• Title, Summary, Keyword: Boar Spermatozoa

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Effects of Cryo-extenders for Spermatozoa Sorted by Percoll on In Vitro Fertility of in Miniature Pigs (Percoll 분리된 미니돼지 정액의 체외 수정 능력에 있어서 동결보존액의 영향)

  • Lee, Sang-Hee;Yoo, Han-Jun;Lee, Yong-Seung;Cheong, Hee-Tae;Yang, Boo-Keun;Km, Dae-Young;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.35 no.1
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    • pp.85-91
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    • 2011
  • The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

Effects of Ginsenoside-$Rg_1$ on Post-thawed Miniature Pig Sperm Motility, Mitochondria Activity, and Membrane Integrity

  • Hwang, You Jin;Kim, Dae Young
    • Journal of Embryo Transfer
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    • v.28 no.1
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    • pp.63-71
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    • 2013
  • In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activity in post-thawed sperm that was supplemented with ginsenoside-$Rg_1$. Varying concentrations of ginsenoside-$Rg_1$ (0, 25, 50 and $100{\mu}M/ml$) were used in the extender during cryopreservation to protect the DNA of thawed sperm, thereby increasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. The results derived from CASA were used to compare the fresh, control, and ginsenoside-$Rg_1$ groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the $50{\mu}M/ml$ ginsenoside-Rg1 group ($61.0{\pm}4.65%$) than in the control ($46.6{\pm}7.02%$), $25{\mu}M/ml$ ($46.2{\pm}4.76%$), and $100{\mu}M/ml$ ginsenoside-$Rg_1$ ($52.0{\pm}1.90%$) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membrane integrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher ($91.6{\pm}0.82%$) in the $50{\mu}M/ml$ ginsenoside-$Rg_1$ group than in the other groups. Here, we report that ginsenoside-$Rg_1$ affects the motility and viability of boar spermatozoa. Moreover, ginsenoside-$Rg_1$ can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.

Studies on the Liquid and Freezing storage of Boar Semen III. Effects of Dilutor on Livability and Fertility of Liquid Semen and Effects of Dilution and Vessel on Livability of Frozen Semen (돈정액의 액상 및 동결보존에 관한 연구. III. 보존액이 액상정액의 정자생존성과 수태율에 미치는 영향과 희석방법과 용기가 동결정액의 생존성에 미치는 영향)

  • 임경순;정장용
    • Korean Journal of Animal Reproduction
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    • v.3 no.1
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    • pp.30-35
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    • 1979
  • A, B and C dilutors were used to make Ka (A plus B (1 : 1)) and Na (B plus C(1 : 1)) dilutors in this experiment. Three aliqots of semen were respectivly diluted 1 : 1 and 1 : 2 (semen: dilutor) with Ka, Na and C dilutors and stored at 5$^{\circ}C$ for 7 days in order to study their livability during storage. Fertility was checked for the diluted semen with Ka, Na and C dilutors. Whole semen and extended semen with Na dilutos with and without DMSO were cold shocked at various temperatures for 10 min. Effects of different 1st and 2nd dilution with A, B, C and Na dilutors and of vessels on freezability of spermatozoa were investigtigated. 1. Extended semen 1 : 2 with Na and C dilutors showed highest live sperm index during storage for 7 days at 5$^{\circ}C$. 2. The components of Na dilutor per 100$m\ell$ were skim milk 2.5g, trisaminomethane 0.54g, citric acid 0.265g, glucose 2.835g, fructose 1.5g, sodium lauryl sulfate, 0.08g, penicillin 0.06g, streptomycin 0.075g, and egg yolk 10$m\ell$. 3. Fertility of diluted semen was higher than that of whole semen. Ka dilutor showed higher fertility than Na and C dilutors, and there was no difference in the fertility between Na and C dilutors. 4. Na dilutor with DMSO showed slightly higher livability than Na dilutor without DMSO during storage for 7 days at 5$^{\circ}C$. 5. Cold shock at 1$0^{\circ}C$ for 10 min. decreased greatly the sperm livalility of whole semen but not of extended semen with Na dilutor. Addition of DMSO to Na dilutor has no effect in prevention of cold shock. 6. The extended semen with C. C dilutor (1st and 2nd dilution with C and C dilutor) showed higher post-thawing sperm livability than A.A and Na. B dilutors. Na. B dilution shwed higher post-thawing sperm livability than A.A dilution. There was no difference in the post-thawing livability between semen in 1$m\ell$ straw and 10$m\ell$ aluminium package.

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Effects of PZM Media on In Vitro Development of Porcine IVM/IVF Embryos (PZM 배양액이 돼지체외수정란의 배발달에 미치는 영향)

  • 한만희;천행수;김종화;박병권;서길웅;이규승
    • Reproductive and Developmental Biology
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    • v.28 no.2
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    • pp.113-117
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    • 2004
  • The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

Use of a Xanthine-Xanthine Oxidase System on in vitro Maturation and Fertilization in the Pig (돼지난자의 체외성숙과 수정에 있어서 Xanthine-Xanthine Oxidase System의 이용)

  • Sa, S. J;H. T. Cheong;Lee, S. Y.;Lee, J. H.;I. S. Ryu;B. K. Yang;Kim, C. I.;Park, C. K.
    • Korean Journal of Animal Reproduction
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    • v.27 no.1
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    • pp.77-85
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    • 2003
  • This study was undertaken to evaluate the effects of catalase using xanthine (X)-xanthine oxidase (XO) system on in vitro maturation and fertilization in the pig. When follicular oocytes were cultured with X or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obtained in culture with X-XO-catalase system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without that than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, but were not different between the medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with none (P<0.05), XO and X+XO. On the other hand, when sperm were treated with none, X, XO and X+XO, lipid peroxidation were produced with higher rates in medium without that than with catalase, and consequently the changes in sperm penetration and lipid peroxidation showed opposite patterns. Under the above all conditions, however, sperm-SH group were higher detected by catalase. When the activity of sperm binding to zona pellucida was evaluated through binding to salt-stored porcine oocytes, sperm binding to zona pellucida in control group were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO-catalase system may be caused stimulating in vitro maturation and fertilization in the pig.

Effects of Fetal Calf Serum and Gonadotropins Supplemented to the Medium on Maturation and Fertilization In Vitro of Porcine Follicular Oociytes (성선(性腺)자극호르몬과 우태아혈청(牛胎兒血淸)첨가가 돼지난포란(卵胞卵)의 체외성숙(體外成熟) 및 체외수정(體外受精)에 미치는 영향)

  • Kim, Kyu Hyon;Jung, Bum Sik;Park, Soo Bong;Park, Hang Kyun
    • Current Research on Agriculture and Life Sciences
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    • v.8
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    • pp.45-50
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    • 1990
  • This study was carried out ot investigate the effects of fetal calf serum (FCS) and gonadotropins supplemented to the medium on maturation and fertilization in vitro of porcine follcular oocytes. Ovaries were obtained from gilts at local slaughter-house. Oocyte-cumulus complexes were recovered by puncturing the ovarian follicles(3~5 mm in diameter). The complexes from individual ovaries were pooled in a $0.4m{\ell}$ droplet of medium covered with paraffin oil, then washed twice in fresh droplet and cultured for 36hrs in culture media according to experimental conditions. Boar epididymal spermatozoa were capacitated by preincubation for 4hrs in m-KRB medium and the preincubated spermatozoa were insemenated in the fertilization medium containing the cultured oocytes. The results obtained in this study are summarized as follows: 1. The maturation rates of oocytes cultured in m-KRB and m-KRB supplemented to 10% FCS were 82 and 37%, respectively. When PMSG, hCG. and PMSGt hcG($10Iu/m{\ell}$) were added to the media supplemented to 10% FCS, the maturation rates were 66, 58 and 68%, respectively. 2. Expansion of cumulus cells was not occured in m-KRB and m-KRB supplemented to 10% FCS. However, when PMSG, hCG and PMSG+hCG($10Iu/m{\ell}$) were added to m-KRB supplemented to 10% FCS, the expansion rates of cumulus cell layers were 92, 13 and 91%, respectively. 3. When oocytes were mltured in m-KRB, the rates of penetration and formation of male pronucle: were 93 and 7%, respectively. By adding FCS and gonadotropin to m-KRB, the penetration and formation of male pronuclei were 100 80%, respectively.

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Studies on In Vitro Fertilization and Development of In Vitro Matured Porcine Follicular Oocytes I. Effect of Various Media and Co-culture with Porcine Cumulus Cellsor Mouse Fetal Fibroblast Cells on In Vitro Development of In Vitro Fertilized Oocytes (체외성숙 돼지난포란의 체외수정과 배발달에 관한 연구 II. 각종 배양액, 돼지난구세포 및 생쥐태아간세포와의 공동배양이 체외수정 돼지 난포란의 체외발달에 미치는 영향)

  • 정형민;엄상준;승경록;이상준;이훈택;정길생
    • Korean Journal of Animal Reproduction
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    • v.17 no.2
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    • pp.113-120
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    • 1993
  • To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

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Association Study of Zygote Arrest 1 on Semen Kinematic Characteristics in Duroc Boars (두록 정자 운동학적 특성과 Zygote arrest 1 유전자 변이와의 연관성 분석)

  • Lee, Mi Jin;Ko, Jun Ho;Kim, Yong Min;Choi, Tae Jeong;Cho, Kyu Ho;Kim, Young Sin;Jin, Dong Il;Kim, Nam Hyung;Cho, Eun Seok
    • ANNALS OF ANIMAL RESOURCE SCIENCES
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    • v.29 no.4
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    • pp.150-157
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    • 2018
  • The Zygote arrest 1 (ZAR1) gene is known to affect early embryonic development in various vertebrates. In this study, we performed the association analysis to check whether there is any significant relationship between semen kinematic characteristics and the ZAR1 gene. To determine semen kinematic characteristics, we measured motility (MOT), straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF) of spermatozoa in boars. In order to detect single nucleotide polymorphisms (SNPs), we extracted genomic DNA from multiple Duroc boars, and then subsequently used them in sequencing reactions. As a result, three SNPs were detected in the intronic region of ZAR1 gene (g.2435T>C in intron 2, g.2605G>A and g.4633A>C in intron 3 ). SNPs g.2435T>C and g.2605G>A were significantly associated with MOT (p<0.01) and VSL (p<0.05), and g.4633A