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Yu J.,Nankai University | Zhao Q.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Sun M.,Nankai University | Zhao X.,Nankai University
Proceedings of the World Congress on Intelligent Control and Automation (WCICA)

Donor cell injection is a significant technique in nuclear transfer and many other biological operations. Recent achievements in biology and biomedicine demonstrate great potential of these micromanipulation technologies. Traditionally, these operations are done manually, which require high professional skills and are of low repeatability. To overcome these disadvantages, automatic operation of this procedure is required. This paper presents a robotic donor cell injection process in Somatic Cell Nuclear Transfer (SCNT). In this process, some techniques including target somatic cell selection, motion control of the injection pipette and the somatic cell, and oocyte penetration are discussed. Finally, experiments are conducted, and the results show that the proposed operating process is capable of performing donor cell injection with comparable speed of human operators. Over 98% detection success rate and 3 um positioning error verify practicability and applicability of the robotic donor cell injection procedure. © 2014 IEEE. Source

Cui M.-S.,China Agricultural University | Cui M.-S.,Tianjin Institute of Animal Science | Liu Z.-X.,China Agricultural University | Wang X.-L.,China Agricultural University | And 4 more authors.
Agricultural Sciences in China

The developmental competence of porcine parthenotes cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 (NCSU-23) media was investigated. After in vitro maturation oocytes were electro-activated, and the subsequent developmental competence, rates of apoptotic, fragmented and arrested embryos from the two media were evaluated. Also, the ratio of mRNA expression of Bcl-2 and Bax gene was determined. Results demonstrated that cleavage, blastocyst, hatched blastocyst rates, and blastocyst cell numbers were significantly higher in PZM-3 than in NCSU-23. The rate of fragmented embryos in PZM-3 was lower than in NCSU-23 on d 1 and 3 (P<0.05) while the percentage of arrested embryo was lower in PZM-3 than in NCSU-23 on d 4 and 5 (P<0.05). TUNEL positive signals were higher in NCSU-23 than in PZM-3 from d 3 to 7 (P<0.05). The ratios of Bcl-2 and Bax mRNA expression in embryos were higher on d 5 than on d 3 and 1 in PZM-3 (P<0.05). In contrast, the ratios of Bcl-2 and Bax mRNA expression in embryos on d 1 were higher than on d 3 and 5 in NCSU-23 (P<0.05). Additionally, the ratios of Bcl-2 and Bax mRNA expression in embryos in PZM-3 were higher than in NCSU-23 on d 3 and 5 (P<0.05). In conclusion, lower apoptotic embryo rates and down-regulating Bax together with up-regulating expression of Bcl-2 transcripts may be responsible for the better developmental competence of porcine parthenotes in PZM-3. © 2011 Chinese Academy of Agricultural Sciences. Source

Yu J.,Nankai University | Cui M.,Tianjin Institute of Animal Science | Sun M.,Nankai University | Zhao X.,Nankai University
Chinese Control Conference, CCC

As an effective approach to manipulate intracellular material exchange, such as injection of transgenic solutions or donor cells into receptor cells, microinjection possesses the incomparable advantage of introducing precisely defined amounts of substances into the cytoplasm without altering other components of the cell. Accurate quantification is generally obtained by calibrating the relationship between the injection volume and injection pressure in advance. Shift in the injection pressure and difference of the initial condition causes large deviation of the results. This paper presents a control method that can achieve the required injection volume with fast speed and high stability based on the flow characteristics of fluids inside a micropipette. First, a targeted control strategy has been brought up to resolve the problem of difficulty in microfluid dynamics modeling and instability of conventional control methods in settling this issue. Next, gray moment operators are employed to locate target edge to subpixel values in order to obtain higher precision of the injection volume. At last, the proposed method is applied to the automated transgenic injection tasks. Regulating time is approximately from 6 to 12 seconds, control accuracy of the injection volume is 0.0182 picoliter, and average operating speed is 1.5 cells/min. © 2015 Technical Committee on Control Theory, Chinese Association of Automation. Source

Deng S.,CAS Institute of Zoology | Yu K.,China Agricultural University | Wu Q.,Beihang University | Li Y.,China Agricultural University | And 5 more authors.
Oxidative Medicine and Cellular Longevity

Toll-like receptor 4 (TLR4) is an important sensor of Gram-negative bacteria and can trigger activation of the innate immune system. Increased activation of TLR4 can lead to the induction of oxidative stress. Herein, the pathway whereby TLR4 affects antioxidant activity was studied. In TLR4-overexpressing sheep, TLR4 expression was found to be related to the integration copy number when monocytes were challenged with lipopolysaccharide (LPS). Consequently, production of malondialdehyde (MDA) was increased, which could increase the activation of prooxidative stress enzymes. Meanwhile, activation of an antioxidative enzyme, glutathione peroxidase (GSH-Px), was increased. Real-Time PCR showed that expression of activating protein-1 (AP-1) and the antioxidative-related genes was increased. By contrast, the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) were reduced. In transgenic sheep, glutathione (GSH) levels were dramatically reduced. Furthermore, transgenic sheep were intradermally injected with LPS in each ear. The amounts of inflammatory infiltrates were correlated with the number of TLR4 copies that were integrated in the genome. Additionally, the translation of γ-glutamylcysteine synthetase (γ-GCS) was increased. Our findings indicated that overexpression of TLR4 in sheep could ameliorate oxidative injury through GSH secretion that was induced by LPS stimulation. Furthermore, TLR4 promoted γ-GCS translation through the AP-1 pathway, which was essential for GSH synthesis. © 2016 Shoulong Deng et al. Source

Li Y.,China Agricultural University | Lian D.,Benedictine University | Deng S.,CAS Institute of Zoology | Zhang X.,Tianjin Institute of Animal Science | And 11 more authors.
Journal of Animal Science and Biotechnology

Background: Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth. Results: In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference. Conclusions: Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep. © 2016 The Author(s). Source

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