• 한국수정란이식학회지
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• 제21권3호
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• pp.263-271
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• 2006

본 연구는 미니 돼지와 일반 돼지(Duroc)의 동결융해 후 정자의 수정 능력을 비교하여 동결 보존체계의 기틀을 확립하고자 하였다. 정액 제조는 수압법으로 정액 채취하여 1차 희석하였다. 동결은 LEY (1차: 11% ${\alpha}$-lactose+egg yolk, 2차: 1차 동결액+glycerol+OEP) 동결액을 이용하여 동결을 실시하여 동결 보존하였다. 동결 정액의 융해는 0.5 ml straw를 각각 20, 37 및 $50^{\circ}C$ water bath에서 1분, 40 초 및 10초간 융해하여 세척 과정 후 BTS 5 ml를 첨가하여 $37^{\circ}C$에서 배양하였다. 정자 성상 검사로는 기형율(Rose Bengal staining), 첨체율(Chlortetra-cycline staining) 및 생존율(SYBR-14/PI staining)등을 배양 후 0, 3, 6, 9 및 12시간에서 검토하여 다음과 같은 결과를 얻었다. 정자의 보존 시간에 따른 기형율은 동결 융해 직후 miniature pig가 $19.5{\pm}1.7%$ 로 Duroc의 $13.9{\pm}0.3%$에 비해 유의적으로 높았다. (p<0.05). 첨체 검사에서 수정능 획득이 일어나지 않은 F pattern은 동결 융해 후 miniature pig와 Duroc 종 정액이 $24.1{\pm}2.8%$와 $37.9{\pm}1.1%$로 Duroc 종에서 유의적(p<0.05)으로 높게 나타났으며, 동결 융해후 miniature pig와 Duroc 종의 AR pattern은 $21.1{\pm}1.6%$ 및 $15.5{\pm}2.2%$로 miniature pig가 유의적(p<0.05)으로 높게 나타났다. 융해 온도별 생존율에서는 20과 $37^{\circ}C$에서는 두 종간에서 유의적 차이는 없었으나, $50^{\circ}C$에서는 miniature pig가 $63.8{\pm}3.6%$로 $47.4{\pm}3.2%$인 Duroc 종에 비해 유의적(p<0.05)으로 높게 나타났다. 본 연구의 결과로부터 첨체율과 기형율에 대한 연구를 보완함으로써 miniature pig정액의 안정적인 동결 체계를 확립할 수 있을 것으로 판단된다.

• 한국수정란이식학회지
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• 제9권1호
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• pp.49-52
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• 1994

This study was carried out to compare farrowing rate and litter traits for European and American lines with boar sperm frozen in straws. Farrowing rate, litter size and mean pig weght at birth and 21 days were investigated. A total of 36 gilts Landrace, Large white and Duroc were investigated at the Chungnam Provincial Animal Breeding Station. We obtained higher farrowing rate and litter traits for European line boars compared to American line boars.

• Animal Bioscience
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• 제36권11호
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• pp.1647-1654
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• 2023

Objective: Aging roosters typically exhibit subfertility with decreasing semen quality, furthermore Thai native roosters reared in rural areas are raised for a longer duration than their usual lifespan. The present study therefore aimed to assess the effect of selenium supplementation as an antioxidative substance in diets to improve the semen cryopreservation of aged roosters. Methods: Semen samples were collected from young (n = 20) and aged (n = 20) Thai native roosters (Pradu Hang Dum) at 36 and 105 weeks of age when starting the experiment, respectively. They were fed diets either non-supplemented or supplemented with selenium (0.75 ppm). Fresh semen quality and lipid peroxidation of fresh semen was evaluated before cryopreservation using the traditional liquid nitrogen vapor method. Post-thaw sperm quality and fertility potential were determined. Results: Advancing age is unrelated to decreasing fresh semen quality (p>0.05). However, lipid peroxidation in rooster semen depended on age, and the malondialdehyde (MDA) concentration increased in aged roosters (p<0.05). Selenium supplementation in diets significantly decreased the MDA concentration and increased the sperm concentration (p<0.05). In contrast, cryopreserved semen was affected by advancing rooster age, and selenium influenced sperm quality (p<0.05). Younger roosters had higher post-thaw sperm quality and fertility potential than aged roosters (p<0.05). Likewise, diet selenium supplements improved post-thaw sperm quality and fertility compared with the non-supplement group. Conclusion: Rooster's age does not influence the rooster sperm quality of fresh semen, while sperm cryotolerance and fertility were greater in young roosters than in aged roosters. However, sperm of aged roosters could be improved by dietary selenium supplementation.

• 한국가축번식학회지
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• 제23권2호
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• pp.127-132
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• 1999

본 연구는 소형견 정액의 원정액과 정장제거 정액의 일반성상, 채취분획별 및 단기보존시 정자의 생존성과 아울러 동결보존시의 정자의 생존성에 대해 조사하고자 수행하였다. 1. 원정액과 정장제거 정액에 saline 및 Tris-buffer를 희석 후 각각 일반성상 검사를 실시했을 때 원정액의 경우 정자농도는 5.07$\pm$2.32$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 95.42$\pm$2.65%, 기행정자수는 4.42$\pm$0.15%로 나타났으며, RSP(saline 및 Tris-buffer)군의 경우 정자농도는 4.69$\pm$3.27~4.25$\pm$3.65$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 91.17$\pm$3.85~88.52$\pm$3.85%, 기형정자수는 6.57$\pm$0.43~5.54$\pm$0.52%로 나타났다. 2. 분획별 채취 전정액중 제 1 분획에서는 정액량이 0.92$\pm$0.7$m\ell$, 정자농도는 4.57$\pm$0.78$\times$$10^{6}$cells/$m\ell$, 정자활력은 10.72$\pm$3,21%, 기형정자수는 5.50$\pm$0.70%로 나타났으며, 제 2분획에서는 정액량이 2.14$\pm$0.19$m\ell$, 정자농도는 2.01$\pm$0.12$\times$$10^{6}$cells/$m\ell$, 정자활력은 95.44$\pm$4.21%, 기형정자수는 4.31$\pm$0.53%로 나타났다. 또한, 제3 분획에서는 정액량이 2.66$\pm$0.23$m\ell$, 정자농도는 2.35$\pm$0.21$\times$$10^{6}$cells/$m\ell$, 정자활력은 90.71$\pm$2.63%, 기형정자수는 6.33$\pm$0.91%로 나타났다. 3. 원정액과 정장제거 정액을 4$^{\circ}C$ 와 2$0^{\circ}C$ 및 37$^{\circ}C$ 에서 각각 보존했을 때 보존시간별 정자의 활력은 2$0^{\circ}C$ 의 경우 1, 6, 13, 24, 30 및 40 시간에서 각각 98.51%와 98.32%, 86.32%와 92.15%, 83.71% 와 89.20%, 74.29% 와 82.08%, 52.98% 와 72.07%, 15.45% 와 60.02%, 2.41% 와 37.19% 의 정자활력을 나타내어 4$^{\circ}C$와 37$^{\circ}C$에 비해 높은 정자운동성을 나타냈다. 4. 제 2 분획 정액과 정장제거 정액을 각각 제 1차 및 제 2차 희석액으로 희석 평형 시킨 후 동결 융해했을 때 정자의 생존율은 각각 33.3$\pm$8.7, 54.7$\pm$9.5%로서 대조군의 15.4$\pm$5.2% 에 비해 높게 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권4호
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• pp.486-494
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• 2006

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1119-1126
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• 2018

Objective: Present investigation was aimed to study the Single Nucleotide Variants of the luteinizing hormone beta ($LH{\beta}$) gene and to analyze their association with the semen quality (fresh and post-thawed frozen semen) and luteinizing hormone (LH) concentrations in Murrah buffalo bulls. Methods: Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and Sanger sequencing method is used to study genetic variability in $LH{\beta}$ gene. LH assay was carried out using enzyme-linked immunosorbent assay method. A fixed general linear model was used to analyze association of single nucleotide polymorphism (SNP) of $LH{\beta}$ gene with semen quality in 109 and LH concentrations in 80 Murrah bulls. Results: $LH{\beta}$ gene was found to be polymorphic. Total six SNPs were identified in $LH{\beta}$ gene g C356090A, g C356113T, g A356701G, g G355869A, g G356330C, and g G356606T. Single Stranded Conformational Polymorphism variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on sperm concentration (million/mL), percent mass motility, acrosome integrity and membrane integrity in fresh and frozen semen whereas significant (p<0.05) effect was observed on percent live spermatozoa. SSCP variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on luteinizing hormone concentrations too. Conclusion: The observed association between SSCP variants of $LH{\beta}$ gene with semen quality parameters and LH concentrations indicated the possibilities of using $LH{\beta}$ as a candidate gene for identification of markers for semen quality traits and LH concentrations in Murrah buffaloes.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.33-40
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• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

• Animal Bioscience
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• 제35권12호
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• pp.1827-1838
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• 2022

Objective: The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom Equine Genotyping Array. Results: We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds. As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME/NM23 family member 8 (NME8), olfactory receptor family 2 subfamily AP member 1 (OR2AP1), and olfactory receptor family 6 subfamily C member 4 (OR6C4). Potential implications of effects of these genes on sperm motility are herein discussed. Conclusion: The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system.

• 한국수정란이식학회지
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• 제20권1호
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• pp.43-48
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• 2005

본 실험은 돼지정액의 동결보존을 위한 희석액과 냉각속도 및 동해방지제의 적정농도를 결정하기 위해 실시하였으며, 얻어진 결과는 다음과 같다. 1. $25^{\circ}C$에서 $5^{\circ}C$까지의 냉각속도에서는 LEY 희석액에서 분당 $0.17\~0.22^{\circ}C$로 냉각하는 것이 생존율과 정상 첨체율에서 가장 높은 결과를 얻었다. 2. LEY 희석액이 BF5와 M-Soejima 희석액보다 정자를 동해로부터 보호하는 능력이 우수하였다. 3. LEY 희석액에 첨가하는 glycerol의 농도는 3 또는 $4\%$가 $2\%$의 glycerol을 첨가한 것보다 4. LEY 희석액에 15mM의 fructose와 $3\%$ 또는 $4\%$의 glycerol 첨가구가 가장 높은 생존율과 정상첨체율을 나나냈다(p<0.001).

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.149-154
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• 2005

The purpose of this study was the analysis of sperm ability in Specific Pathogen Free (SPE) miniature pig for production of bio-organ. The collected semen was diluted with extender and stored at $17^{\circ}C$t for up to 7 days. The semen samples were evaluated at 0, 1, 3, 5, and 7 days of storage for analysis of sperm ability. Sperm ability was evaluated by examining viability, progressive motility, sperm abnormality and intensity of the sperm membrane. Also, the semen was processed according to the convenient freezing method, and frozen-thawed sperm was evaluated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) staining. Motility of spermatozoa of SPF miniature pig was significantly (P<0.05) lower on 3 days or later compared to the Duroc, Yorkshire and Landrace in domestic boar. The percentage of abnormal spermatozoa of Landrace were significantly (P<0.05) higher than in SPF miniature pig, Duroc and Yorkshire that had a similar percentage on 5 or 7 days of sperm storage. The percentage of spermatozoa with coiled tail decreased during the storage period but there were no significant difference. On the other hand, viability of frozen-thawed spermatozoa had a significantly (P<0.05) lower in SPF miniature pig than in other domestic boars. CTC patterns had no significant difference, but SPF miniature pig had higher percentage of capacitated spermatozoa and lower percentage of acrosome-reacted it than domestic boars. Therefore, this study suggest that it is necessary to develop the suitable extender and freezing methods methods for the high viable rate and fertilizing ability in vitro.