• Title/Summary/Keyword: Semen Extender

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Effect of bicarbonate and progesterone on plasma membrane integrity, acrosome reaction and proportion of fatty acids in boar sperm

  • Park, Choon-Keun;Lee, Sang-Hee
    • Journal of Animal Reproduction and Biotechnology
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    • v.37 no.3
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    • pp.202-208
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    • 2022
  • This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

Studies on Frozen Semen Characteristics Following Pentoxifylline Treatment and Artificial Insemination in Dog (개에서 Pentoxifylline 첨가에 따른 동결정액 성상과 인공수정에 관한 연구)

  • Ji, D.Y.;Kim, C.K.;Lee, J.H.;Park, S.J.;Ryu, L.S.;Ryu, J.W.;Lee, J.H.;Jeong, Y.C.;Pang, M.G.
    • Journal of Animal Science and Technology
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    • v.47 no.6
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    • pp.925-936
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    • 2005
  • The present study evaluated whether pentoxifylline added to the freezing extender could improve the sperm characteristics and function in canine frozen semen. Also the conception rate following AI with frozen-thawed semen was investigated. The beneficial effects of pentoxifylline supplementation were visible in motility, viability, acrosome reaction, and tail swelling patterns. Especially, highest sperm viability and function were obtained in the forzen semen supplemented with 1mM pentoxifylline. The follicle size measured by ultrasonography was 6.48 mm, 11.52 mm and 8.9 mm on 11, 13 and 15 days after the onset of natural estrus, respectively and ovulation occurred on 13 and 15 days. The pregnancy rates in bitches inseminated with frozen semen on natural and induced estrus were 71.4% and 75.0%, respectively. There was no significant difference between the pregnancy rates in bitches inseminated with frozen semen following natural and induced estrus, but the litter size was slightly increased in natural cycle.

Changes of Frozen-Thawed Semen Characteristics in Miniature Pig and Duroc (Miniature Pig와 Duroc 종간의 동결-융해 후 정액 성상 비교)

  • Lee, Y.S.;Choi, W.C.;Lee, S.H.;Cheong, H.T.;Lee, S.Y.;Yang, B.K.;Park, C.K.
    • Journal of Embryo Transfer
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    • v.21 no.3
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    • pp.263-271
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    • 2006
  • The purpose of this study was undertaken to compare ability of frozen-thawed sperm characteristics between two strains (miniature pig and Duroc). The semen was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle. The semen was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. The frozen-semen straw were thawed at 20, 37 and $50^{\circ}C$ for 1 min, 45 sec and 10 sec within water-bath. The semen sample were evaluated at 0, 3, 6, 9, and 12 h after incubation at $37^{\circ}C$ for analysis of sperm ability. Abnormality of spermatozoa in miniature pig was significantly (p<0.05) higher than that in Duroc at 0, 9 and 12 h of post-thawing incubation after frozen-thawing. The percentage of F-patterned spermatozoa in miniature pig was significantly (p<0.05) lower, while the percentage of AR (acrosome reacted spermatozoa) pattern was higher in the miniature than in the Duroc. On the other hand, there was no significant difference in the viability of spermatozoa thawed at different temperature ($20^{\circ}C\;and\;37^{\circ}C$) between two species, but the viability in miniature pig was higher (p<0.05) than in Duroc when sperm was thawed at $50^{\circ}C$. In conclusion, this study suggest that suitable freezing method for miniature pig semen is required for increasing post-thawing viability and fertilization capacity.

Effects of ice-binding protein from Leucosporidium on the cryopreservation of boar sperm

  • Park, Sang Hyoun;Oh, Keon Bong;Ock, Sun-A;Byun, Sung June;Lee, Hwi-Cheul;Kumar, Suresh;Lee, Sung Gu;Woo, Jae-Seok
    • Journal of Embryo Transfer
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    • v.33 no.3
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    • pp.185-194
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    • 2018
  • The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted ($1.5{\times}10^8/ml$) in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h. The cooled semen was then diluted ($1{\times}10^8/ml$) in LeIBP containing LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen ($LN_2$) vapors for 20 minutes and then plunged into $LN_2$. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

Effects of Taurine and $\alpha$-Tocopherol Treatment during freezing on Sperm Characteristics and Function in Frozen-Thawed Porcine Semen (돼지 정액의 동결시 Taurine과 $\alpha$-Tocopherol 첨가가 동결$\cdot$융해 정자의 성상과 기능에 미치는 영향)

  • Shin H. A.;Kim C. K.;Chung Y. C.;Pang M. G.
    • Reproductive and Developmental Biology
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    • v.29 no.3
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    • pp.155-162
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    • 2005
  • The present study evaluated whether an exogenous antioxidants, taurine and $\alpha$-tocopherol, could, when added to the freezing extender, improve the post-thaw sperm characteristics, function, the level of reactive oxygen species (ROS) generation, and the level of lipid peroxidation (LPO) in frozen-thawed porcine semen. CASA (computer-aided sperm analysis), HOST (hypoos-motic swelling test), chemiluminescence using luminol and lucigenin and the detection of malondialdehyde for LPO was performed in frozen-thawed porcine sper-matozoa. The results obtained in these studies are as follows. While no beneficial effects of taurine and $\alpha$-tocopherol supplementation were visible in motility, viability, acrosome reaction, tail swelling patterns, and the generation of $O^{2-}$ of frozen-thawed porcine sper-matozoa, $H_{2}O_{2}$ was decreased by all treatments except taurine 50mM treatment. In conclusion the taurine and $\alpha$-tocopherol treatments during freezing reduced generation of reactive oxygen species and production of malondialdehyde in frozen-thawed porcine semen, and the ROS savangers may minimize various damages of spermatozoa during freezing.

Analysis of Frozen-Thawed Sperm Characteristic in Alpha 1,3-Galactosyltransferase(GalT) Knock-out Cloned Miniature Pig (Alpha 1,3-Galactosyltransferase (GalT) Knock-out 복제 미니돼지 정액의 동결-융해 후 정액 성상 분석)

  • Woo, Jea-Seok;Lee, Yong-Seung;Yoo, Han-Jun;Hwang, Seong-Soo;Oh, Keon-Bong;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Soo-Bong;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.35 no.3
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    • pp.385-390
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    • 2011
  • The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

Effect of Glycerol and Ethylene Glycol on Post-Thawed Sperm Function in Jeju Horse (제주마의 동결정액 제조에 있어 Glycerol과 EthyleneGlycol이 동결 융해 후 정자의 기능에 미치는 영향)

  • Oh, Shin-Ae;Ko, Min-Hee;Ko, Moon-Suck;Lee, Chong-Eon;Park, Yong-Sang;Kang, Tae-Young;Ko, Jae-Hyoung;Cho, Won-Mo
    • Reproductive and Developmental Biology
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    • v.36 no.3
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    • pp.199-206
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    • 2012
  • Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol ($39.85%{\pm}11.41$) than in 5% glycerol treatment ($18.08%{\pm}1.61$). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol ($34.12%{\pm}11.02$) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

Effect of Sugar Combination in Tris-buffer on the Viability of Post-thaw Spermatozoa in Canine

  • Yu, D.J.;Jeong, S.R.;Oh, I.S.;Bae, I.H.;Cho, S.G.;Kong, I.K.
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.90-90
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    • 2002
  • The purpose of this study was to investigate the effect of kind and combination of sugars on the viability and acrosome damage of post-thaw spermatozoa in canine. The extender used was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as single (fructose, xylose, trehalose), two combinations (Fruc+Tre, Fruc+xyl, Tre+xyl) and three combinations (Fruc+Tre+Xyl). The concentration of sperm collected were adjusted of 50${\times}$10$\^$6/ per straw for freezing. The frozen spermatozoa were thawed at 37$^{\circ}C$ for 1 min and then analysis for CASA program in Livestock Improvement Main Center, NACF. The motility of post-thaw spermatozoa in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (79% vs. 63, 66, 70, 71, 74 and 75%). The progressive motility after CASA analysis in Fuc+Tre group was also higher than those in fructose, trehalose, xylose, Fru+xyl, Tre+xyl and Fru+Tre+Xyl (67% vs. 53, 57, 60, 61, 62 and 64%). The acrosome damage of post-thaw spermatozoa stained was not significantly different among treatment groups such as fructose, trehalose, xylose, Fru+Tre, Fru+xyl, Tre+xyl and Freu+tre+xyl (17.7, 18.3, 28.0, 17.0, 19.7, 20.0 and 19.0%). The results indicated that the motility and progressive motility of post-thaw spermatozoa in Fru+Tre group was better, and acrosome normality was not different among all groups. The use of Tris-buffer supplemented with Fru+Tre as sugar for frezing of canine spermatosoa could be better and apply to semen banking and artificial insemination.

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Effect of Sheath Fluid with HEPES on Viability of Sex-sorted Sperm in Hanwoo (Korean Native Cattle) (한우 정자와 성 분리 시 HEPES를 첨가한 Sheath Fluid가 생존율에 미치는 영향)

  • Lee, Ji-Eun;Lee, Kyung-Jin;Yoo, Han-Jun;Park, Joung-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.26 no.3
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    • pp.181-186
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    • 2011
  • Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

Effects of α-Linolenic Acid in Frozen-thawed Boar Spermatozoa (돼지 정자의 동결보존 시 α-Linolenic Acid의 효과)

  • Lee, Won-Hee;Hwangbo, Yong;Lee, Sang-Hee;Yang, Jin-Woo;Kim, Hwa-Young;Lee, Yu-Rim;Park, Ji-Eun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.40 no.3
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    • pp.27-31
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    • 2016
  • The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.