• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.23-30
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• 1990

These studies were carried out to investigate the effects of fetal calf serum(FCS), estrous porcine serum(EPS), porcine follicular fluid(PFF), hormone and matured cumulus cell(MCC) on in vitro maturation and fertilization of porcine follicular oocytes. The ovaries and testes were obtianed from slaughtered Landrace sow and boars, respectively. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5 mm and the semen were prepared from boar's epididymal cauda. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, EPS, PFF and MCC for 48hrs. in a incubator with 5% CO2 in air at 36$^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with $1.5\times$106/ml motile capacitated sperm in the modified Tyroide solution containing 100$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS and PMSG＋HCG were 55.6~64.5% and 33.3~37.1%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in the TCM-199 medium supplemented with 20% EPS and PMSG＋HCG were 50.0~55.0% and 30.3~33.3%, respectively. 3. The maturation rate(59.0~64.2%) and fertilization rate(34.8~39.3%) of follicular oocytes cultured in TCM-199 medium supplemented 20% FCS and 50% PFF were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and 10% and 50% PFF. 4. The maturation rate(60.0%) and fertilization rate(40.0%) of follicular oocytes cultured in TCM-199 medium supplemented with 20% FCS and granulosa cell (1$\times$106/ml) were significantly higher than those of fiollicular oocytes cultured in TCM-199 medium supplemented with 5%, 10% and 15% FCS and granulosa cell.

• Korean Journal of Veterinary Service
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• v.31 no.4
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• pp.547-554
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• 2008

Bacteriospermia is a frequent finding in fresh boar semen and can result in detrimental effects on semen quality and longevity. The objectives of this study was to evaluate types of bacterial contaminants in porcine fresh semen and the reducing effect of antibiotic and density gradient with percoll on the bacterial contaminants. Fresh semen was collected by gloved-hand method into a pre-warmed($37^{\circ}C$) thermostable bottle, and was inoculated onto blood agar and MacConkey agar, respectively. After incubated for 48 hour, 7.5% $CO_2$ at $37^{\circ}C$, bacterial colonies were selected and identified by Gram staining, oxidase test, catalase test and finally identified using API kits and Vitek system. Aerobic culture yielded a variety of bacteria from different genera. The most prevalent contaminant of fresh semen were Leclecia adecarboxylata, Acineobacter banmanni, Staphylococcus epidermidis, Staphylococcus cohni spp urealyticus, Proteus mirabilis. Most of identified bacteria were Gram(-) and non-pathogenic bacteria. It seems that bacterial contaminants in fresh semen were seem originated from multiple sources at the stud/farm, and were from animal and non-animal origins. Gentamicin treatment did not eliminate the bacterial contaminants completely but 3 step-density gradient with percoll completely removed the bacterial contaminants in fresh semen. Therefore, future study is necessary to prove that density gradient method with percoll can eliminate bacteria in fresh semen without significantly affecting sperm viability or function.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.7
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• pp.925-937
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• 2016

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive $F_1$ piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive $F_1$ boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive $F_1$ sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.255-262
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• 2000

The aim of this work was to study the effects of ascorbic acid (Asc) and/or ferrous sulfate (Fe$^{2+}$) and spernatozoa preincubation on the in vitro fertilization in porcine. Porcine follicular oocytes matured in culture were inseminated with frozen-thawed boar spermatozoa preincubated for 0, 1, 2, 3, 4 and 5h. The penetration rates (37~51%) were not significantly different between durations of spermatozoa preincubation in medium with 0.1 mM Asc. The addition of 1.0 mM Fe$^{2+}$ during spermatozoa preincubation were not significantly affecting the penetration rates (41~56%). When spermatozoa were preincubated with Asc and Fe$^{2+}$, the penetration rates had a tendency to increase with time of spermatozoa preincubation, and were significantly (P<0.05) higher in spermatozoa preincubated with that than without Asc and Fe$^{2+}$ for 5 h. On the other hand, when spermatozoa were preincubated in fertilization medium without Asc and/or Fe$^{2+}$, the penetration rates were significantly (P<0.05) higher in medium with Fe$^{2+}$ than with Asc or Asc and Fe$^{2+}$ for in vitro fertilization. The rate of polyspermy in penetrated oocytes in medium with Asc and Fe$^{2+}$ decreased with the period of spermatozoa preincubation. Despite different culture conditions for spermatozoa preincubation, no differences were observed in polyspermy rates in the presence of Asc and/or Fe$^{2+}$ These results indicate the advantage of preincubating spermatozoa with Asc and Fe$^{2+}$ and an addition of Fe$^{2+}$ during in vitro fertilization with spermatozoa preincubated maintain penetration potential without increased polyspermy rates on in vitro fertilization in porcine oocytes.on in porcine oocytes.