This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

The acrosome cap allows sperm to penetrate the egg membrane and produce male pronuclei within female chicken eggs, facilitating successful fertilization. Given this, it is important to establish practical methods for evaluating the integrity of the acrosome cap and thus the quality of the rooster’s sperm. There are several established methods for evaluating the acrosomes of mammalian sperm, but none of these methods are suitable for evaluating the acrosome status of rooster spermatozoa. Therefore, a simplified method for evaluating the rooster acrosome is needed. Here we evaluated the usefulness of CBB (coomassie brilliant blue) staining of the acrosome at concentrations of 0.04%, 0.08%, and 0.3% CBB solutions. Our data revealed a clear staining pattern for intact acrosome caps at 0.04% and 0.08% CBB but not at 0.3% CBB. This protocol revealed differences in acrosome integrity between fresh and frozen rooster sperm smears suggesting that CBB staining may facilitate easier semen evaluation in roosters. This protocol allows for the accurate differential staining of acrosome cap in rooster spermatozoa.

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozenthawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezingthawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

Iron is required for cell viability but is toxic in excess. While the iron-mediated malfunction of testicular cells is well appreciated, the underlying mechanism(s) of this effect and its relationship with fertility are poorly understood. Ferritin is a ubiquitous intracellular protein that controls iron storage, ferroxidase activity, immune response, and stress response in cells. Ferritin light chain protein (FTL) is the light subunit of the Ferritin. Previously, we had identified the FTL in bovine spermatozoa following capacitation. In present study, to investigate the role of Ferritin in sperm function, mice spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of sodium nitroprusside (SNP), an iron donor. SNP was increased Ferritin levels in a dose-dependent manner. The Ferritin was detected on the acrosome in spermatozoa by immunocytochemistry. Short-term exposure of spermatozoa to SNP increased tyrosine phosphorylation and the acrosome reaction (AR). Finally, SNP affected a significant decrease in the rate of fertilization as well as blastocyst formation during early embryonic development. On the basis of these results, we propose that the effects of Ferritin on the AR may reduce overall sperm function leads to poor fertility in males and compromised embryonic development.

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.

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.

The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into LN2. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol (72.5±5.00%, 54.88±0.66% and 46.00±2.40%; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol (34.69±4.64% vs 46.00±2.40%; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: 55.81±2.94, 55.19±3.34 vs 47.94±3.48%; p<0.05 and membrane integrity: 44.94±3.51, 46.06±2.25 vs 40.38±1.03%; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN. The presented straws were examined the viability and motility after thawed at 37 water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to , and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

본 실험은 돼지정액의 동결보존을 위한 희석액과 냉각속도 및 동해방지제의 적정농도를 결정하기 위해 실시하였으며, 얻어진 결과는 다음과 같다. 1. 에서 까지의 냉각속도에서는 LEY 희석액에서 분당 로 냉각하는 것이 생존율과 정상 첨체율에서 가장 높은 결과를 얻었다. 2. LEY 희석액이 BF5와 M-Soejima 희석액보다 정자를 동해로부터 보호하는 능력이 우수하였다. 3. LEY 희석액에 첨가하는 glycerol의 농도는 3 또는 가 의 glycerol

본 연구는 개의 동결정액제조 시 동결보호 희석액 내에 첨가되는 당의 종류와 조합이 동결융해 후 정자 두부의 첨체의 손상과 생존율 및 운동성 등에 미치는 영향에 대하여 조사하고자 실시하였다. 당의 종류에 따른 정자의 첨체손상 정도는 정상 정자비 율은 Fru+Tre, 처리구가 Fructose, Trehalose, Fru+Tre+Xyl, Fru+Xyl, Tre+Xyl, Xylose구에 비해 가장 높았다(83.05.6%, 82.3 3.1%, 81.72.1%,

본 연구는 수정능획득유기물질로 알려진 카페인 헤파린을 병행처리하여 한우 정자의 첨체 반응율과 생존율을 알아보고 수정능획득과정 중에 단백질의 변화상을 전기영동방법으로 조사하였다. 동결융해후 정자의 생존율은 90%이상이었으나 전배양처리후 0.5시간에 70%로 감소하고 2시간 이후에는 35%로 감소하였다. 정자의 첨체반응율은 동결융해후 정상정자가 85.7%였으나 전배양시간에 따라 53.4%에서 14.3%로 감소하였다. 동결융해후 첨체가 소실된 생존정자는 9.

The present study was performed to investigate the effects of caffeine and heparin on capacitation and acrosome reaction of bovine spermatozoa, effects of antisperm antibodies on acrosome reaction of bovine spermatozoa. The rates of acrosome reaction in control group, caffeine treated group, heparin treated group, caffeine-heparin complex treated group were 40.3, 54.3, 63.3, 72.3%, respectively and there were significant differences among the groups(p<0.01), especially higher in caffeine-heparin complex treated group than the others. The rates of acrosome reaction of antisperm antibodies serum supplemented groups(5, 10 and 20%) were 60.4, 48.9 and 37.1%, respectively and there were significant differences among the groups(p<0.0l), and the more increases in serum concentrations, the more decreases in acrosome reaction, but this phenomenon was not seen in fetal calf serum supplemented group and heifer serum group. When the serum concentration was 5%, the rates of acrosome reactions were significantly lower in fetal calf serum supplemented group than heifer serum group and in antisperm antibodies serum group(p<0.01), and there were no significant differences between heifer serum group and antisperm antibodies serum group(p<0.01). When the serum conecntrations were 10%, 20%, the rates of acrosome reactions were significantly lower in antisperm antibodies serum supplemented group than in fetal calf serum group and in geifer serum group(p<0.01), and there were no significant differences between fetal calf serum group and heifer serum group(p<0.01). These results indicate that caffeine-heparin complex treatment is very effective for inducing acrosome reaction of bovine spermatozoa and that antisperm antibodies block acrosome reaction.

Effects of caffeine, heparin and caffeine-heparin treatments for in vitro capacitation of Korean Native Cattle sperm on acrosorne reaction and viability were studied using the methods of Wells-Awa and Dual stain. The results were summerized as follows: 1. The acrosome reaction of sperm when treated with caffeine after 0 to 4 hrs of preincubation were 11.0~75.7% for Wells-Awa stain, and 14.3~75.55% for Dual stain. True acrosome reaction of sperm for Dual stain was 3.0~29.2%. The viability of sperm was 62. 2~27.2%. 2. The acrosome reaction of sperm when treated with heparin after 0 to 4 hrs of preincubation were 17.0~81.2% for Wells-Awa, and 14.3~75.5% for Dual Stain. True acrosome reaction of sperm for Dual stain was 1.5~26.6%. The viability of sperm was 58.6~35. 8%. 3. The acrosome reaction of sperm when treated with caffeine-heparin after 0 to 4 hrs of preincubation were 13.0~83.2% for Wells Awa, and 11.0~78.5% for Dual stain. True acrosome reaction of for Dual stain was 5.1~26.3%. The viability of sperm was 60.5~30.1%.