REPRODUCTIVE BIOTECHNOLOGY IN SWINE

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1 REPRODUCTIVE BIOTECHNOLOGY IN SWINE

2 References * Animal breeding and infertility by M. J. Meredith * Controlled reproduction in pigs by I. Gordon * Reproduction in farm animals by E.S.E. Hafez * Progress in reproductive biotechnology in swine Theriogenology 56: * Transgenic technology and application in swine Theriogenology 56:

3 Factors affecting reproductive efficiency * Early development of the pig conceptus * Genetic and environmental factors * Nutrition and sow reproduction * Breeds * Fertility and temperature * Sow culling strategies * Stress, records and reproduction * Effect of antibiotics and hormones

4 Control of estrus Breeding pigs at younger ages Fixed-time artificial insemination Embryo transfer Controlled reproduction in pigs Pregnancy testing Increasing litter size Control of farrowing More frequent farrowings

5 Biotechnology in livestock * Reproductive biotechnological procedures * Molecular genetics

6 Reproductive biotechnological procedures Artificial insemination (AI) Estrus synchronization Induction of parturition Embryo transfer (ET) Cryopreservation of oocytes and embryos Sperm sexing In vitro production of embryos (IVMFC) Embryo bisection Nuclear transfer Microinjection of DNA constructs

7 Molecular genetics Genome analysis e.g. sequencing, mapping and determination of polymorphisms of porcine genes Molecular diagnostics e.g. identify genetic disorders, identity and/or diversity Functional genomics e.g. expression patterns, interaction of genes Transgenic modification e.g. gain or loss of function

8 Animal biotechnology the first live born offspring in swine Technology AI (fresh) AI (frozen/thawed) ET Embryo freeze Sexing IVF Embryo bisection Blastomere proliferation Nuclear transfer Transgenic Year of publication (contr. fr., hatch. bl.); 1995 (z.p.i.) 1991 (SI); 1997 (IVF) 1985; 1989 (IVM) 1985 (4-cell); 1988 (mo./bl.) (blastomere); 2000 (somatic cells) 1985 (microinjection) Authors Rodin and Lipatow Polge et al. Kvasnickii Hayashi et al. Nagashima et al. Johnson; Rath et al. Cheng; Mattioli et al. Polge; Nagashima et al. Saito and Niemann Prather et al. Polejaeva et al. Hammer et al.

9 Factors affecting semen quality and sex driven in boars Effects of nutritional and environmental factors Space requirements Light programes and boar reproduction Fertility problems in the boar Boar culling rate Efficiency of the mating process Overuse and underuse of the boar Boar stimuli at mating time Endocrine factors in boar sperm production

10 Reproductive biotechnological procedures Artificial insemination (AI) Estrus synchronization Induction of parturition Embryo transfer (ET) Cryopreservation of oocytes and embryos Sperm sexing In vitro production of embryos (IVM/F/C) Embryo bisection Nuclear transfer Microinjection of DNA constructs

11 Advantages of AI Genetic improvement e.g., widespread use of outstanding sires; improving accuracy of selection through progeny test; permitting crossbreeding; introduction of new genetics Control of venereal diseases Availability of accurate breeding records Economic service Safety through elimination of dangerous male Use of deep-frozen semen after a donor is dead Gender control

12 Sex of offspring Genetic sex Nuclear transfer Parthenogenetic activation of ova Fusion of two oocytes

13 Differences between X and Y sperm * DNA size * Shape of sperm * Weight * Density * Identity motility * Surface charge/biochemistry * Internal biochemistry

14 The degree of differences may be affected by age of semen repeat breeding co-twin with heifers

15 Valid laboratory methods to separate X and Y sperm * Albumin separation yielding 75-80% Y sperm * Sephadex filtration yielding 70-75% Y sperm

16 Methods used : transcervical insemination surgical insemination AI is facilitated with estrus synchronization programs * Extending the luteal phase * Shortening the luteal phase

17 The basic requirements for embryo transfer A source of embryos A reliable method of transferring the embryos Suitably synchronized recipients

18 The advantage of embryo transfer Disease control Breeding improvement

19 The advantage of cryopreservation of embryos * Embryos contain the complete genome * Enables breeding centers to carry a wider range of stocks * Save space and money e.g., transportation * Afford protection against loss e.g., fire, disease and other hazards * Preserves special genetic combinations, inbred strains and mutations * Research in animal genetics

20 The variable degrees of success in cryopreservation of embryos in different mammalian species Varied responses of certain stages of embryonic development to different biophysical and physiochemical parameters The nature of and concentration protocol of the cryopreservation used The type of programmable freezer The thawing rate The dilution protocol of the concentration of the cryoprotectant after thawing

21 Embryos damaged caused by The formation of large intracellular ice crystals The increased intracellular concentration of solutes and accompanying changes

22 Removing cytoplasmic lipids from early embryos Freezing of the hatching and hatched blastocysts Vitrification Cryopreservation of pig embryos Three approaches to the successful cryopreservation of pig embryos

23 In vitro production of pig embryos An insufficient cytoplasmic maturation of the oocyte An unusually high degree of polyspermic fertilizstion Low numbers of viable blastocysts Too few cells resulting in low development in vivo

24 Improvement of IVP of pig embryos 2-step protocol of IVM of pig oocytes Addition of glutathione is beneficial for the formation of male pronucleus Capacitated semen; epididymal semen after freezing and thawing Co-culture of oocytes with epithelial cells; addition of oviductal fluid; preincubation of sperm with FF to reduce the high proportion of polyspermy Addition of hyaluronic acid to increase monospermic fertilization in IVF

25 Generation of identical multiples Blastomeres from the 4- and 8-cell stage have potential to grow into blastocysts and to undergo in vivo development blastomere-derived blastocysts have lower ratio of ICM/TE than intact blastocysts Nuclear transfer produces larger numbers of genetically identical animals of a single genotype methods: electrofusion or microinjection donor cells: blastomere, blastocyst-derived cells, fetal fibroblast small fibroblasts with a smooth sufrace recipient oocytes: in vivo derived oocytes activation: Ca2+ dependent and Ca2+ independent approaches

26 The aims of transgenic technology in swine Enhance growth and development Increase disease resistance Produce foreign proteins in milk For xenotransplantation * similarity of anatomy and physiology to human organs * possible to be raised under the highest hygienic standards

27 The methods to produce transgenic animals Retroviral gene transfer methods Pronuclear injection of foreign DNA into fertilized ova Injection of pluripotent or totipotent embryonic cells Sperm-mediated exogenous DNA transfer during IVF Nuclear transfer with embryonic and adult somatic cells

28 Embryos or oocytes are exposed in vitro to concentrated virus solutions or incubated over a single layer of virus-producing cells. The virus enters the perivitelline space through a slit in the ZP The injection of virus under the ZP of oocytes with subsequent IVM and IVF.

29 The main advantage of retroviral-mediated gene transfer into animlas The technical ease The disadvantage of retroviral-mediated gene transfer into animlas the sequence of DNA transferred is limited by size the inserted gene is not always expressed in the second generation many founders are mosaic, with multiple insertion sites (breeding experiments required)

30 Pronuclear injection of DNA into a fertilized ovum

31 The primary advantage of microinjection of cloned DNA The higher efficiency compared to other methods The disadvantage of microinjection technique the method could not be used with embryos of later developmental stage (>4-cell stages) difficult to screen the insertion sites, multiple copies with configuration of tandem head-to-tail matter or array livestock species have a much lower frequency of intergatrion of foreign DNA into their chromsomes

32 The porcine ES cells (8-15 mm diameter) exhibit a high nuclear/cytoplasmic ratio and contain several prominent nucleioli. The ES cells usually grow in colonies with colony diameters ranging from mm.

33 Isolation of ES cells, transformation/transfection of ES cells and injection of ES cells into blastocysts to produce chimeric animals

34 The main advantages of using ES cells for production of transgenic animals ES cells may be grown in vitro for many generations ES cells could be transformed in vitro with foreign DNA ES cell line can be isolated and derived from a single cell transformed ES cells can be screened and selected for incorporation of the foreign DNA before injected ex., tissue specific expression offspring arisen from the ES cells carry the transgene in an identical site in the genome allow large numbers of genetically identical animals to be established efficiency of transgenic animal production may be increased it might be possible to replace existing livestock genes

35 The efficiency for production of ES cell lines from porcine blastocysts is low

36 Nuclear transfer with a karyoplast of a derived from blastomere from a preimplantation embryo or an adult somatic cell

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