The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21～22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05<P<0.1) nuclear maturation when compared with oocytes matured in M199 (76.9% vs. 83.8%) but no significant differences were found in embryo cleavage, blastocyst formation, and mean number of cells in blastocyst (73.8% vs. 74.6%, 11.1% vs. 12.1%, and 28.4 cells vs. 30.1 cells, respectively). The oocyte degeneration was not reduced by CB treatment during ICSI (11.9%) when compared with no treatment control (11.3%) while the treatment showed detrimental effects (P<0.05) on embryonic cleavage (40.0%) and blastocyst formation (1.8%) rates when compared with control (60.0% and 11.5%, respectively). For activation of ICSI oocytes, additional electric stimulus has no positive or negative effect on in vitro development of preimplantation stage ICSI porcine embryos. Our results demonstrate that CB treatment during ICSI inhibits embryonic development of ICSI oocytes and additional electric activation after ICSI has no effect in improving ICSI embryonic development in pigs. Further studies are needed to improve ICSI efficiency by investigating factors influencing embryonic development after ICSI in pigs.

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

Artificial insemination (AI) has been performed widely in swine industry using fresh liquid sperm instead of frozen type of sperm. However fresh sperm are not able to preserve more than three days with optimal motility and other sperm parameters for the successful fertilization, since in vitro stored sperm has an oxidative stress that resulted increase of abnormality and acrosome reation. To overcome these major problems, novel preservative formulation is needed to neutralize the oxidative stress and to provide suitable physiological environment for sperm in in vitro. In this study, naturally derived substances such as Poncirus trifoliate (Trifoliate orange), Garcinia mangostana (Mangosteen), pig placenta and testis extracts were tested as sperm preservative agents. Placenta extracts (PE), trifoliate orange extracts (TOE), testes extracts (TE) and mangosteen extracts (ME) were applied to analyze specific parameters for sperm motion characteristics individually and combinatorial. Each individual extract treatment can accelerate the sperm motility but noticeably TOE, TE and ME treatments exhibited the considerable and significant preservation of sperm motility. PE, TE and ME showed a significant (p<0.05) increase in ALH after one week. Further we evaluated the five different combinations of these extracts on sperm motility and its motion characteristics. Surprisingly even after one week ME, TOE and TE combination significantly preserved the sperm motility about 75%. It is noteworthy that unlike individual extract treatment, combination of ME, TOE and TE simultaneously protect the sperm motility and its motion characteristics. Taken together these data conclude that addition of ME, TOE and TE can be effective for preservation of pig sperm.

In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activity in post-thawed sperm that was supplemented with ginsenoside-Rg1. Varying concentrations of ginsenoside-Rg1 (0, 25, 50 and 100 μM/ml) were used in the extender during cryopreservation to protect the DNA of thawed sperm, thereby increasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. The results derived from CASA were used to compare the fresh, control, and ginsenoside-Rg1 groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the 50 μM/ml ginsenoside-Rg1 group (61.0±4.65%) than in the control (46.6±7.02%), 25 μM/ml (46.2±4.76%), and 100 μM/ml ginsenoside-Rg1 (52.0± 1.90%) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membrane integrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher (91.6±0.82%) in the 50 μM/ml ginsenoside-Rg1 group than in the other groups. Here, we report that ginsenoside-Rg1 affects the motility and viability of boar spermatozoa. Moreover, ginsenoside- Rg1 can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

본 연구는 돼지의 난포란을 체외성숙하여 세포질내정자주입(ICSI)에 의해 생산된 체외수 정란의 체외발달율을 평가하기 위하여 실시하였다. 세포질내정자주입에 의한 체외수정란의 발달율은 서로 다른 보존상태의 정자인 신선정자, 액상정자 및 동결-융해된 정자를 이용하 더라도 수정율과 배발달율에는 영향을 미치지 않는 것으로 나타났다. 세포질내정자주입 후 난자의 전기적 활성화를 처리한 실험군이 활성화를 처리하지 않은 실험군에 비해 수정율과 배반포기배로의 발달율에 있어서는 높은 경향을 나타내었으나, 체외수정 실험군 및 전기적 활성화를 처리한 실험군과 전기적 활성화를 처리하지 않은 실험군간 배반포기배의 할구수는 유의적인 차이를 나타내지 않았다. 또한 각각의 실험군에서 얻은 배반포기배의 염색체를 분 석한 결과, 정상 이배체 염색체상의 비율에 있어서도 유의성을 나타내지 않았다.

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.

Germ cell-specific hyaluronidases such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5) are in part responsible for dispersal of the cumulus cell mass, which is a critical step in establishing fertilization in mammals. In this study, we identified two testis-hyaluronidases, SPAM1 and Hyal5, in hamster and rat. These two genes were expressed specifically in the testis. At the protein level, hamster SPAM1 and Hyal5 display 78.7% and 75.4% identity with mouse SPAM1 and Hyal5. Further, the activity of the enzymes with respect to cumulus cell dispersion did not differ, although we observed that the enzymatic activity differed in pH range. These studies suggest that different sperm hyaluronidases are capable of dispersing the cumulus cell mass despite differences in enzyme activity.

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.

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ( in glycerol vs. in glycerol + trehalose) and progressive ( in glycerol vs. in glycerol + trehalose). A significant (p<0.05) increase in VAP ( vs. ), VSL ( vs. ), VCL ( vs. ), STR ( vs. ), and LIN ( vs. ) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

The objective of this study was to assess the effect on post-thawed sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks. The Sperm rich fraction of ejaculates from three Duroc boars were collected by a glove-hand technique. Samples with more than 80% motile sperm were used for this experiment. Semen was diluted with freezing extender (LEY) containing 11% (v/v) lactose, 20% (v/v) hen egg yolk with 3.5% (v/v) glycerol, and 0.5% (v/v) Orvus Es Paste(OEP, Nova Chemical Sales Inc., Scituate, MA. USA) to yield a final sperm concentration of 5×108 cells/ml. Following complete dilution, semen samples were loaded in 0.5 ml French medium straws (IMV technologies, France) and transferred to programmable semen freezer (SY-LAB Gerate GmbH, Austria). For freezing the semen samples, each straw was cooled from 5℃ to — 5℃ at 6℃/min, auto-seeding at — 5℃ and held for 60sec, samples were then cooled from — 5 to — 80℃ at 40℃/min, and thereafter from — 80℃ to — 150℃ at 60℃/min. The yolks used were sourced from fresh chicken and duck eggs. To evaluate the post-thaw sperm quality, semen was thawed at 38℃ for 20 sec and sperm motility, viability and acrosome integrity were assessed. Motility was assessed for %motile cell characteristics using computer-assisted semen analysis (CASA; SAIS SI-100, Medical supply, Korea). The percentage of sperm viability was assessed using LIVE/DEAD® sperm viability kit (Molecular probes, Eugene, OR, USA). The acrosome integrity was assessed by FITC-PNA staining. Sperm quality in terms of motility, viability and acrosome integrity showed higher after freezing in medium containing duck yolk than chicken yolk. However, there was no significant difference in sperm quality for the different types of yolk(p>0.05). * The result of this study showed that there was no significant difference between the egg yolk types when considering the sperm motility, viability and acrosome integrity of boar semen frozen in the freezing extender with chicken or duck egg yolks.

This study evaluated a method of sorting X and Y chromosomes based on size using the forward angle light scatter related refractive index (FSC) of a flow cytometer. Hanwoo bulls sperm were separated to X and Y chromosomes by the parameters of FSC or Hoechst 33342 intensity. As a result, using monitor program linked flow cytometry during sorting processing, the purities were or for the X-fraction and or for the Y-fraction in the two sperm sorting methods. There were no differences in the X and Y ratios (X and Y %) between the sperm sorting methods based on FSC or DNA content. The proportions of female and male embryos used for in vitro fertilization and development were or , and or when sperm were processed using the sex sorting method by FSC or Hoechst 33342. In conclusion, further study is needed to determine the optimum procedure and improve the nozzle to enhancing sorting accuracy or efficiency. Also, the findings of this study do not negate the possibility that the difference method of sperm sorting cannot use a UV laser beam.