Effect of Monosaccharide L-fucose and Polysaccharide Fucoidan on Sperm ${\alpha}$-L-fucosidase Activity and Relation to Sperm-oocyte Interaction in Pig

Song, X.X.,Park, C.K.,Piao, Y.J.,Niwa, K. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.3

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that ${\alpha}$-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm ${\alpha}$-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm ${\alpha}$-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm ${\alpha}$-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of ${\alpha}$-L-fucose on sperm binding and penetrating into ZP caused sperm ${\alpha}$-L-fucosidase inhibited by ${\alpha}$-L-fucose.

Percoll에 의한 미니돼지 정액내 세균 제거가 정자 성상과 수정란 분할에 미치는 영향

유한준,전준명,이용승,정희태,양부근,김대영,박춘근 한국동물생명공학회(구 한국동물번식학회) 2009 Reproductive & developmental biology Vol.33 No.1

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.

Liquid Boar Sperm Quality during Storage and In vitro Fertilization and Culture of Pig Oocytes

Park, C.S.,Kim, M.Y.,Yi, Y.J.,Chang, Y.J.,Lee, S.H.,Lee, J.J.,Kim, M.C.,Jin, D.I. 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at 4℃ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and 10×10^(6) sperm/ml than in 0.2 and 1×10^(6) sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in lO×10^(6) sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in 1×10^(6) sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4℃ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend 1×10^(6) sperm/ml concentration for in vitro fertilization of pig oocytes.

Effect of aromatase on sperm fertility and relative quantity of sperm aromatase protein in pig

Jong-nam Oh,Jae Yeon Hwang,Dong-Kyung Lee,Kwang-hwan Choi,Chang-Kyu Lee 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Aromatase is an enzyme that converts testosterone to estrogen. This enzyme, present in the sperm as well as various tissue and cells, has been considered to be related to the fertility of human and mouse sperm. Therefore, we examined effect of aromatase inhibitor on viability and fertility of sperm, and quantity of aromatase in sperm groups with different density in pig. To analyze the effect of aromatase on sperm viability, we treated aromatase inhibitor to the sperm with different concentrations (0, 10, 20, 50, 100, 200, 500 μM) at different time (0.5, 1, 2, 4, 8 hours). After the treatment, the sperm viability was calculated by hypo-osmotic swelling test. We selected 0, 50, 100 μM concentration during 0.5 hour as inhibitor treatment condition before in vitro fertilization. Next, we examined fertility and quantified aromatase protein in sperms with different density. In the first experiment, viability of sperm was decreased following the increasement of inhibitor concentration. The aromatase inhibited sperm showed lower penetration rate and cleavage rate than those of non-treated sperm. Concentration of 50 μM inhibitor had no significant effect on the sperm viability, but it significantly reduced sperm fertility. Second, sperms with low density showed higher penetration rate, but no significant difference between sperms with high density. In conclusion, aromatase is responsible for viability and fertility of porcine sperm similar to mouse and human, however, density of sperm has no correlation with quantity of aromatase protein.

Liquid Boar Sperm Quality during Storage and In vitro Fertilization and Culture of Pig Oocytes

Park, C.S.,Kim, M.Y.,Yi, Y.J.,Chang, Y.J.,Lee, S.H.,Lee, J.J.,Kim, M.C.,Jin, D.I. 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at 4℃ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and 10×10^6 sperm/ml than In 0.2 and 1×10^6 sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10×10^6 sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest In 1×106 sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4℃ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend 1×10^6 sperm/ml concentration for in vitro fertilization of pig oocytes.

Liquid Boar Sperm Quality during Storage and In vitro Fertilization and Culture of Pig Oocytes

Park, C.S.,Kim, M.Y.,Yi, Y.J.,Chang, Y.J.,Lee, S.H.,Lee, J.J.,Kim, M.C.,Jin, D.I. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.10

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and $10{\times}10^6$ sperm/ml than in 0.2 and $1{\times}10^6$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in $1{\times}10^6$sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at $4^{\circ}C$ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

Effects of Astaxanthin on Miniature Pig Sperm Cryopreservation

Eunjoo Lee,Daeyoung Kim 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

The purpose of this study is to evaluate the effects of astaxanthin added to freezing buffer on semen parameters, total sperm oxidation stress after post-thawing of boar sperm and Lipid peroxidation (LPO) which is caused by reactive oxygen species (ROS) in sperm membrane. Varying concentrations of astaxanthin (0, 10, 50, 100 and 500 μ M) were used in the freezing buffer during cryopreservation to protect the DNA of thawed miniature pig sperm. Semen parameter was measured using computer assisted sperm analysis (CASA) for sperm motility and determine ROS rate, oxidative stress of boar sperm using fluorescence-activated cell sorting (FACS). Sperm motility was higher (p<0.05) in the astaxanthin group than in the control group. Sperm motility and the number of progressive motile sperm was higher (p<0.05) in the astaxanthin 500 μM group (66±1.7%) than in the control group (49.8±4%). In ROS evaluation, the astaxanthin group lowered intracellular O2 and H2O2 in viable sperm. The Yo-Pro-I/HE and PI/ H2DCFD a staining as revealed using flow cytometry was lower in astaxanthin groups than in the other groups. As the result, we found that astaxanthin could protect the sperm plasma membrane from free radical and LPO during boar sperm post-thawing.

Cryopreservation with Trehalose Reduced Sperm Chromatin Damage in Miniature Pig

박철호,김성원,황유진,김대영 사단법인 한국동물생명공학회 2012 한국동물생명공학회지 Vol.27 No.2

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 WST1assays 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 (57.80 ± 4.60% in glycerol vs. 75.50 ± 6.14% in glycerol + trehalose) and progressive (51.20 ± 5.45% in glycerol vs. 70.74 ± 8.06% in glycerol +trehalose). A significant (p<0.05) increase in VAP (42.70 ± 5.73 μm/s vs. 59.65 ± 9.47 μm/s), VSL (23.06 ± 3.27μm/s vs. 34.60 ± 6.58μm/s), VCL (75.36 ± 11.36 μm/s vs. 99.55 ± 12.91μm/s), STR (54.4 ± 2.19% vs. 58.0 ± 1.63%), and LIN (32.2 ± 2.05% vs. 36.0 ± 2.45%) 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.

Effects of L-Carnitine and Nicotinic Acid on Sperm Characteristics in Miniature Pigs

Yeon-Ju Lee,Sang-Hee Lee,Yu-Jin Kim,Yong Hwangbo,Seunghyung Lee,Hee-Tae Cheong,Boo-Keun Yang,Choon-Keun Park 한국동물생명공학회(구 한국동물번식학회) 2016 Reproductive & developmental biology Vol.40 No.1

This study investigated the effects of L-carnitine (LC) and nicotinic acid (NA) on sperm viability during liquid storage at 18℃ in miniature pigs. 10 μM LC and 30 mM NA, combined LC and NA (LN) were treated in fresh semen for 3, 7, and 10 days. In results, sperm survival increased in NA- and LN-treated semen on 7 and 10 days (p<0.05), mitochondrial integrity of live sperm increased in LN-treated semen on 7 days (p<0.05), but not NA-treated semen. In addition, we examined the acrosome reaction of sperm in miniature pigs. LC and NA did not influence on acrosome reaction of boar sperm. In conclusion, LC and NA effectively maintained the viability and quality of sperm during long-term storage in miniature pigs, suggesting that the combined LN may be useful for improving the semen extender for long-term liquid storage in pigs.

Effects of Fertilization Time and Culture Medium of Pig Oocytes Matured In Vitro by Liquid Boar Sperm Stored at 4℃

Park, C.S.,Yi, Y.J.,Kim, M.Y.,Chang, Y.J.,Lee, S.H.,Jin, D.I 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23℃) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800×g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0×10^(9) sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4℃. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10μg/ml insulin, 2μg/ml vitamin B_(12), 25 mM HEPES, 10μg/ml bovine apotransferrin, 150μM cysteamine, 10IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75μg/ml sodium penicillin G, 50μg/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5℃, 5% CO₂in air. Oocytes were inseminated with liquid boar sperm stored at 4℃ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 μl mTBM fertilization media with 1.0×10^(6) sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 μl NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4℃ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 μl TBM fertilization medium with 1×10^(6) sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.