This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed. Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as 6～14×107cells/ml. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 cm above LN2 gas for 5 or 10 min. Survival rates of pre-frozen sperm were 65.0±13.2%, 68.3±10.4%, 66.7±11.5% and post-frozen were 53.3±23.1%, 45.0±15.0%, 50.0±18.0% in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were 36.7±10.4%, 40.0±7.1%, 30.0±13.2% at 3 cm－5 min and 33.3±11.5%, 31.7± 2.9%, 21.7±10.4% at 3 cm－10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were 43.3±15.3%, 32.0±17.9%, 22.3±15.7% at 5cm－5 min and were 47.5±15.0%, 43.3±12.6%, 48.3±15.3% at 5cm－10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3～7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above LN2 gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

This study was performed to investigate the characteristics within ages and freezing tolerance of spermatozoa in Jindo Dog. Experimental animals were selected 12 herds within 1～8 year’s old and collected semen for 2 times in a week. Collected semen was evaluated whole volume and sperm number with CASA system (SIAS, Medical Supply, Korea). Then seminal plasma were separated and diluted with modified Tris-egg yolk extender and added 4, 6 and 8% glycerol for 4 times to final concentration and equilibrated for 1.5 hrs. Before and after freezing, equilibrated semen were evaluated the survival rates. Total volume of sperm at 1～2 year old group is as 5.2×108 cells/ ml largest and there were no significance among groups. The motility of 1～2 year old group is highest as 90.9% and there were significance among groups. Abnormal sperm showed similar among groups. The survival rate in terms of pre-freezing and post-freezing were decreased all levels of glycerol and reveled 87.0% to 64.5% in 4%, 87.5% to 51.9% in 6% and 73.4% to 29.7% in 8%, there were significant difference among the groups (p<0.05). These results suggest that the optimal sperm-freezing methods in Jindo Dog are utilized with modified Tris egg-yolk extender with 4% glycerol and were improve the reproductive activity by these methods.

The objective of this study was investigate the superovulation treatment and to relate concentrations of blood urea nitrogen(BUN) in Hanwoo donors. Thirty six, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg PGF2α was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100 μg GnRH at the time of 1st insemination. Embryos were recovered 7 or 8 days after the 1st insemination. Cows with BUN ＜10, 11～18 and ≥19 mg/dl had return of estrus of 34.6, 30.5 and 30.4 days respectively. Return of estrus after superovulation treatment was not significantly lower for cows with blood urea nitrogen (BUN) above 10 mg/dl than for cows with BUN below 10 mg/dl. Cows with BUN ＜10, 11～18 and ≥19 mg/dl had number of transferable embryos of 3.2±1.2, 5.4±1.9 and 4.1±2.1 respectively.

This studies were conducted to investigate the survival rate of frozen-thawed spermatozoa of Jindo Dog by monosaccharide and freezing rates. Experimental animals were prepared 12 males within 1~8 year's old and collected once in a couple of weeks by digital manuplation methods. Collected semen was diluted 1:1 with Tris-egg yolk extender and added 4, 6 or 8% of glycerol and none, 4 mM glucose or 4 mM fructose as cryoprotectant and was equilibrated for 2 hrs in . In monosaccharide groups, the freezing rate was 5 cm-5 min. above . The survival rates without monosaccharide were , , in 4, 6 or 8% glycerol, respectively. In addition of glucose, the survival rates were , , in 4, 6 or 8% glycerol, respectively and in fructose, were , , in 4, 6 or 8% glycerol, respectively. There showed significantly different between glycerol groups and monosaccharides groups (p<0.05). The survival rates of freezing rate in 5 cm-5 min. group was , , and in 10 cm-10 min. group was , , in 4, 6 or 8% glycerol, respectively. There were significantly different between freezing rates (p<0.05). These results suggest that the addition of fructose with 6%-glycerol and slow freezing improve the survival of frozen-thawed sperm in Jindo Dog.

We investigated the cleavage rate and blastocyst yield for each culture condition to enhance tolerance of cryo-preservation of bovine IVF embryo with relatively lower cryo-tolerance compared to in vivo embryo. The cleavage rate and blastocysts yield for CR1aa, IVMD, IVD, CR1aa+10% FBS were 73.2, 69.3, 72.8, 68.5% and 44.1, 30.8, 33.3, 48.0%, respectively. The values did not differ among each treatments without serum. For embryo vitrification, In vivo and In vitro blastocysts were exposed to VS1(10% glycerin, 0.1 M glucose, 0.1 M sucrose, PEG 1%) for 5 min, and VS2 (10% glycerin, 10% EG, 0.2 M glucose, 0.2 M sucrose, PEG 2%) for 5 min and then VS3 (10% glycerin, 30% EG, 0.3 M glucose, 0.3 M sucrose, PEG 3%) for 1 min. The exposed embryos were then loaded into the 0.25 ml plastic straws and then plunged into liquid nitrogen. The straws were held for period of 1 to 2 weeks before thawing. In embryo viability, no differences in blastocyst re-expansion rates were found between in vivo and in vitro embryos. whereas expansion-BL rates was significantly higher for in vivo-derived embryos (72.7%) when compared to in vitro-derived embryos (51.4%), respectively (P<0.05). In conclusion, our results indicate that combined use of CRIaa culture medium with vitrification might enhance tolerance of cryopreservation for bovine IVF embryo production.

The objective of this study was to investigate the effects of abnormal ovarian cycles after superovulation treatment of Hanwoo donors. Thirty six, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received GnRH at the time of Ind insemination. Embryos were recovered 7 or 8 days after the 1st insemination. The cows were considered to have resumed ovarian cyclicity on the day of ovulation if followed by regular ovarian cycles. 50.0 percentage of the cows (18/36) had normal resumption of ovarian cyclicity (resumption within 40 days after superovulation), and 50.0% (18/36) had delayed resumption(resumption did not occur until>40 days after superovulation). Delayed resumption Type II (first ovulation did not occur until 40 days after superovulation, i.e. delayed first ovulation 33.3%) were the most common types of delayed resumptions. The mean numbers of total ova from < 10 and 10 of corpora lutea (CL) was 7.3 and 13.9, respectively. The number of transferable embryos differed between < 10 and 10 CL was 4.2 and 5.1, respectively. 11.1 percentage of the cows (4/36) did not resumption their ovarian cyclicity until 60 days after superovulation treatment.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was performed to investigate whether oxygen consumption reflects morphological grade of in vivo derived bovine blastocyst-stage embryos (blastocyst). The oxygen consumption of in vitro produced blastocyst was compared to its total cell number. In addition, pregnant rate was measured after transplantation of in vivo blastocysts with different oxygen consumption. The quality of blastocyst collected on day 7 after artificial insemination was categorized as grade I and II (G I and G II) based on microscopic observation of the morphology. Oxygen consumption of blastocyst was measured using a scanning electrochemical microscopy (SECM) and total cell number of in vitro blastocyst was enumerated by counting cells stained by propidium iodide. Pregnancy of recipient cow was confirmed with rectal palpation after 60 days of embryo transfer. The oxygen consumptions of G I blastocysts were significantly higher than those of G II blastocysts ( versus , p<0.05). Total cell numbers of in vitro blastocysts were 74.8, 90.7, and 110.2 in the oxygen consumption of below 10.0, 10.0~12.0, and over respectively. Total cell number was significantly increased in embryos with high oxygen consumption (p<0.05). Pregnant rate in recipient cow was 0, 50, and 85.7% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0, and over , respectively. These results suggest that measurement of oxygen consumption may help increase the pregnant rate of bovine embryos.

In vivo embryo produced from Hanwoo donor cows were collected and transferred to Hanwoo recipients. Cows, at random stages of the estrous cycle, received Progesterone Releasing Intravaginal Device (CIDR-plus, InterAg, New Zealand) together with injection of 1 mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment began 4 day later. For superovulation, a total of 28 mg FSH was intramuscularly injected twice a day in the way of decreasing doses 4 day (5, 5, 4, 4, 3, 3, 2 and 2 mg). Twenty one Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered 7 days after the second insemination by flushing the uterus with Embryo Collection Medium. The results obtained were as follows: The rates of transferable embryos were 50.3%, and 78 fresh embryos at morulae and blastocysts stage were transferred into Hanwoo recipients on day 7 of estrus cycle. The pregnancy rates were first embryo transfer 55.6%, 2nd 62.9% and 3rd 57.9%, respectively. In conclusion, These results suggest that CIDR-based superovulation protocol may be effectively used for production of superior Hanwoo embryos. Also, since it seems the condition of recipient cows greatly affect pregnancy rate, it is very important to evaluate recipient for effective cattle production.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption of embryos. Here, we measured oxygen consumption of bovine embryos at various developmental stages was measured using a scanning electrochemical microscopy (SECM). We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell-stage to morula-stage), indicating that oxygen consumption reflects the cell number ( versus , p<0.05). In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos ( versus , p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro-derived bovine blastocyst-stage embryos (p>0.05). In the frozen-thawed blastocyst-stage embryos, live embryos showed significantly higher oxygen consumption than dead embryos ( versus , p<0.05). These results indicate that the measuring oxygen consumption by SECM can be used to evaluate bovine embryo quality.

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of by doubling in every 10 minutes at cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to . The presented straws were examined the viability and motility after thawed at water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen (). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above ( and 70.7% vs, 33.18% and vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 porcine FSH, 0.5 equine LH, 1.0 17 -estradiol () and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at in humidified atmosphere of 5% in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

It is not easy for porcine embryos produced by in vitro systems to develop into blastocysts with high quality. To solve this problem, many researchers have developed novel culture methods. However, the formation of blastocysts with high quality is still low. In this study, we aimed to produce piglet following transfer of in vitro produced early embryos ( cell stage embryos) or morula and blastocyst. The cell stage embryos were transferred to five estrus-synchronized recipients (200 embryos per recipient). One of the five sows farrowed three piglets, which contain two live piglets and one dead piglet, 114 days after embryo transfer. However, two recipients transferred with morula and blastocysts did not farrow. Microsatellite analysis confirmed that the genomic DNA of two live piglets were not genetically identical to that of the recipient. These results indicate that it is possible to obtain piglets by transfer of early embryos produced by in vitro production (IVP) systems.