• 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.