• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.377-383
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• 2011

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into $LN_2$. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol ($72.5{\pm}5.00%$, $54.88{\pm}0.66%$ and $46.00{\pm}2.40%$; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol ($34.69{\pm}4.64%$ vs $46.00{\pm}2.40%$; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: $55.81{\pm}2.94$, $55.19{\pm}3.34$ vs $47.94{\pm}3.48%$; p<0.05 and membrane integrity: $44.94{\pm}3.51$, $46.06{\pm}2.25$ vs $40.38{\pm}1.03%$; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycerol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

• Journal of Animal Science and Technology
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• v.55 no.1
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• pp.41-49
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• 2013

In vitro and in situ incubation studies were conducted to determine effects of yeast culture supplements (Saccharomyces cerevisiae) on cellulolytic bacterial function and fiber digestion in rice straw. In vitro dry matter digestibility of rice straw gradually increased according to supplemental levels of yeast culture (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0%). Digestibility of rice straw started to increase apparently when yeast culture was added more than 0.6% level (p<0.05). Also, we reconfirmed that in vitro dry matter digestibility was significantly increased by 0.6% of yeast culture addition in 4% NaOH treated and non-treated rice straws (p<0.05). When in situ dry matter digestibility was tested in Korean native goats fed basal diet or experimental diet which contained 1.0% of yeast culture, the yeast culture feeding improved in situ dry matter digestibility in both 4% NaOH treated and non-treated rice straws (p<0.05). In case of real-time PCR monitoring cellulolytic bacterial function, the bacterial population attached on rice straw showed the increasing trends with higher level of yeast culture spraying on rice straw. F. succinogenes and R. flavefaciens were significantly increased in accordance to spraying levels of yeast culture (0.0, 0.1 and 0.3%) at both 12 and 24 hrs of in situ incubation (p<0.05). R. albus was significantly higher population in yeast culture spraying than non-soraying at 12 hrs of in situ incubation (p<0.05). These bacterial populations were showed the increasing trends with digestibility enhancement of rice straw according to the higher levels of yeast culture supplement. Overall, these results clearly suggest that the presence of yeast culture result in noticeable increase of rice straw digestion, which is modulated via good effect on cellulolytic bacterial attachment to fiber substrates.

• 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.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.1-8
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• 2017

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under $5^{\circ}C/2min$ or $37^{\circ}C/40sec$ with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in $5^{\circ}C$ than in $37^{\circ}C$ (P < 0.05). However, the activity of viable sperm thawed at $5^{\circ}C$ was significantly decreased before refreezing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1997.05a
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• pp.27-34
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• 1997

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours($38^{\circ}C$, 5% $CO_2$) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at $0^{\circ}C$, cooled from $0^{\circ}C$ to $-6^{\circ}C$ at $-1^{\circ}C$/minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in $30^{\circ}C$ water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at -0.3^{\circ}C$ vs. $-0.5^{\circ}C$/minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of $0.3^{\circ}C$/minute(64.6%), 31/48) than at $-0.5^{\circ}C$/minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.223-227
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• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.898-908
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• 1998

The aim of this experiments were to assess the time-interval change of motional characteristics in frozen-thawed semen of Korean native cattle (KNC) by using computer aided semen analysis (CASA) technology. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system (SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber (Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The measurement parameters in SIAS were as follows ; frame rate = 30 frames per sec, image capture = 1 sec, minimum motile speed = $10{\mu}m/s$, maximum countable sperm number = 400. Statistical analysis was done by Student t-test with use of the Sigma plot program on a IBM personal computer. The dancemean(DNM) and hyperactivated sperm(HYP) of frozen-thawed KNC semen kinematics were significantly decreased(p < 0.05) after 10 min of incubation at $37^{\circ}C$ water bath. But, wobble(WOB) of same sample semen was significantly increased(p < 0.05) after 10 min of incubation and significantly decrease(p < 0.05) after 60 min of same incubation. And, after 30 mim of incubation, significantly differences were found most of motion kinematics, motifity(MOT), curvilinear velocity(VCL), straight line velocity(VSL), average path velocity(VAP), amplitude of lateral head displacement(ALH), beat cross frequency(BCF), mean angular displacement(MAD), dance(DNC), on same sample semen. The DNM of KNC semen sample was variable kinematics after 30 min of incubation. Also, the linearity(LIN) and straightness(STR) was significantly decreased(p < 0.05) from 60 min of incubation. In conclusion, the AI within 30 min after thawing of frozen semen can be an effective method for obtaining high fertility rate in KNC reproductive program.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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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.

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.57-71
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• 1983

1. Diluted chicken semen can be preserved at 2 to 5$^{\circ}C$ for 24 to 48 hr with resultant fertility of greater than 90% of that of fresh semen. Turkey semen can be preserved at 10 to 15$^{\circ}C$ for 6 to 24 hr and provide economical fertility. 2. Frozen chicken semen has given variable results; a 21 to 93% fertility ranges as compared to 92 to 94% expected with fresh semen. Highest fertility levels obtained with frozen turkey semen intravaginally inseminated have been 61 and 63% using DMSO and glycerol, respectively, as cryoprotectants. 3. The use of glycerol as a cryoprotectant reauires that its concentration in semen be reduced to less than 2% either by dialysis or centrifugation after thawing and before intravaginal insemination if optimal fertility is to be obtained. 4. The temperature at which cryoprotectants are added to semen and the time allowed for equilibration are important for subsequent fertility pre- and post-freezing. 5. The type of container used for packaging the semen, freeze or cooling rates, thaw rates and level of cryoprotectant all interact in affecting cell survival. 6. Plastic freeze straws as a packaging device for semen offers the following advantages: easy to handle, require minimal storage space, offer a wide range of freeze and thaw rates, and insemination can be made directly from them upon thawing. 7. Controlled slow cooling rates of 1 to 8$^{\circ}C$/min have thus far provided the best results for cooling chicken semen throught the transition phase change (liquid to solid) or critical temperature range of ＋5 to -20 or -35$^{\circ}C$. 8. Highest fertilities have been achieved with frozen chicken semen where a slow thaw rate (2。 to 5$^{\circ}C$) has been used regardless of the freeze rate. 9. To maintain a constant high level of fertility throughout a breeding season with frozen semen, a higher absolute number of spermatozoa must be inseminated (2 to 3 times as many) as compared to fresh semen since a, pp.oximately 50% are destroyed during processing and freezing. 10. The quality of semen may vary with season and age of the male. Such changes in sperm quality could be accentuated by storage effects. Thus, the correct number of spermatozoa may very well vary during the course of a breeding period. 11. As to time of insemination, it is best to avoid inseminating chicken hens within 1-2 hr after or 3-5 hr before oviposition; and turkey hens during or 7-10 hr before oviposition. 12. The physiological receptiveness of the oviduct at the time of insemination is a very important biological factor influencing fertility levels throughout the breeding season.

• 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.