 활성산소계(reactive oxygen species; ROS)의 발생 정도 및 지질 산화(lipid peroxidation, LPO)에 미치는 영향을 구명하기 위하여 시행하였다. 1차 및 2차 희석액내 taurine과 $\alpha$-tocopherol이 첨가된 돼지 동결정액의 융해 후 정자 운동성 양상, 정자 생존성, 정자 기법을 적용하여 다음과 같은 결과를 얻었다. Taurine(25mM, 50mM), $\alpha$-tocopherol($500{\mu}M,\;1,000{\mu}M$ 단일처리군 그리고 taurine과 $\alpha$-tocopherol의 혼합처리군($25mM\~500{\mu}M\~1,000{\mu}M$)은 동결$\cdot$융해 후 대조군과 비해 정자 운동성과 생존성은 유의적인 차이를 나타내지 않았다. Taurine과 $\alpha$-tocopherol의 혼합처리군 $50mM\~1,000{\mu}M$에서 HOST, 점체 반응이 대조군과 비교하여 유의차는 인정되지 않았으나 다소 증가하였다. 동결$\cdot$융해 정자의 ROS 발생 억제를 위한 taurine과 $\alpha$-tocopherol의 모든 처리군은 대조군과 비교하여$\cdot\{O_2}{^-}$ 발생을 유의적으로 완화시키지 못하였다. 그러나 taurine 단일처리군(25mM), $\alpha$-tocopherol 단일처리군($50mM,\;1,000{\mu}M$)과 혼합처리군($25mM\~500mM,\;50mM\~1,000{\mu}M$은 $H_{2}O_{2}$의 발생을 대조군과 비교하여 유의적으로 완화시켰다(P<0.05). 동결$\cdot$융해 정자의 malondialdehyde의 생산은 taurine과 $\alpha$-tocopherol의 모든 처리군에서 대조군과 비교하여 유의적인 감소를 보였다(P<0.05). 이상의 결과를 종합해 보면 돼지 정액의 동결보존시 taurine와 $\alpha$-tocopherol 같은 항산화제의 처리는 ROS 발생과 LPO을 효과적으로 완화시킴에 따라 돼지 정액의 동결보존 효율을 증진시킬 수 있는 유용한 방법이라 사료된다.

• 한국임상수의학회지
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• 제34권2호
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• 한국수정란이식학회지
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• 제30권3호
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• pp.137-142
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• 2015

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen post-thawed in boar. Recently, polymorphism (g.158T>C) of phospholipase C zeta (PLCz) gene reported to be significant association with MOT. This study was conducted to evaluate the PLCz gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.158 T>C SNP of PLCz was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.0001, p=0.0002, p<0.0001 and p<0.0001, respectively). Therefore, we suggest that the intron region of the porcine PLCz, may be used as a molecular marker for Duroc boar post-thawed semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

• 대한수의학회지
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• 제40권2호
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• pp.403-414
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• 2000

To improve the semen production, the selenium(Se) and vitamin E(Vit. E) were administrated into Hanwoo young sire for intensification an antioxidant system and the taurine were supplemented into semen extender for improving the semen characteristics. The 16 heads ranging from twenty to thirty two months of age were randomly assigned to control group, Se-admistrated group(Se-group), Vit. E-administrated group(Vit. E-Group) and Se and Vit. E administrated group(Se and Vit. E-group). Se and Vit. E dministrated 3 times every 30 days by intramuscular injection. The administration of Se, Vit. E, and Se and Vit. E didn't affect on semen volume, sperm concentration, and total sperm number among all groups(p>0.05). Adiministration of Se improved sperm motility and viability. Motility of Se-group and control were 26.01% and 19.20%, respectively(p<0.05). Viability of Se-group(47.07%) was higher than control group(35.73%), Vit. E group(36.55%)(p<0.05). The administration of Se and Vit. E didn't affect sperm capacitation and acrosome reaction. The 100mM taurine supplement into semen extender increased the motility of frozen/thawed semen in the Vit. E-group(p<0.05) and had a beneficial effects on decreasing abnormality of frozen/thawed semen in all groups(p<0.05). These results indicate that Se administration improve sperm motility and viability, and the taurine supplement into semen extender decrease abnormality in Hanwoo young sire.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• 한국수정란이식학회지
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• 제26권3호
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• 한국수정란이식학회지
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• 제25권4호
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• pp.291-295
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• 2010

Four estrus-induced Himalayan tahrs (Hemitragus jemlahicus) were inseminated with frozen-thawed semen by laparoscopic or transcervical insemination techniques with no regard to the site of ovulation in non-breeding season. In June and July, 2009, estrus was synchronized by Eazi-Breed $CIDR^{(R)}$ (Controlled internal drug release; Pfizer Animal Health, New Zealand) insertion for 16 days and PG 600 (PMSG 400IU, hCG 200 IU; Intervet, Netherlands) injection (IM) a day before removing $CIDR^{(R)}$. Forty eight hours later, laparoscopic or transcervical insemination was done to each of two tahrs under anesthetic condition inducted by ketamine (1.5 mg/kg) and medetomidine (0.09 mg/kg). For examination of estradiol and progesterone, blood was collected right before $CIDR^{(R)}$ insertion, PG 600 injection, $CIDR^{(R)}$ removal and insemination. Estradiol levels of four tahrs (No. 1, 2, 3, 4) before $CIDR^{(R)}$ insertion and insemination were 13.3, 8.8, 14.3, 12 pg/ml and 23.5, 25.5, 21.1, 11.5 pg/ml, respectively. Progesterone levels of four tahrs (No. 1, 2, 3, 4) before $CIDR^{(R)}$ insertion and insemination were 1.8, 0.05, 0.63, 0.61 ng/ml and 1.03, 0.37, 1.48, 2.12 ng/ml. Except for No. 4 tahr, cervices showed cervical mucus and opened enough to penetrate with embryo transfer gun sheet usually used for cows. Therefore, No.4 was laparoscopically inseminated together with No. 1. In conclusion, none of four Himalayan tahrs was pregnant. However, we proved that estrus could be induced by CIDR and PG 600 injection in non-breeding season, and laparoscopic or transcervical insemination with frozen-thawed semen could be one of assisted reproductive techniques in Himalayan Tahr.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.155-159
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• 2013

본 연구는 제주흑우의 정액의 동결과 보존기술 개발을 위해 AndroMed와 Trialdyl를 사용하여 보다 우수한 희석제와 우수 종모우 선정을 위해 실시되었다. 종모우 1번부터 4번까지 개체 정자의 성상비교에서 수정능 획득과 첨체반응의 비율은 36.8%, 26.8%, 37.8% 그리고 38%의 결과를 나타내었다. 2번 개체 정자에서 수정능 획득과 첨체반응의 비율이 유의적으로(p<0.05) 낮은 결과를 보였다. 제주흑우 정액을 동결하기 전 정자의 생존율은 약 $93.27{\pm}1.62$, 동결 융해 후 생존율은 $73.34{\pm}3.27%$로 나타나 동결전 정액의 생존율이 유의적으로(p<0.05) 높은 결과를 나타내었으나, 사멸 정자의 비율은 약 $7.35{\pm}2.63%$와 $13.71{\pm}2.85%$의 결과를 나타내었다. 그리고 $Triladyl^{(R)}$ 동해 방지제를 사용하였을 때, 정자의 운동성은 $72.86{\pm}2.83%$, $AndorMed^{(R)}$를 사용하였을 때 운동성은 $81.47{\pm}2.48%$로 $AndorMed^{(R)}$ 동해방지제를 사용하였을 때 운동성이 다소 높은 경향을 보였으나, 유의적인 차이는 나타나지 않았다. 마찬가지로 정자의 사멸율에서도 $Triladyl^{(R)}$ 동해 방지제는 $18.41{\pm}3.42%$, $AndorMed^{(R)}$ 동해방지제는 $17.26{\pm}4.25%$의 결과를 보여 AndroMed를 사용한 동해방지제가 정자의 생존율을 향상시키는 것으로 나타났다. 결론적으로 제주흑우 정액의 동결 보존을 위해 보다 향상된 동결보호제 선정과 동결보존 기술 개발이 필요할 것으로 사료된다.