• Journal of Embryo Transfer
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• v.30 no.4
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.235-240
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• 2004

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.57-57
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• 2003

수정란의 체외생산(IVP)기술은 동물생산기술의 적용과 생리학이나 세포생물학의 기본 연구에 새로운 biotchnologies의 발생에 있어 매우 중요하고 흥미로운 사건으로서 여러 종류의 포유류에 적용되고 있다. 이러한 기술은 3가지의 절차를 밟아야 하는데 In vitro maturation(IVM), In vitro fertilization(IVF) 및 In vitro development(culture)(IVD)가 그것이다. 그런데 돼지는 다태동물로서 난소의 난포가 성장할 때 난포간 meiotic competence가 난자에 의하여 진행되므로 난포내의 난포액에 의하여 난자의 발육이 좌우된다고 보고 있다 돼지WP에 사용되는 난자는 난포의 직경이 3~5mm에서 수집하고 cumulus-oocyte complexes(COCs)의 형태에 따라 선택하는 것이 보편화되어 있다. 실험목적은 돼지 난소의 antral follicles이 성장할 때 크기에 의하여 oocyte meiotic competence의 방출에 어떤 영향을 주는지를 난자의 체외성숙과 체외수정 및 체외발육 비율을 각각 조사하여 미성숙 난자의 체외배양 기술을 개선할 목적이었다. 수행내용은 도축돈 난소의 난포크기별 3mm<, 3~5mm, 5mm)로 나누어, 다시 말해서 preantral stage, antral stage, postantral stage으로 구분하여 채취한 COCs를 IVM, IVF, IVC 상태에서의 COCs 형태, cell cleaved rate, developmental rate 등을 조사하였다.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.351-358
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• 2007

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.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.43-49
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• 1997

The objective of this study is to determine developmental pattern and cell allocation to the ICM and TE in haploid and diploid of embryos following parthenogenetic activation and in vitro fertilization. The incidence of development to blastocyst was lower in the combined treatments of ethanol stimulation and cytochalasin B as compared to the control. However, the combined ethanol stimulation and cytochalasin B treatment (diploid) accelerated development to the blastocyst as compared to the ethanol treatment alone (haploid). Significantly reduction in the average number of total cells and ICM was observed in the parthenotes alone as compared to fetilized embryos, but those of combined ethanol stimulation and cytochalasin B treatment embryos were significantly increased as compared to ethanol alone embryos. These results suggested that the ploidy affects preimplantation developmental patters and cell allocation to the ICM and TE in the porcine.

• Asian Journal of Turfgrass Science
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• v.3 no.2
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• pp.77-82
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• 1989

This experiment was carried out to determine the value of turfgrass clippings as a source of protein for animal feed. Levels of crude protein(CP) and in vitro dry matter digestibility(JVDMD) were determined for Korean lawngrass (Zoysia Japonica Steud.) under three levels of nitrogen fertilizer ( 0 , 350 and 700kg ha year and three levels of mowing interval between cuts (lO, 20, and 30 days). Sampling was done on the three-year old turffield on which nitrogen and mowing treatments were Imposed. Clippings were collected after mowing, and CP levels were calculated from a measurement of total N(CP = N >< 6.25) l)y Auto-analyser system-2 procedure and IVDMD by Maeng's method which was partly modified with Tilley and Terry's procedure. With the increased nitrogen legime the CP content of leaf clippings varied from 6.6 to 13.6% and that Of stem clippings from 4 to 10.3%. The CP levels of clippings were directly proportional to the N fertilization rate. The highest CP content was recorded in early July when it was 14.1% at 35OkgN ha -and I 18.l%kgN ha-1 with 10-wcek mowing interval. Mowing interval had little effect on the CP content in both leaf and stem clippings. The level of IVDMD in the Korean lawngrass clippings varied with mowing interval. The highest level measured was 47%, hut this varied during the growing season and occurred in the following order : early summer > summer > fall. This experiment has shown that Korean lawngrass clippings can he helpful source of protein for use in some animal feeds and that a high concentration of CP can occur depending upon N fertllizer and that of I VDM I) upon mowing practices.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.235-241
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• 1994

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + $\beta$ estradiol, PMSG + $\beta$ estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.