• 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.

• Journal of Animal Environmental Science
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• v.20 no.2
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• pp.77-84
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• 2014

This study was conducted to investigate the bacterial capability to accumulate phosphorus during liquid composting process of pig slurry. Storage liquid compost and pig slurry were analyzed by using MALDI-TOF technique, which showed the colonies of Acinetobacter towneri and Bacillus licheniformis. In addition, bacterial colonies were isolated under high phosphoric acid conditions using X-phosphate MOPS medium with the addition of 2 mM $K_2HPO_4$. Microbial growth was observed in high and low phosphoric conditions due to the growth of bacterial diversities in the liquid fertilizer and slurry. The colonies isolated in the high phosphoric acid medium were uncultured bacterium clone and Acinetobacter sp. were identified by analysis of 16S rRNA gene sequences. Uncultured bacterium showed higher growth rate and excellent phosphorus ability then Acinetobacter sp.. In addition to Paenibacillus sp. AEY-1 isolated from pig slurry performed excellent phosphorus utilizing capability.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.46-49
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• 2010

In this study, we investigated the number of follicles, oocyte recovery rate and oocyte competence after in vitro maturation according to the size of follicle. And equine oocyte competence after in vitro maturation was investigated in terms of the diameter of follicle with criteria of maturation: nuclear stage after Hoechst staining. The average number of follicles per ovary with middle size (11-20 mm, 2.68) was higher than those of small (5-10 mm, 0.74) and large size follicle (> 21 mm, 1.63), therefore medium follicle (53.1%) had higher proportion than other size of follicles. The average numbers of follicle per ovary was 5.05. The rate of oocyte recovery in small (54.5%) and middle follicle (50%) was higher than that in large follicle (40.9%). After culture for 48 h in Medium 199, 50%, 45.5%, and 44.4% of oocytes from the follicles with diameters of 5-10, 11-20, > 21 mm, respectively reached the metaphase II stage. This is the first report showing number of follicle, oocyte recovery rate according to follicular size, and in vitro oocyte maturation in Jeju mare in Korea. To fulfill in vitro equine embryo production, further studies such as the seasonal effect, in vitro fertilization etc is need.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.192-203
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• 1986

Two experiments in this study were designed to compare the potential for in vitro capacitation and in vitro fertilization of ejaculated sperm among individual rabbit bucks. In experiment 1, for in vitro capacitation, the ejaculated sperm were preincubated in DM for 12 hr or 18 hr after HIS treatment, then 12 hr -or 18 hr- preincubated sperm were incubated with superovulated rabbit ova in a 5% CO2 incubator for 36 hr at 38$^{\circ}C$, and a part of cleaved ova was transferred to the recipient does for implantation of embryo. In experiment 2, effect of lysolecithin addition to preincubation medium on induction of accelerated in vitro capacitation and in vitro fertilization of individual rabbit sperm was studied. Experiment 1; 1. Percent acrosome reaction of sperm, noted after staining, after 12 hr or 18 hr preincubation ranged from 52.5 to 76.0% and from 67.5 to 90.0%, respectively and sperm motility index of these sperm ranged from 20.0 to 47.5 for 12 hr-preincubated sperm and from 15.0 to 37.5 for 18 hr- preincubated sperm. There was no a certain relation between percent acrosome reaction and sperm motility index. 2. In vitro fertilization rate (cleavage rate) of in vitro capacitated sperm varied widely among individual bucks, ranging from 0 to 47.8% for 12 hr - preincubated sperm and from 0 to 60.9% for 18 hr -prein- cubated sperm. Cleavage rate of 18 hr - preincubated sperm was higher and faster than that of 12 hr - preincubated sperm. 3. Eight of 44 in vitro fertilized embryos transferred into 6 recipients were implanted in 4 recipients (66.7%) up to day 15 and implnatation rate was 18.2%. Experiment 2; 1. The percent acrosome reaction of sperm before and after 4 hr preincubation in DM without lysolecithin varied significantly among individual bucks, ranging from 0.4 to 18.4% and from 1.7 to 37.4%, respectively and percent acrosome reaction of sperm at 30 min after addition of 60${\mu}$g/ml lysolecithin also was significantly different among bucks, ranging from 19.2 to 67.1%. 2. Effect of accelerated acrosome reaction following lysolecithin addition was more considerable in the individuals showed less percent acrosome reaction before and after 4 hr preincubation. Percentage of motile sperm and motility score showed a trendency towards a decrease with increase of preincubation time and time after lysolecithin addition. 3. In vitro fertilization rate (cleavage rate) at 24 hr postinesmination with pooled sperm were treated to 60 $\mu\textrm{g}$/ml lysolecithin for 30 min after 4 hr preincubation was 24.6%, a higher rate than 13.2% for control. While 80 $\mu\textrm{g}$/ml lysolecithin-added sperm showed a lower cleavage than control and 60$\mu\textrm{g}$/ml-added sperm at both 24 hr and 48 hr postinsemination. These results from 2 experiments suggest that more useful preincubation time for the in vitro capacitation of ejaculated rabbit sperm is 18 hr in DM after HIS treatment, although there is wide variation in vitro capacitation and in vitro fertilization rate among individual bucks, and lysolecithin addition to at least 4 hr - preincubated sperm in DM can result in almost same in vitro fertilization rate as that of 18 hr - preincubated sperm in the experiment 1.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.219-227
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• 2004

This study was conducted to develop a serum-free, defined medium of IVM of pig oocytes. The TCM-199 with supplemented with polyvinylalcohol(PVA), polyvinylpyrrollidone(PVP) and porcine follicular fluid(pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development under in-vitro fertilization and in vitro culture were examined and subsequently considered on the possibilities be sustituted for the bovine serum albumin(BSA). Maturation rate of pig oocytes in IVM media containing PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly higher(P<0.05) than that of PVP(78.6％). Cleavage rate after IVF of PVP(64％) was significantly lower(P<0.05) than these of PVA(73％), pFF(77％) and BSA(73％) supplements. in vitro development rates to morulae and blastocyst on PVP(54％) were also significantly lower(P<0.05) than these of the supplements of PVA(63％), pFF(69％) and BSA(65％). In comparison of maturation and fertilization rates of pig oocytes in each supplements, the maturity rates of PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly lower(P<0.05) than that of PVP(72.4％) and while, the fertilization rates of pFF(87.1％) and BSA(89.1％) were significantly higher(P<0.05) than these of PVA(78.0％) and PVP(70.6％). It may be concluded that PVA and pFF can be substituted far BSA in medium for culturing pig oocytes; however, it may be considered that PVP were limited to for BSA in the in vitro culture of the embryos.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.86-86
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. (omitted)

• Journal of the Korean Society of Tobacco Science
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• v.17 no.2
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• pp.109-113
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• 1995

Nicotiana repanda has resistance to many important tobacco diseases but no tobacco cultivars are currently available that carry risistance genes derived from this species. Numerous attempts to hybridize N, repanda with commercial tobacco cultivars have been largely unsuccessful because of cross incompatibility or the uncovering of lethal genes. In vitro pollination of placenta attached ovules was useful in by passing prezygotic barriers for interspecific hybrid combination between N.tabacum cv. NC82 and N. repanda. Six days after in vitro pollination of N. tabacum cv. NC82×N. repanda, enlarged ovules on plancenta were removed and transferred into ovule culture medium of kitsch and kitsch (1969). Within 15 days of ovule culture, germination occurred. Most of the hybrid seedlings obtained had poor root system and finally died, while few of them had good root system and grew well.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.41-47
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• 1992

1. In vitro system, LR and FSR accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus. 2. Caffeine (2mM) in the fetilization medium was required not only for inducing zona penetrating ability of boar also for developing to the male pronucleus of the penetrat- ing spermatozoa in vitro. 3. The germinal vesicle (GV)stage was observed for the first 17.6 hr;germinal vesicle break-down (GVBD)stage between 17.6~26.4 hr ;metaphase I (M-I)from 26.4 - 30. 9hr;anaphase I(A-I)ranged from 30. 9~33.4hr;telophase I(T-I) at 33.4~34.4hr; and metaphase II(M-II) at 34.4-48hr. 4. The addition of 10%(v /v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (p<0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. 5. The presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.

• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.420-430
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• 2000

These studies were preformed to investigate the freezing conditions to achieve good post-thaw viability of spend and the practical methods of artificial insemination frozen canine semen. Semen were collected from nine male dogs which had been proved to be fertile in the past and the semen were treated for freezing procedure. Post-thaw motility and viability of canine sperm were evaluated to investigate individual tolerance of freezing, difference among freezing extenders, dif-ference among freezing equipments and freezing conditions, difference between fast and slow cooling rate, difference according to different glycerol concentration, effect of seeding on post-thaw viability, difference according to cutting part of straw, difference according to thawing temperatures, and dif-ference according to media added to thawed semen. Thawed semen for insemination were added with equal volnme of canine capacitation medium (CCM) and the volume of semen and the number per insemination were adjusted as 2-3 ml and $20-30 {\times}10^7,$ respectively. The semen were inseminated in vagina using balloon catheter and en17ryos were cellected from 9 to 11 days after the second Al to d determine fertilization.