• Korean Journal of Animal Reproduction
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• v.8 no.1
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• pp.22-28
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• 1984

This experiment was carried out to investigate effects of DMSO or ethylene glycol as a cryopotectant and of freezing methods on survival rate of forozen-thawed rat 2-cell embryos by morphological observation. 2-cell embryos were recovered from oviducts of Sprague Dawley females mated with males of same strain on day 2 of pregnancy after inducing superovulation by intrapertioneal injection of PMSG and HCG. In slow freezing and thawing groups, embryos were frozen to -79$^{\circ}C$ or -196$^{\circ}C$ in a glass test tube containing 0.2ml PBI with 1.5M DMSO or 1.2M ethylene glycol at a rate of 0.3-1.0C/min. and thawed slowly. When samples were frozen to -79$^{\circ}C$, higher survival rate was obtained in the medium containing DMSO (43.9%) than ethylene glycol (41%). And similar result was obtained (32.5% in DMSO vs. 31.4% in ethylene glycol) when samples were frozen. In rapid freezing and thawing groups, embryos were frozen to -79 or -196$^{\circ}C$ in a glass test tube containing 0.2ml of PBI with 1.5M DMSO or 1.2M ethylene glycol by rapid cooling, and thawed rapidly. When samples were frozen to -79$^{\circ}C$, 1.5M DMSO (13.2%) was more effective than 1.2M ethylene glycol (6.1%). When the storage temperature was -196$^{\circ}C$, survival rates were 9.8% in 1.5M and 5.4% in 1.2M ethylene glycol.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.91-100
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• 2002

The experiment was divided into two phases. In phase-I fresh embryos were transferred and in Phase-II frozen embryos were transferred. Embryos were collected by using Dulbecco's phosphate buffered saline. In phase-I total of 65 ova were collected out of 107 ovulation in 18 goats. Recovery of ova was 60.74％, of which 51 (78.46％) was fertilized. Sixteen embryos were transferred to 10 recipient goats and kidding was observed in 6 goats, that produced 10 kids. Thus, 62.50％ embryo survival and 60％ kidding were achieved in phase-I. In phase-II of the experiment, 17 regular cyclic Black Bengal goats were used. The main purpose was to study the viability of caprine embryos after cryopreservation. In this phase the embryos were collected and frozen using Bio-cool freezers. A two step addition of cryoprotectants (5％ glycerol and 10％ glycerol) and three-step dilution of cryoprotectants with 1mole (M) sucrose was used. Embryos were preserved for 10 to 45 days. Out of 27 embryos preserved, 18 were recovered after freezing and thawing (37$^{\circ}C$ water bath) with 33.33％ embryonic loss. Seventeen frozen and thawed embryos were transferred in 9 recipient goats, out of which kidding was observed in 6 goats and 7 kids were produced, giving a 66.66％ kidding and embryo survival of 41.17％. The technique utilized for fresh and frozen embryo transfer can be successfully utilized to produce goats of superior genetic merits. The protocol used for addition of cryoprotectant, freezing, thawing and dilution was found suitable for caprine embryo freezing.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.29-35
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• 2012

The purpose of this study was to examine the effect of growing stages of the Korean Native Striped Bull (KNSB) on the freezability and fertility of frozen-thawed semen. First, we investigated the total motility (TM) and progressive motility (PM) according to the diluent used for semen freezing. Second, we examined the effect of the age of KNSB on semen volume, TM and PM of fresh and frozen-thawed semen. Third, we examined the effect of frozen semen from the different age of KNSB on the $in-vitro$ fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl$^{(R)}$ and Tris-egg yolk extender (EYE). Semen was collected from 5 KNSB in the growing stage (15 months) and 5 adult KNSB (36 months). When Triladyl or Tris-EYE extender was used for semen freezing, there was no difference of the mean TM and the mean PM. However, the mean TM was significantly higher in Bull No. 1885 than Bull No. 4283 ($p$ <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ($p$ <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ($2.1{\times}10^9$ spermatozoa/ml) and 36-month-old ($1.8{\times}10^9$ spermatozoa/ml) bulls. For the 15-month-old and 36-month-old bulls, the mean TM of fresh semen were 93.7% and 88.3%, respectively, and the mean PM were 97.0% and 88.3%, respectively; the 15-month-old bulls showed a particularly high PM ($p$ <0.05). For the 15-month-old and 36-month-old bulls, the mean TM (56.0% and 58.0%, respectively) and the mean PM (64.0% and 70.7%, respectively) of frozen-thawed semen did not differ. The development rates of embryos after $in-vitro$ fertilization and the pregnancy rate after artificial insemination using frozen-thawed semen did not differ according to the bull's age. In summary, semen volume differed according to the bull's age, but semen concentration and survival rate, the $in-vitro$ fertilization rate, and the pregnancy rate did not differ according to the stripe bull's age. Accordingly, semen from bulls in the growing stage can be collected and frozen for the preservation and multiplication of rare livestock.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.291-295
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• 2010

Four estrus-induced Himalayan tahrs (Hemitragus jemlahicus) were inseminated with frozen-thawed semen by laparoscopic or transcervical insemination techniques with no regard to the site of ovulation in non-breeding season. In June and July, 2009, estrus was synchronized by Eazi-Breed $CIDR^{(R)}$ (Controlled internal drug release; Pfizer Animal Health, New Zealand) insertion for 16 days and PG 600 (PMSG 400IU, hCG 200 IU; Intervet, Netherlands) injection (IM) a day before removing $CIDR^{(R)}$. Forty eight hours later, laparoscopic or transcervical insemination was done to each of two tahrs under anesthetic condition inducted by ketamine (1.5 mg/kg) and medetomidine (0.09 mg/kg). For examination of estradiol and progesterone, blood was collected right before $CIDR^{(R)}$ insertion, PG 600 injection, $CIDR^{(R)}$ removal and insemination. Estradiol levels of four tahrs (No. 1, 2, 3, 4) before $CIDR^{(R)}$ insertion and insemination were 13.3, 8.8, 14.3, 12 pg/ml and 23.5, 25.5, 21.1, 11.5 pg/ml, respectively. Progesterone levels of four tahrs (No. 1, 2, 3, 4) before $CIDR^{(R)}$ insertion and insemination were 1.8, 0.05, 0.63, 0.61 ng/ml and 1.03, 0.37, 1.48, 2.12 ng/ml. Except for No. 4 tahr, cervices showed cervical mucus and opened enough to penetrate with embryo transfer gun sheet usually used for cows. Therefore, No.4 was laparoscopically inseminated together with No. 1. In conclusion, none of four Himalayan tahrs was pregnant. However, we proved that estrus could be induced by CIDR and PG 600 injection in non-breeding season, and laparoscopic or transcervical insemination with frozen-thawed semen could be one of assisted reproductive techniques in Himalayan Tahr.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.167-172
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• 1990

This study was carried out to clarify the effects of various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. Mouse embryos were collected by hyperstimulation induction of ICR mouse. The samples were slowly cooled ($l^{\circ}C/min$) to temperatures between $-7^{\circ}C$ and $-30^{circ}C$ before direct transfer to liquid nitrogen ($-196^{\circ}C$) and thawed rapidly ($-500^{\circ}C$/min). As cryoprotectants, Glycerol, DMSO, Ethylene glycol and Propylene glycol were used and applied each 2 cell, 8 cell, morula in embryo stage. After normal mouse embryos developed to blastocyst by in vitro culture, we observed recovery rate and developing rate of embryos at thawing. The results obtained in these experiments were as follows : 1. The in vitro development rate from the frozen-thawed 2 cell embryos to the blastocyst were 67.7% in ethylene glycol, 65.7% in Propylene glycol, 55.2% in glycerol and 50.0% in DMSO respectively. 2. The in vitro development rate from the frozen-thawed 8 cell embryos to the blastocyst were 83.6% in DMSO, 75.7% in glycerol, 52.2% in propylene glycol respectively. 3. The in vitro development rate from the frozen-thawed morula to the blastocyst were 84.2% in glycerol, 80.0% in DMSO, 66.6% in propylene glycol and 55.2% in ethylene glycol respectively.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.175-181
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• 1986

These experiments were carried out to clarify the effects various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. As cryoprotectant, glycerol, DMSO and methanol were used and the procedures of adding them in medium were practiced by one-step or six-step adding method. Morphologically normal mouse embryos developed to blastocyst by in vitro culture after freezing and thawing were transferred to pseudopregnant recipients by surgical procedures. The results obtained in these experiments were summarized as follows: 1. The survival rates of the frozen-thawed 8-cell embryos, morulas and blastocysts following one-step addition of glycerol were 83.6, 80.3 adn 70.3%, respectively, while following six-step addition of glycerol, 69.2, 56.3 and 66.7% respectively. 2. When glycerol, DMSO and methanol were used as cryoprotectant under the same condition of freezing and thawing, the survival rates of frozen-thawed embryos were 74.0, 76.1 and 37.6%, respectively. 3. The implantation rate of embryos transferred to pseudopregnant recipients after freezing and thawing was 49.2%.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.151-159
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• 1997

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.5
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• pp.612-616
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• 2004

This study was carried out to compare the semen characteristics, frozen-thawed sperm viability and testosterone concentration and in vitro fertilization (IVF) and development of in vitro matured pig oocytes between two Yorkshire boars. Semen and blood samples were collected once per week from October to November 2002 from two adult Yorkshire boars at 18 months of age with 170 kg body weight. Sperm were deep frozen in 5 ml maxi-straws with lactose-egg yolk and N-acetyl-D-glucosamine (LEN) diluent and stored in liquid nitrogen. Blood samples were obtained at 10 a.m. by inserting a 21 gauge, hypodermic needle attached to 10 ml syringe into surface veins in the ear. The concentration of testosterone was determined by Competitive Enzyme Immunoassay. Ovaries were collected from prepubertal gilts at a local slaughter house. Cumulus oocyte complexes were aspirated from antral follicles (3 to 6 mm in diameter). The medium used for oocyte maturation was modified TCM 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at $38.5^{\circ}C$, 5% $CO_2$ in air. For IVF, one frozen 5 ml straw was thawed at $52^{\circ}C$in 40 sec and was diluted with 20 ml Beltsville thawing solution at room temperature. Sperm were washed 2 times in mTLP-PVA and inseminated without preincubation after thawing. Oocytes were inseminated with $2{\times}10^7$/ml sperm concentration. Oocytes were coincubated for 6 h in 500 ${\mu}$l mTBM fertilization medium. At 6 h after IVF, oocytes were transferred into 500 ${\mu}$l NCSU-23 culture medium for further culture of 48 and 144 h. There were no significant differences in the semen volume, motility, normal acrosome morphology and sperm concentration of raw semen between A and B of Yorkshire boar. However, motility and normal acrosome of boar A were higher than those of boar B at 0.5, 2, 3, 4, 5 and 6 h incubations of frozen-thawed sperm. Testosterone concentration (3.75 ng/ml) of boar A was higher than that (2.34 ng/ml) of boar B. The rate of blastocyst formation (15.1%) of boar A was higher than that (10.4%) of boar B. In conclusion, serum testosterone concentration of boar showed very important role for the frozen-thawed sperm viability and the blastocyst formation of pig oocytes matured in vitro.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.203-208
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• 1995

This study was carried out to efficiently use the ultrarapid freezing method in the cryopreservation of mouse ova. For this, the effects of dehydration method, oval vigour and $0^{\circ}C$ controlling method on post-thawing viability were investigated. Fresh mouse ova were dehydrated in mPBS with 3.5M DMSO and /or 0.25M sucrose, and directly immersed in L$N_2$ for ultrarapidly freezing. The frozen ova were thawed at 37$^{\circ}C$, rehydrated in mPBS with 0.25M sucrose, and then repeatedly washed in HAM's Fl0 before evaluating the morphological normality of frozen-thawed ova. The results obtained showed that there was difference between treatments in a experiment. 1) The post-thawing viability of ova dehydrated in multi-step (48.4$\pm$13.8%) was higher than that of ova in two-step (40.9$\pm$14.0%). 2) The post-thawing viability of fertilized ova (87$\pm$14.0%) was significantly(p<0.0l) higher than that of unfertilized ova (5.4$\pm$5.4%). 3) The post-thawing viability of ova dehydrated and rehydrated using a cooling machine (95.8$\pm$4.2%) was significantly(p<0.05) higher than that on ice(84.1$\pm$9.9). In conclusion, in order to efficiently cryopreserve ova in vitro with ultrarapidly freezing method, highly viable embryos should be selected, heavy osmotic shock to the dehydrating ova should be avoided, and embryos in high osmotic condition were dehydrated and rehydrated in a constantly low temperature.