The objective of this study was to investigate the efficiency of nicotinic acid during in vitro fertilization (IVF) in frozen-thawed bull sperm . The ejaculated semen was diluted with Triladyl containing 20% egg-yolk and cryopreserved in liquid notrigen. The frozen sperm was thawed for 45 seconds in the 38℃ water bath. Sperm was diluted with IVF medium (Bovine-Oviduct medium; BO) containing 0, 15, 30 and 60 mM nicotinic acid (NA), which were incubated at 39℃, 5% CO2 for 0, 0.5, 1, 2 and 4h. The characteristics of frozen-thawed sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI for outer acrosomal membrane damage and Rhodamine123/PI for mitochondrial integrity using flow cytometry. And the sperm ability was analysed by Coomassie brilliant blue (CBB) staining for acrosome reaction state and Rose bengal staining for abnormality. Acrosome reaction and abnormality were analyzed using a microscope. In results, sperm viability was significantly higer in 30 mM group than 0 and 15 mM groups at 1 and 2 h (p<0.05). Outer acrosomal membrane damage was significantly lower in 30 mM group than 0 and 15 mM groups at 1, 2 and 4 h (p<0.05). And mitochondrial integrity was significantly higher in 30 mM group than 0 and 15 mM groups at 2 and 4 h (p<0.05). Also, acrosome reaction was significantly lower in 30 mM than 0 and 15 mM groups at 1 and 2 h (p<0.05) and abnormality was lower NA groups than 0 group at 1 h (p<0.05). In couclusion, we suggest that using the thawing medium containing NA for sperm dilution can be benefical for IVF in bulls

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen Post-thawed in boar. Recently, polymorphism (g. 35756 T>C) of Estrogen Receptor 1 (ESR1) gene reported to be significant association with MOT. This study was conducted to evaluate the ESR1 gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.35756 T>C SNP of ESR1 was significantly associated with frozen semen motility and kinematic characteristics. The g.35756 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.001). The SNP was also significantly associated with ALH (P<0.05). Therefore, we suggest that the g. 35756 T>C polymorphism in the intron 1 region of the porcine ESR1 gene could potentially be applied in frozen semen programs to improve MOT trait, but only after validation in other populations.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen post-thawed in boar. Recently, polymorphism (g.158T>C) of phospholipase C zeta (PLCz) gene reported to be significant association with MOT. This study was conducted to evaluate the PLCz gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.158 T>C SNP of PLCz was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.0001, p= 0.0002, p<0.0001 and p<0.0001, respectively). Therefore, we suggest that the intron region of the porcine PLCz, may be used as a molecular marker for Duroc boar post-thawed semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

The purpose of this study is to produce wanted sex progeny of genetically confined White Hanwoo (albinism) with preselected sex sperm. One bull of White Hanwoo was chosen for semen donor and X sperm was sorted by MoFlo XDP cell sorter. To compare the pregnancy and birth rates, KPN straw was used as control, total number of unsorted sperm was 20×106/straw. Sexed X frozen semen with 2×106 cells or 4×106 cells per straw were in seminated twice on Hanwoo heifers. The abnormality of the sexed X semen was 24.9 ± 7.31% and distal reflex abnormality of mid piece was significantly (p<0.05) higher (11.7%) compared with that of KPN 768 (5.6%). There were no differences on the pregnancy and birth rates between 2×106 cells or 4×106 cells of X-sperm but KPN semen showed significant differences (p<0.05). The pregnancy rates of KPN 768, 2×106 cells and 4×106 cells X-sperm of White Hanwoo cattle were 85.0%, 26.3% and 50%. The birth rates were 80.0%, 15.8% and 21.4%, respectively. The female offspring rates of KPN 768, 2×106 cells and 4×106 cells X-sperm of White Hanwoo cattle were 43.8%, 100% and 100% (p<0.05). These results indicated that sex sorted White Hanwoo could be used for the production of wanted progeny with 2×106 cells/straw for AI. To increase the efficiency of calf production, the sperm number of sex sorted semen will be optimized for sex selection of White Hanwoo progeny.

Sexed semen is commonly used for the production of calves of the desired gender. Gender selection is important in animal production industries. For example, female cattle are required for the dairy industry while males are preferred in the beef cattle industry. The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58±8.31 and 13.25±7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75±1.98 vs. 8.23±6.07, P＜0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P＜0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p＜0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

The objective of this study was to investigate the relationship between fertility and protein pattern change using in vitro fertilization, analysis of sperm characteristics and two-dimensional gel electrophoresis in different pig types. In results, the viability and mitochondria integrity of sperm were higher significantly (p<0.05) but the portions of ac-rosome reaction was lower significantly (p<0.05) in Duroc and F1 (potbellied×PWG miniature pig) than PWG minia-ture. On in vitro fertilization to investigate fertility, the fertility of F1 semen war higher significantly (p<0.05) than in Duroc and PWG miniature pig. On the other hand, protein patterns showed similar function among the different boar semen. Especially, the heat shock 70 kDa 1-like and G patch domain-containing protein 4 were significantly (p<0.05) higher expressed in F1 than in Duroc and PWG miniature pig. The proteins associated with mitochondria in Duroc were significantly (p<0.05) higher expressed than in F1 and PWG miniature pig. The developmental rates to blastocyst stage of oocytes fertilized with sperm of F1 pig were significantly (p<0.05) higher than in PWG miniature pig. However, phosphoglycerate kinase 2 and zinc finger protein 431 were significantly (p<0.05) higher expressed in PWG miniature pig than in F1 and Duroc pigs. In conclusion, the results of the present study indicate that different proteins were expressed in different pig types, and were associated with a sperm functions and embryo develop-ment.

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of 2×108/ml by doubling in every 30 minutes at 4℃ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at 37℃ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm (93.27±1.62%) than frozen-thawed sperm (73.34±3.27%). However, there were no significant differences between fresh and frozen-thawed dead cell rate (7.35±2.63 vs, 13.71±2.85). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different (72.86±2.83 vs, 81.47±2.48), similarly, the dead cell rates was similar (18.41±3.42% and 17.26±4.25). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under 30℃ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in 0.3℃. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group (43.3±4.7%) was significantly (p<0.05) higher than other treatment groups (Tissue: 16.3±2.7% and Water: 27.5± 3.1%), dying sperm (SYBR+/PI+) in Air treatment group (55.6±4.7%) was significantly lower than other treatment groups (Tissue: 77.6±3.2% and Water: 67.6±3.3%) (p<0.05). Acrosome reaction in Air treatment group (0.2±0.1%) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: 0.7±0.2% and Water: 0.5±0.1%), the acrosome reaction in Air treatment group (28.6±2.8%) within all sperm also was significantly lower than other treatment groups (Tissue: 44.2±1.8% and Water: 36.2±2.0%) (p<0.05). And mitochondrial intact in Air treatment group within live (97.1±0.4%) and all (61.9±3.3%) sperm were significantly higher than other treatment groups (Tissue: 85.2±3.3%, Water: 87.8±2.9% within live sperm and Tissue: 49.28±3.7%, Water: 42.0±3.1% within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

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% ±11.41) than in 5% glycerol treatment (18.08%±1.61). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol (34.12%±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.

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22～24 hr incubation from counting agar plate in which sperm dilute to 10 ～106 in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome- intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 (77.24±6.47, p<0.001) and 7 days (77.24±6.47, p<0.001) after preservation compared to 1 (15.71±7.18) and 3 days(18.39±7.22) after preservation, respectively. Sperm viability was significantly lower (53.25± 35.03, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 (15.71±7.18) and 3 (18.39±7.22) days compared to 5 (21.84±7.91) and 7 (22.59±9.93) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY (; EG and ; 7% G) than 8% LDL (; EG and ;G). Treatment of 4% LDL + 5% EY-EG () has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG () among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

닭 정액의 주요성분인 glutamine은 정장의 주요 성분으로 정자의 생리 활성에 매우 중요 한 것으로 알려져 있으며 조류 정액 희석액의 주요성분으로 이용되어져 왔다. 그러나 glutamine은 액상의 배양액에서 변성이 일어날 수 있어 세포배양에는 alanyl glutamine을 이용하 여 장기 세포배양에 이용되고 있다. 본 연구에서는, alanyl glutamine을 이용하여 개발된 희 석제를 이용하고 닭 정액의 냉장보존에 이용하였으며, 닭 정자의 장기 생존성에 미치는 영 향을 조사하였다. 국립축산과학원 가축유전자원시험장에서 사양관리중인 오골계 수탉 또 는 레그혼에서 복부 마사지법으로 채취된 정액을 이용하였다. 채취한 정액은 96 mM L- Glutamine 또는 Alanyl Glutamine을 함유하는 희석액에 각각 1:8의 부피비율로 상온에서 희석하 여 상업용 냉장고와 4℃로 조절된 BOD 배양기에 보존을 실시하였다. 냉장되는 온도의 저 하 속도를 낮추기 위하여 상온에서 희석하는 온도와 동일한 온도의 증류수 300 ml을 비이 커에 담고 물위에 1.5 ml E-tube에 담아서 파리필름으로 밀봉하였다. 희석된 정자의 활력은 상업용 냉장고에 보존할 경우, 4일차까지 75.6%의 활력정자가 유지 되었으나 5일 이후에는 급격히 생존성이 저하되었으며 6일째에는 활력 정자의 수가 18%로 낮게 나타났다. 한편, BOD 배양기에서 보존된 동일한 정자는 9일까지 약 73%의 활력정자가 유지되었으며, 10일 이후에는 활력 정자의 수가 급격히 감소되는 현상을 관찰하였으며, 15일째에는 약 20%의 정자 활력이 유지되는 현상을 관찰하였다. 이러한 정자의 활성 유지는 정자의 미토콘드리 아 에 필요한 글루타민 에너지 공급원을 안정적으로 공급하는 기능에 의하여 유지 되는것으로 추정되며 특히 hyper activated sperm의 비율을 줄여주어 정자군의 노화를 방지할 수 있는 효과가 있는 것으로 추정되었다. 그러므로 조류 정액을 냉장 보존할 때, 알라닐글루타민을 희석액의 주요 성분으로 이용될 수 있음을 보여주고 있다.