Index > Division of Animal Technology
Division of Animal Technology

The key mission of the Biotechnology Division is applied biotechnology for improving the competitiveness of the animal industry, conducting fundamental research and developing bio-products and/or platforms. To enhance the competitiveness of the animal industry, this year the Institute has developed a biomarker detection kit for egg production and improving the reproductive performance of the Taiwan native chicken (TNC); and has measured the nutritional components of sow milk to improve both piglet growth and sow reproductive performance. Meanwhile, the Institute has invited Finnish expertise to exchange experience and foster international cooperation. In terms of basic research, the Institute has studied the interaction of intestinal cells with porcine immune-cells, the interaction of Chinese herbs and adipocyte, and the ameliorate xenograft-rejection via pig transgenesis. In developing biological products and platforms, the Institute has initiated a new project focused on developing biological products for use in commercial animal husbandry, including anti-serum, viral pathogen detection kits, and bacterial and viral vaccine (single / multi-valent); meanwhile, the Institute has also developed a high efficiency DNA extraction kit. In relation to bio-safety issues, the Institute has established a RT-PCR detection technique for Theiler's Murine Encephalomyelitis virus, and been officially recognized as complying with Good Laboratory Practice. In the transgenic pig isolated-testing station, human FIX (factor IX) activity was enhanced by the addition of Vitamin K to sow rations. The streptozotocin-induced pig for diabetic mellitus animal model has been established for non-clinical trials involving the effect on wound healing of thermoplastic polyurethane dressing with negative pressure.

  • Enhancing Animal Production using Biotechnology
    • 1.Product development of egg-laying biomarker detection for Taiwan native chicken
      • Taiwan native chickens (TNC) are a popular source of meat in Taiwan. However, the reproductive performance (egg production) of TNC has gradually declined. In a project supported by COA, the Institute thus has focused on establishing a standard operating process for producing and purifying apoVLDL-II (apo II) antigen and antibody. Using these key components, a prototype competitive ELISA kit has been assembled for detecting chicken apo II (Fig. 15). The concentration of serum apo II was measured and the correlation of apo II concentration and egg production was analyzed based on a TNC field trial conducted at National Chiayi University (Table 4). The results revealed a significant negative relationship between the concentrations of apo II at 16, 20, 24 weeks and the first laying days. This result demonstrated that apo II biomarker could predict egg production during the premature stage. Furthermore, the concentrations of apo II at 20 and 24 weeks were positively and significantly correlated with total and normal egg number at 32, 40 and 55 weeks. The results identify apo II as a potential biomarker for predicting high laying groups. This platform technology can be further developed as a rapid test strip for egg-productivity to improve the reproductive performance of TNC and thus benefit the community.
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    • 2.Sow milk composition and energy supply
      • Sow reproductive performance selection was commonly applied to increase numbers of pigs produced and reduce production costs, thus helping pig producers survive in the periods of low pig prices. Currently, the sow selection index includes two important traits that require improvement, number of piglets born alive and weaning weight. Measuring piglet weaning weight is both labor intensive and time consuming, and is further complicated by weaning ages varying among litters and the existence of recursive fostering. All of these indicate large variation in sow reproduction performance, which can be improved by selection. For above mention, sow reproductive performances, including number of live births, birth weights, number of piglets weaned, weaning weight, and time from weaning to estrus, were recorded. Litter size was standardized to ensure each sow nursed the same number of piglets. Sow milk samples were taken for composition analysis. Since sow milk was the only energy source available to piglets, milk intake volume and quality strongly impact piglet growth and health. The analytical results show significant differences in composition and total energy, indicating potential for selection to improve piglet weaning weight and thus sow reproductive performance.
    • 3.International swine breeding experience exchange program
      • Swine breeding in most European countries is organized and operated by a single nationwide institute. Exchange programs between these institutes can help realize economic or research cooperation between the respective countries. Successful cooperation may take time to build up but can be very rewarding. With support from the Council of Agriculture (COA), an exchange program was initiated between the Insititute and Nordic Genetics. Nordic Genetics is a company owned by Swedish Quality Genetics and Finnish Finnpig, and is cooperating with the Norwegian Norsvin company in pig breeding. Dr. Timo Serenius from Nordic Genetics was invited to Taiwan in September to lecture as part of breeding workshops in Kaohsiung, Changhua and Taipei. Total 136 attendees at these workshops included breeding staff, government officers, research staff, college staffs and students. Topics ranged from ‘introduction of an effective breeding program' to ‘how to build up a modern breeding program'. The workshop participants suggested establishing a data analysis unit above the recording and testing system to provide breeding value estimates for selection and thus benefit the industry. The workshops were followed up by a short internship program in Sweden and Finland to allow Taiwanese interns to see firsthand how the breeding program was implemented in these countries. Per capita consumption of pork in Sweden and Finland is  around 35kg annually, accounting for 40% of total meat consumption. Although locally produced pork is considered better quality than imported, pig farmers in Sweden and Finalnd are still losing money due to over production and global competition. Animal welfare and environmental protection regulations are also contributing to increasing production costs, but are unavoidable and associated with positive broader social developments. Reducing labor and feed costs, or increasing pig productivity, thus are key priorities for pig farmers seeking profits. In both countries, pig feeding is mechanized (reducing labor requirements and opening the pig farming workforce to women). Feed typically uses locally grown crops to minimize the transportation cost, with use of soybeans being an exception. Pig health is maintained at the minimum acceptable level to save vaccine and drug expenses. Production costs have been further reduced with help from animal breeding. The number of days required for pigs to reach 100kg in Finland and Sweden now decrease from 180 to 150 days shorter than in 1995, and the number of piglets born alive has also increased by one over the same period, with these gains resulting primarily from swine breeding. Sweden and Finland have achieved promising improvements in pig feeding, disease control and performance. In the future, the pig production environment in Taiwan will become increasingly difficult as Taiwan follows overseas trends. By learning from the experience of Sweden and Finland, Taiwanese pig producers thus will become more cooperative and establish integrated production chains to increase production efficiency.
  • Research and Development of Basic Biotechnology
    • 1.Co-culturing human intestine Caco-2 cells with porcine blood mononuclear cells
      • The development of a cell-based model to screen drugs or high throughput efficacy assays for further fine tuning of animal test design is currently a major research trend. Caco-2 cells were seeded on to cell culture inserts (0.4 µm pore size) in 24 well plates at 3x104, 5x104 and 1x105 cells/cm2 and reached confluence separately around days 10, 9 and 7. Caco-2 monolayer integrity in transwells was assessed by measuring TEER using a Millicell-ERS device (Millipore). The TEER values for Caco-2 seeding 5x104 and 1x105 cells/cm2 peaked at approximately 386 Ω.cm2 on days 13, 10 and 17. However, this reading cannot be reached in the 3x104 cells/cm2 seeding condition. Furthermore, 1x105 cells/cm2 Caco-2 cells were co-cultured with porcine blood mononuclear cells (2x106 cells/well) for 24 hours. The TEER values for this coculture condition increased from 320 Ω.cm2 to 350 Ω.cm2. In contrast, the TEER value for the control decreased from 300 Ω.cm2 to 250 Ω.cm2. No notable changes in morphology were observed under phase microscope. Furthermore, Caco2 cell co-cultured with PBMC did not alter ZO-1 protein expression assessed by immunofluorecence (Fig. 16). These preliminary results suggest that the Division has successfully developed a platform for screening drugs for improving intestine immune function.
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    • 2.Inhibitor of adipogenesis
      • Acorus calamus (Araceae) has been applied in both traditional medicines and food supplements. Calamus oil was found to exert anti-adipogenic effects on the differentiation of 3T3-L1 preadipocytes into adipocytes. The major anti-adipogenic component was purified and identified as β-asarone. β-Asarone significantly inhibited intracellular lipid accumulation during adipocyte differentiation in a concentration-dependent manner. Additionally, the protein and mRNA expression levels of C/EBPβ, C/EBPα, and PPARγ were decreased in 3T3-L1 cells treated with β-asarone during differentiation. Phosphorylation of ERK1/2, which is known to regulate early adipogenesis, was attenuated via β-asarone treatment. The above results suggest that β-asarone exerts anti-adipogenic activity, partly by suppressing adipogenic transcription factor expression.
    • 3.Different expressions of human leucocyte antigen-DR in different aged transgenic pigs
      • Increasing the matching number used in typing of human leucocyte antigen (HLA)-II can enhance graft survival in allotransplantation. HLA-DR transgenesis may humanize pig organs and is expected to help overcome xenograft rejections. However, transgenic (Tg) pigs still have endogenous gene (swine leucocyte antigen, SLA) which is codominantly expressed with transgene. This study measured both endogenous (SLA-DR) and transgenes (HLA-DR) on the peripheral blood mononuclear cells (PBMC) in Tg pigs of different ages. Blood samples were collected from the vena cava of 12 pigs (9 Tg and 3 NTg) ranging in age from 42 days to 24 months old. The PBMCs were stained using SLA-DR (FITC, mouse anti-porcine SLA Class II DR, USBiological) and HLA-DR (PE, mouse anti-Human HLA-DR, BD) antibodies, then measured using flowcytometry (B.D.). Some 10,000 PBMCs were inspected and the percentages of SLA-DR and HLA-DR positive PBMCs were recorded. Observation results revealed that Tg pigs at 42 days old expressed HLA-DR positive PBMCs were 11.00±1.90% (mean ± s.e.) and those who more one year old were 6.91±1.56%. Furthermore, only one 42 day old Tg pig expressed 6.51% PBMC only with HLA-DR (Fig. 17 D3), and this phenomena did not appear in adult (>one year old) pigs (Fig. 17 B1 and C1 to C4; A1 to A3 are NTg).
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  • Bio-product Development and Platform Construction
    • 1.Development of veterinary biologic reagent
      • Pets are increasingly important in the world-wide. The "companion animals" is replacing the "pet" that impacts the intimate relationship between human and animal is increasingly. Not only are they gaining increasing legal protection, but the pet-related industry is also strongly growing. The development of the pet-related industry has created ​​a huge market. According to the ITRI IEK report (2009) the United States, Japan and Europe collectively spend US$ 75.3 billion annually on pets. Meanwhile, the pet market in Taiwan represented NT$ 25.0 billion as of 2007.This year, the Institute implemented the "Veterinary Biologics Development Project", a Technology Development Program supported by the Ministry of Economic Affairs. The project involved developing biological reagents for canine distemper virus, canine parvovirus, infectious canine hepatitis virus, Leptospira and Bordetella. Developed items include therapeutic antisera/antibodies, disease detection reagents and single / multi-valent vaccine. These items can be utilized in veterinary clinics, improving disease diagnosis and treatment in the Taiwanese pet industry. As part of the project, pigs were immunized using canine distemper virus-fusion cells to induce anti-serum / antibodies, and thus producing serum with antibody titer exceeding 1,000 folds. The Division also established a purification process and QC analysis methods for producing F(ab')2 antibodies using this anti-serum. For detection kit development, the Division constructed 12 viral protein fragments in various expression vectors and purified the expressed proteins for rabbit immunization, thus producing antisera with antibody titter exceeding 32,000 folds. The antisera of canine distemper virus could recognize canine virus distemper particles. Three strains of Leptospira genome sequence were also fully sequenced. Finally, nine antigens of Leptospira were identified and the associated genes cloned and expressed.
    • 2.High performance DNA extraction Kit
      • Distinct from fish or bird spermatid, spermatid from pig and eutherian mammals has a specially organized nucleus where genomic DNA is caged by covalently cross-linked protamines. Therefore, it is very difficult to isolate genomic DNA by traditional protocols. The Institute has developed a reagent, named HS-1, to solve this problem. Adding merely one twentieth volume of HS-1 to the homogenization buffer of any commercially available genomic DNA isolation kits, either by a precipitation method, such as GeneDirex and Promega, or by a column method, such as Fermenta and QIAGEN, eliminates the need to change the original protocols. Figure 18 shows an example in which no detectable quantity of pig spermatid DNA was obtained using the genomic DNA purification kit from Fermenta, but 6 to 9 micrograms of genomic DNA was routinely extracted from 0.1 mL boar sperm in the presence of HS-1.
    • 3.Establishing real time RT-PCR for Theiler's Murine Encephalomyelitis Virus
      • Theiler's murine encephalomyelitis virus (TMEV) is a natural mouse pathogen that belongs to the Picornaviridae family. TMEV may infect rodents, replicates in the gastrointestinal tract and is transmitted via the fecal-oral rphotooute. The World Health Organization (WHO) regulatory guideline mandates testing of manufacturers of mouse-derived vaccines to verify the absence of TMEV and thus ensure product purity and safety. To help meet this requirement, the Institute has developed a real time RT-PCR and further validated it for specificity, reproducibility and detection limits. The assay directly detected specific amplified products of TMEV by monitoring the increase in emitted fluorescence intensity during Taqman RT-PCR reaction. The reliable detection range was from 10 to 105 PFU/mL with high reproducibility. Encephalomyocarditis virus (EMCV), xenotropic murine leukaemia virus (X-MuLV), and mammalian orthoreovirus serotype 3 (Reovirus 3) were found to verify the specificity of this RT-PCR for TMEV. The limit of detection (LOD) was 10 PFU/mL (Table 5). Moreover, the proposed method is also broadly applicable to bio-technology products derived from murine cell lines, and for monitoring laboratory animal health.
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    • 4.Bio-safety assessment at the transgenic animal testing center
      • The law requires that transgenic animals undergo bio-safety assessment at authorized testing stations before commercial use. Studies of newly developed transgenic animals at qualified testing stations should fully comply with good laboratory practice (GLP). The Department of Health (DOH) administrates GLP certification for medical producers. The DOH suggested that the Transgenic Animal Testing Station be closely integrated with the Biotechnology Laboratory as a functional unit for bio-safety assessment. The new functional unit, Transgenic Animal Testing Center (TATC), was later set up and approved by the Institute. To obtain GLP certification, TATC must demonstrate its ability to perform a GLP compliant study. TATC offered two studies to the DOH for auditing, and an additional two transgenic animal studies were taken. Totally, four studies thus were performed, of which three were completed. To update knowledge of the development and management of transgenic technology among high school science teacher, an introductory seminar was administered to 61 participants during summer. Simultaneously, testing facility web site performance was analyzed and network traffic monitored to ensure the delivery of fresh content. The TATC website has served over 6,490 persons. Improvements in facility and quality were made to ensure TATC can better serve the biotechnology industry and thus increase the competitiveness of biotechnology businesses.
    • 5.Effect of supplementation with 220 ppm rational Vitamin K on homologous human FIX transgenic sows
      • Production of recombinant human factor IX (rhFIX) from hFIX transgenic (Tg) pigs found that pigs fed vitamin K exhibited increased activity (Fig. 19) and γ-carboxylation ratio of rhFIX in Tg sow milk. Following National Research Council (NRC) recommendation, the supplemental dose of vitamin K in pig diets was 500 μg / kg; however, excessive supplementation with vitamin K may cause blood systemic disorder.   This study fed 220 ppm vitamin K (menadione) to Tg sows to examine the effects on physiology, reproduction and offspring. Four homozygotic Tg pigs were tested; in the first parity two sows were randomly selected for the addition of vitamin K and the remaining two sows were fed commercial feed without supplementation; meanwhile, in the second parity the sows fed feed supplemented with vitamin K were swapped over (i.e. the sows previously fed commercial feed received supplemented feed and vice versa). Following parturition, during days 3 to 35, an extra 220 ppm of vitamin K was added to the feed; on days 3, 7, 14, 21, 28 and 35, 15 mL of blood samples were collected via the vena cava for analysis of the RBC, MCV, HCT, PLT, MPV, WBC, HGBTP, ALB, AST and ALT. Litter size at birth, piglet body weight at birth, weekly and at weaning were recorded. The rebreeding performances of the sows were also observed in terms of estrus interval and farrowing rate, number of piglets born and number of stillbirth. Results revealed no differences in blood physiologic and biochemical data, piglet performance and rebreeding performance of sows between fed feed supplemented with and without vitamin K. The results also failed to reveal any effects of excessive vitamin K supplementation.
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    • 6.Effects of thermoplastic polyurethane dressing on negative pressure wound therapy in streptozotocin-induced diabetic pig model 
      • The incidence of diabetes mellitus (DM) is gradually increasing. Due to growing associated public health costs, DM has become an important public health issue in the 21st Century. Owing to similarities between human and porcine characteristics, including physiological, anatomical, and metabolic characteristics, as well as a tendency towards obesity, porcine DM animal model is attracting increasing attention within humanoid animal model research on cardiovascular, endocrine and metabolism issues. Negative pressure wound therapy (NPWT) (Fig. 20) is often applied to the feet of diabetics, as well to pressure ulcers, skin ulcers, burns and in surgical wound care. The NPWT improves healing efficacy and involves treatment using various negative pressure dressings. The ideal dressing should help keep the wound moist, remove excess exudates and toxic components, provide insulated ventilation, and minimize the risk of secondary infection. Thermoplastic polyurethane (TPU), with excellent biocompatibility, is widely applied in various disposable products and implantable medical devices. This investigation uses pig as an animal model for diabetes mellitus and assesses the efficiency of TPU dressing in NPWT. The experimental pig could induce DM syndromes successfully after intravenous infection of doxorubicin followed by surgical removal of a portion of skin on the back. Three types of negative pressure therapy dressings were tested, including: TPU dressing (treatment group), physiological salt foam dressing (control group one) and soaked sterilized water gauze (control group two). Skin samples were collected at the end of the trial for pathological examination. Pathological examination indicated that granulation tissue mainly presented hyperplasia in gross lesions. Microscopic examination found that wound healing surfaces displayed varying degrees of inflammation (infiltration of inflammatory cells and bacteria). The severity of inflammatory response was listed as follows: sterilized gauze had the strongest response, followed by foam dressings, and finally TPU dressing. The granulation tissue mostly comprised fibroblasts and angiogenesis, and the collagen fibers were not obvious. Pathological examination of this phase found no differences between treatment and control groups. It should also be tested by using the earlier organizational samples to understand the early effects of TPU dressing in promoting wound healing. Pathological examination could also involve detailed understanding of negative pressure in the course of treatment. Various mechanisms may be associated with cell proliferation and tissue repair.
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