The objective of this study was to evaluate several types of uterine bacteria in Hanwoo. uterine bacteria from randomly selected 5 uterus was collected by flushing methods into a sterilized 1.5 ml centrifuge tube and was inoculated onto MacConkey agar and blood agar, respectively. After being incubated for 5% CO2, aerobic or anaerobic condition at 37℃ during 48h, bacterial colonies were selected and re-inoculated onto blood agar plates. Re-cultured colonies were identified by Gram staining and finally identified using Vitek system. The identified bacteria were Staphylococcus lentus, Staphylococcus sciuri, Staphylococcus vitulinus, Staphylococcus warneri of Gram (+) and Rhizobium radiobacter, Sphingomonas paucimobilis of Gram () bacteria. Although, pathogenicity of identified bacteria was unclear, the bacteria can have an effect on the uterine microenvironment. Therefore, repetitive research will be required to determine the effects of bacteria in cattle exposed to a various environment.

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability (84±1.15 %, p<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability (79±3 %, p<0.05) than those of the X and Y sperm (44.7±1.67 and 41.7±1.2 %) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities (24.3±1.2 and 25.7±0.9 %, p<0.05) compared to that of the sorted Y sperm (13.7±1.2 %) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher (55.0±1.7 and 45.0±1.5 %) than those of the sorted Y-sperm (32.3±0.9 %, p<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 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 (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter 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.

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen ejaculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution Ⅰ: 11% lactose+egg yolk; solution Ⅱ: solutionⅠ+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until 5℃ for 1 hrs, holding at —102℃ for 10 min; B: until 5℃ for 2 hrs, holding at —102℃ for 10 min; C: until 5℃ for 3 hrs, holding at —80 and —102℃ for 10 min). Semen cooled until 5℃ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1～2 hrs, 1～3% glycerol concentrations and glycerol equilibrium time of 0～10 min.

The objective of this study was to evaluate efficiency of methyl-beta-cyclodextrin (MBCD) in the sperm preservation of bull. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 1, 5, 10 and 20 mM MBCD before freezing process. Analysis of viability in frozen-thawed sperm was estimated by SYBR14/PI double stain, hypoosmotic swelling test(HOST) and acrosome damage with FITC-PNA, and mitochondria activation with Rhodamin123 by flow-cytometry. The sperm viability was significantly higher in 0 mM and 5 mM concentrations of MBCD than other groups (p<0.05). However, the HOST was significantly lower at 20 mM concentration of MBCD than other concentrations (p<0.05). In addition, acrosome damage and mitochondria activation rates were significantly lower at 20 mM concentration of MBCD than other groups (p<0.05). In conclusion, the viability of sperm was not significantly different among concentrations of MBCD 0, 5 and 10 mM, but MBCD 20 mM was significantly lower than other groups. In addition, as concentrations of MBCD was high, HOST, acrosome damage and mitochondria activation rates had a negative effect in bull sperm.

This study was performed to investigate the FSH levels for superovulation procedure in Korean Native Cattle (Hanwoo). The effectiveness of 200 mg and 400 mg of FSH to initiate superovulation was examined in Hanwoo. Donors, at random stages of the estrous cycle, received a CIDR. 7 days later, 200 mg FSH group was treated with 40, 30, 20, 10 mg FSH levels in declining doses twice daily by intramuscular injection for 4 days. Also, 400 mg FSH group was treated with 80, 60, 40, 20 mg FSH levels. On the 3rd day administration of FSH, 25 mg PGF2α was administered and CIDR was withdrawn. Donors were artificially inseminated twice at 12 hr intervals. The donor cattle received 250 μg GnRH at time of 1st insemination and embryos were recovered 8 days after the 1st insemination. As a results, average number of CL treated with FSH 200 mg was higher as 20.9±1.20 than 15.8±0.63 for donors treated with FSH 400 mg, respectively(p<0.05). Treated group of 200 mg FSH level increased (p<0.05) the number of embryos recovered per procedure compared to 400 mg FSH level (18.2±1.18 vs. 12.38±0.52, respectively). When treatment of 200 mg FSH was performed, average transferable embryos/ova increased (p<0.05) to 14.1±1.12 from 6.8±0.33 of treated of 400 mg FSH. Group of 200 mg FSH increased (p<0.05) to 8.3±0.76 from 2.0±0.26 in morula stage compare to 400 mg FSH group. Mean of total early blastocyst and expanded blastocyst stage embryos was similar (p<0.05) between the 200 mg and 400 mg FSH levels group (4.7±1.19 vs. 2.9±0.18 and 1.2±0.40 vs. 1.9±0.17). These results suggest that 200 mg FSH level-based superovulation protocol with CIDR may be effectively used for production of superior embryos in Hanwoo. In other words, the less level of FSH may be effectively applied for Hanwoo (Korean Native Cattle), because Hanwoo was smaller body size than beef or daily cow.

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.

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution Ⅰ: 11% Lactose or Triladyl + egg yolk; solution Ⅱ: solutionⅠ + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CTC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p< 0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed (37℃) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher(83.6±2.0 vs 59.0±4.4%) than un- separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form un-separaed sperm was significantly(p<0.05) higher(13.6±0.8 vs 8.1±0.6%) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher(20.2±0.4 vs 16.8±2.8%) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.