• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.92-92
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• 2002

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to evaluate semen quality after cryopreservation and to evaluate the Pregnancy rate after insemination (AI). Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifene (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. The ejaculated semen to freeze was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM: TES, 209 mM: citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 cm above the surface of liquid nitrogen (LN$_2$) for 23 min. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, the pregnancy rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only. In addition, frozen-thawed semen can be used successfully far artificial insemination in dog.

• 한국동물생명공학회지
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• 제34권4호
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• pp.272-279
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• 2019

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.263-267
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• 2013

Pig producers have been shown keen interest of the number of spermatozoa in a semen dose since pig artificial insemination introduce. However, determining the minimal number of spermatozoa need per AI without detrimental effect on overall reproductive performances is not an easy question to answer. To increase the efficiency of semen utilization in pig AI, optimum number of spermatozoa per dose needed to determine. The objective of this study was to determine the reproductive performance and factors that affect on-farm application of low-dose semen insemination in sows. Data were collected from Darby Genetics AI studs from 4th of June to 7th of July, 2012 (n=401). The numbers of parturition were 84, 234 and 83 in sows inseminated with doses of $1.5{\times}10^9$, $2.0{\times}10^9$ and $2.5{\times}10^9$ spermatozoa in 100ml extender, respectively. There were no significant differences on reproductive performances such as gestation period, total born, total born alive, stillbirth and mummy in sows inseminated with different semen doses. The average number of born alive was 10.5, 11.0 and 10.4 from sows inseminated with $1.5{\times}10^9$, $2.0{\times}10^9$ and $2.5{\times}10^9$ sperms, respectively. Also, number of spermatozoa per dose did not affect litter size (p>0.10). There were no significant differences of maternal genetic line difference on gestation period, total number born, number born alive, born dead and mummy. The estimated correlation coefficients of the different semen doses with total number born, number born alive, born dead and mummy were r=-0.00, -0.01, 0.02 and 0.02, respectively. Taken together, the result of this study suggested that when semen was appropriately inseminated after induced ovulation, insemination with low-dose ($1.5{\sim}2.0{\times}10^9$) semen dose not adversely affect sow's fertility.

• Asian-Australasian Journal of Animal Sciences
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• 제29권5호
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• pp.646-651
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• 2016

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

• Journal of Animal Science and Technology
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• 제49권5호
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• pp.585-592
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• 2007

본 연구는 풍산개 동결정액 제조기술을 정립하기 위하여 정액성상과 정액 동결 시 희석액에 첨가되는 Glycerol 농도, 동결속도, 융해온도와 시간에 따라 정액의 운동성과 생존율 및 CASA를 이용한 운동성 등에 대하여 조사하여 최적의 동결조건을 확립하기 위해 실시하였다.1.풍산개의 평균 정액량 5.9ml, 정액농도 116.3 ×106sperm/ml 총정자수 789.3×106sperm, 운동성 88.7±1.77% 및 생존율 87.6±1.85% 였다. 2.1차 희석액을 상온에서 희석 후 상온에서 4°C까지 하강시킨 후 glycerol 농도가 3%, 5% 및 7% 첨가된 2차 희석액을 희석 후 6일간 운동성을 측정한 결과 5일째 3%일 때 46.2±9.3%, 5% glycerol에서는 48.1±8.5% 및 7%일 때 52.7±8.2%로 glycerol 7%가 유의적으로 높은 운동성을 나타냈다.3. 각기 다른 glycerol 농도를 함유한 동결보존액에서 동결보존 후 융해하였을 때 7%의 glycerol 농도에서 각각 52.7%와 57.7±10.3%로서 다른 처리구에 비해 유의적인(P<0.01) 운동성 및 생존율을 나타냈다.4.정액을 동결함에 있어 예비동결시 57 및 10cm의 높이에서 정치시킨 후 동결을 실시하였을 때 액체질소의 표면 7cm의 높이에서 동결을 실시한 처리구에서 전체적으로 유의한 운동성과 생존율을 나타냈다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권2호
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• pp.180-184
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• 2003

The intrauterine inseminator (IUI) was developed to provide the method of depositing dog semen into the uterine body instead of the vagina. The IUI consists of a vaginal endoscope, a balloon sheath, and injection catheter. When the endoscope is inserted into the vagina and the balloon expanded with air, the cervical os becomes visible so a injection catheter can be inserted through the cervix for deposition of the frozen-thawed semen. The efficacy of the IUI device was compared to intra-vaginal artificial insemination using semen that had been collected and frozen from pooled sperm-rich fraction of ejaculates collected from two Jindo dog donors. Aliquots of semen were extended with a Tris-egg yolk diluent, centrifuged, the seminal plasma removed, the pellet resuspended with the same diluent, and cooled to $5^{\circ}C$ over a 2 h period. A Tris-egg yolk-glycerol extender was added at $5^{\circ}C$; after 1 h, semen was loaded into 0.5 ml straws, and straws were frozen in LN vapor for 5 min, and immersed in LN for storage. The final sperm concentration for freezing was approximately $100{\times}10^{6}cells/ml$. The straws were thawed at $70^{\circ}C$ for precisely 6 sec, 1.5 ml Tris-egg yolk buffer at $38^{\circ}C$ added, and the 2 ml of thawed semen was used for a single insemination using the IUI device. Each bitch was inseminated at optimal insemination point, which was estimated by vaginal epithelial cells staining and progesterone concentration analysis. Use of the IUI device resulted in 21 of 26 females giving birth to 89 pups ($4.2{\pm}1.6$ pups per litter), while intra-vaginal AI resulted in 6 of 15 females whelping a total of 17 pups ($2.8{\pm}1.2$ pups per litter). We believe the IUI device is easier to use than previously described devices used for intrauterine insemination. In our experience the expansion of the balloon has a calming effect on the bitch that aids the inseminator. These results indicate that the IUI device was able to provide high fertility with 50 million frozen sperm per insemination and two inseminations.

• 한국가축번식학회지
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• 제22권4호
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• pp.405-410
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• 1998

본 연구는 독자 개발한 돼지액상정액희석제 Kp(한국생명과학연구소)에 pH를 안정화하여 보존 중 정자의 운동성을 유지시킬 수 있도록 EDTA, Tris, Citrate buffer의 적정 첨가농도를 결정하고자 실시하였다. Basic Kp와 BTS (Mini-tube, Germany; BT-Sg), BTS (Tri-bio, USA; BTSa), Modena (SGI, USA)를 17$^{\circ}C$ 에서 각각 보관한 경우 모든 희석액에서 시간 경과에 따라 pH가 증가하였고, Basic Kp 는 희석 당일의 pH가 다른 희석액에 비해 높게 나타났는데 이것은 돼지액상정액의 생리적인 pH인 6.8~7.5에 비해서도 높은 수준이었다. 정액내의 pH 저하를 방지해 주고 정액의 생리적인 pH를 유지하기 위하여 Basic Kp에 EDTA, Tris, Citrate buffer를 단계적으로 첨가하면서 시간 경과에 따른 pH 변화를 살펴 본 결과 1.25g /L EDTA, 1.42g /L Tris, 1.00g /L Citrate를 첨가한 경우 (Modified Kp) pH는 l일째 6.88에서 6일째 7.33으로 유지되었다. 특히, Modified Kp에 첨가된 buffer 의 농도는 Modena와 다른 희석제에 첨가된 농도에 비해 1/2 에서 1/4 정도로 낮은 수준이었다. Modified Kp 와 Basic Kp, BTSg, BTSa 및 Modena로 냉동-융해된 돼지 정자를 희석하여 보존한 경우 정자의 운동성은 Modified Kp가 다른 희석액에 비해 유의하게 높게 나타났다(87.0% vs. 71.0~48.0% in day 1; 13.3% vs. 6.3~0% of day 6), 이상의 결과를 종합해 볼 때, Modified Kp는 낮은 농도의 EDTA, Tris, Citrate buffer 첨가에도 불구하고 돼지정자의 생리적인 pH 수준을 잘 유지할 수 있었으며, 수입되어 냉동-융해된 돼지 정자에 사용하는 희석제 BTSg, BTSa, Modena 보다도 정자의 운동성에 효과적이었다.

• 한국임상수의학회지
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• 제10권1호
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• pp.11-18
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• 1993

Four extenders such as tris-fructose-citrate, Tris-glucose-citrate, glycine-glucose-citrate and lactose that the more frequently utilized types of semen extenders used for freezing dog semen evaluated with sperm motility, viability and acrosomal score in the processing procedures to prior freezing and after frozen-thawing respectively. Each extender contained 4% glycerol and 20% egg yolk were treated by same methods in dilution, freezing, storage and thawing. The results were obtained as follows; 1. The sperm motility and viability in procedure from dilution to frozen-thawing appeared superiorly with recovery rate of 53.2%, 54.8% in tris-fructose-citrate but appeared inferiorly with recover rate of 8.4%, 8.3% in lactose to others. 2. In the processing procedure course to prior-freezing, glycine-glucose-citrate appeared superiorly with decrease rate of 5.4% in motility, and lactose with decrease rate of 4.6% in viability, but tris-glucose-citrate appeared inferiorly with decrease rate of 12.2%, 11.4% in the sperm motility and viability. 3. During frozen-thawing, tris-fructose-citrate appeared superiorly with decrease rate of 35.2% in motility and 30.7% in viability but lactose appeared inferiorly with decrease rate of 76.7% in motility and 75.7% in viability. 4. The variation of acrosome morphology in the total processing procedures appeared that glycine-glucose citrate were superior with acrosome score of 0.1191$\pm$0.029, that tris-fructose-citrate were inferior with acrosome score of 0.1941$\pm$0.045 to others.

• Asian-Australasian Journal of Animal Sciences
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• 제19권11호
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• pp.1561-1565
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• 2006

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without $250{\mu}M$ $H_2O_2$. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group ($85.1{\pm}0.5$ and $72.4{\pm}0.3$, p<0.05). The sperm motility in taurine with $H_2O_2$ after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with $H_2O_2$ ($75.3{\pm}0.3$ and $69.6{\pm}2.9$, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM ($76.0{\pm}0.6$) and 50 mM taurine groups ($78.0{\pm}0.7$), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (${\geq}48%$ in 9 h and ${\geq}42%$ in 12 h) compared to controls ($43.0{\pm}2.1$ and $31.0{\pm}0.6$, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics(motility, viability, survival and membrane integrity).

• 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.