• 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.

• Journal of Animal Science and Technology
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• 제45권4호
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• pp.517-522
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• 2003

한우의 고환둘레와 정액 생산 및 번식과의 관계를 구명하기 위하여, 농협중앙회 가축개량사업소 보유 종모우 63두의 고환둘레와, 이들의 19,742번의 정액 생산기록(정액량, 농도/ml 및 총정자수) 및 이중 16두가 생산한 1,289두의 자손기록을 이용하였다. 정액량, 정액 농도와 총 정자수 모두 정액채취년도, 정액채취월, 정액 채취 순서에 따라 영향을 받았다(P<0.001). 고환 둘레의 평균은 41.2$\pm$4.54cm 이었고, 1회 평균 사정량은 5.47$\pm$0.89ml 이었으며, 정액 농도는 16.2$\pm$2.78(${\times}10^8$/ml), 사정당 총정자수는88.47$\pm$15.24(${\times}10^8$)이었다. 고환둘레와 정액량 및 총 정자수 간에는 0.14 및 0.15의 정의 상관을 나타내었으며, 임신율과는 0.39, 출산율과는 0.26의 상관을 나타내었다.

• 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.

• 한국동물생명공학회지
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• 제35권4호
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• pp.307-314
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• 2020

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

• Asian-Australasian Journal of Animal Sciences
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• 제13권2호
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• pp.167-175
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• 2000

This paper describes the magnetic orientation of bull sperm separated into the head and the flagellum treated by DTT or heparin in a 5,400G static field. Semen samples collected from four bulls (Japanese Black) were mixed to the same sperm density. One percentage triton X-100 was used to extract the plasma membrane. The intact and demembranated sperm suspensions were treated with 20, 200, 2,000 mM DTT, 100, 1,000 or 10,000 units heparin solutions at $4^{\circ}C$ for 6 days. The decondensation of the sperm nuclei treated by DTT or heparin was examined by measuring the head area at 1, 3 and 6 days. After measuring the area, each sample was exposed to a 5,400G static magnetic field generated by Nd-Fe-B permanent magnets for 24 hours at room temperature. Results showed that the sperms were separated into the head and the flagellum through the DTT treatment. Almost of the separated heads showed that their long axis oriented perpendicularly to the magnetic lines of force, and most of the long axis perpendicularly oriented heads showed that their flat plane oriented perpendicularly in a 5,400G magnetic field. Also, the demembranation of the head tended to increase those perpendicular orientations, while those perpendicular orientations of the head declined with the decondensation of the sperm nuclei. These findings suggest that strong magnetic anisotropy for the perpendicular orientation of the long axis and the flat plane of the head occurs in the sperm nuclei in a 5,400G magnetic field. The separated flagellum showed lower parallel orientation, and the separated and demembranated flagellum showed parallel orientation to the magnetic lines of force in this magnetic field. These findings suggest that weak magnetic anisotropy of the parallel orientation of the flagellum occurs in the inside components in a 5,400G field.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1188-1195
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• 2001

A tetraparental chimeric bull was successfully produced by aggregating bovine IVF embryos of F1 (female Holstein${\times}$male Japanese Black) and F1(female Japanese Brown${\times}$male Limousin) and culturing in vitro without the zona pellucida at Yamaguchi Research Station in Japan. In the microsatellite genotyping, 12% (28/228) microsatellite primer sets ware potentially useful for this parentage analysis in the chimeric bull, 78.6% (22/28) of microsatellite present in the chimeric bull were uniquely contributed from the Japanese Black and 21.4% (6/28) from Limousin. This chimeric bull semen was used in producing IVF embryos. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h of incubation was higher (p<0.01) with the Chimera than with the Holstein and in Japanese Brown bulls. But did not differ from Japanese Black and Limousin bull sperm. Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera sperm were higher (p<0.05) than with Japanese Brown and (p<0.01) than with Holstein sperm, but did not differ from Japanese Black and Limousin sperm. The cleavage rates of IVF oocytes inseminated with Chimera sperm were also higher (p<0.001) compared with Holstein, (p<0.01) Japanese Brown and (p<0.05) Limousin, but did not differ from Japanese Black sperm. The blastocyst rates of IVM oocytes inseminated with sperm were higher (p<0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black. Chimeric cattles were produced by aggregation of parthenogenetic (Japanese Brown) and in vitro fertilized (Holstein) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus) and in vitro fertilized (Holstein) embryos at the St. Gabriel Research Station in Louisiana. The aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding. The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derieved embryos cultured without agar than when cultured with agar. The development rate to blastocysts, however, was not different among the treatment. To verify parthenogenetic and the cells derieved from the male IVF embryos in blastocyst formation, 51 embryos were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zonafree chimeric embryos at 24 h following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP. Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transfer of 40 chimeric embryos at the Louisiana station. Two pregnancies were Jost prior to 4 months and one phenotypically chimeric viable male born.

• Asian-Australasian Journal of Animal Sciences
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• 제5권3호
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• pp.449-454
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• 1992

Eight healthy Holstein bulls, 4-6 years old were used to study the effect of season of the year on the incidence of the morphologically abnormal spermatozoa. Semen was collected twice a week by A. V. over one-year period. The percentage of total abnormal spermatozoa was $14.1{\pm}0.5$. Ejaculates collected during hot summer season had significantly higher incidence of abnormal spermatozoa than those collected during winter time. Warm spring had moderate semen abnormality. In addition to its effect on the total number of morphologically abnormal spermatozoa, season affected significantly the primary as well as the secondary types of abnormalities. The differences between incidence of primary and secondary types of abnormalities were not significant for all seasons and seasons pooled together. The ratio between the total forms of abnormality in the head, mid-piece and tail of spermatozoa was as 1:1.5:1. Head and mid-piece had defected more during summer compared with both winter and spring. There was no variation in tail abnormalities due to season. The significant effect of season on head was observed by large, pyriform, free and detached heads, while that on mid-piece was by swollen, coiled mid-pieces and protoplasmic droplets.

• 한국수정란이식학회지
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• 제15권1호
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• pp.85-94
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• 2000

These experiments were conducted to examine the effects of theophylline, pentoxifylline and heparin on frozen-thawed Hanwoo sperm for enhancing motility and viability of sperm. Frozen-thawed semen collected from one bull was treated in TALP(tyrode-albuminlactate-pyruvate) containing varous concentrations of theophylline and pentoxifylline. After incubated at 5% CO2 in air atmosphere for 6 hours, the motility of sperm after the treatments was characterized by CASA(computer aided semen analysis) system. When monitored notility(MOT) and curvilinear velocity(VCL), theophylline and pentoxifylline exerted their optimal action at the concentration of 30 mM and 3 mM, respectively. No difference of sperm motility was observed when the sperm was treated with both substances compared with a single treatment of each substance. Comparison was then made for evaluating the effect of theophylline and / or pentoxiophylline on the motility and viability of significant treatment effects of each substance, high MOT and VCL values were detected in sperm treated with theophylline. In the case of sperm viability examined by an eosin-nigrosin staining, however, a significant decrease was found after the combined treatment of theophylline+pentoxyphilline than after the treatment with heparin alone or no treatment(P<0.05). In conclusion, theophylline, pentoxiphylline or heparin can be used for enhancing the motional characteristics and viability of frozen thawed Hanwoo semen. Considering characteristics of these substances, theophyline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

• Animal Bioscience
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• 제37권3호
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• pp.428-436
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• 2024

Objective: This study compared five distinct sets of biological pathways and associated genes related to semen volume (VOL), number of sperm (NS), and sperm motility (MOT) in the Thai multibreed dairy population. Methods: The phenotypic data included 13,533 VOL records, 12,773 NS records, and 12,660 MOT records from 131 bulls. The genotypic data consisted of 76,519 imputed and actual single nucleotide polymorphisms (SNPs) from 72 animals. The SNP additive genetic variances for VOL, NS, and MOT were estimated for SNP windows of one SNP (SW1), ten SNP (SW10), 30 SNP (SW30), 50 SNP (SW50), and 100 SNP (SW100) using a single-step genomic best linear unbiased prediction approach. The fixed effects in the model were contemporary group, ejaculate order, bull age, ambient temperature, and heterosis. The random effects accounted for animal additive genetic effects, permanent environment effects, and residual. The SNPs explaining at least 0.001% of the additive genetic variance in SW1, 0.01% in SW10, 0.03% in SW30, 0.05% in SW50, and 0.1% in SW100 were selected for gene identification through the NCBI database. The pathway analysis utilized genes associated with the identified SNP windows. Results: Comparison of overlapping and non-overlapping SNP windows revealed notable differences among the identified pathways and genes associated with the studied traits. Overlapping windows consistently yielded a larger number of shared biological pathways and genes than non-overlapping windows. In particular, overlapping SW30 and SW50 identified the largest number of shared pathways and genes in the Thai multibreed dairy population. Conclusion: This study yielded valuable insights into the genetic architecture of VOL, NS, and MOT. It also highlighted the importance of assessing overlapping and non-overlapping SNP windows of various sizes for their effectiveness to identify shared pathways and genes influencing multiple traits.

• 한국초지조사료학회지
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• 제38권4호
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• pp.320-324
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• 2018

본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).