REPRODUCTIVE

BIOTECHNOLOGY IN SWINE

* 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: 1291-1304 2001* Transgenic technology and application in swine Theriogenology

56: 1345-1369 2001

References

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

Control of estrus

Controlledreproduction

in pigs

Breeding pigsat younger ages

Embryo transfer

Increasing litter size

More frequent farrowings

Control of farrowing

Pregnancy testing

Fixed-timeartificial insemination

Biotechnology in livestock

* Reproductive biotechnological procedures

* Molecular genetics

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

Molecular genetics

•Genome analysis e.g. sequencing, mapping anddetermination of polymorphisms ofporcine 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

Animal biotechnologythe first live born offspring in swine

Hammer et al.1985 (microinjection)Transgenic

Prather et al.Polejaeva et al.

1989 (blastomere);2000 (somatic cells)

Nuclear transfer

Saito and Niemann1991Blastomere proliferation

Polge;Nagashima et al.

1985 (4-cell);1988 (mo./bl.)

Embryo bisection

Cheng; Mattioli et al.1985; 1989 (IVM)IVF

Johnson; Rath et al.1991 (SI); 1997 (IVF)Sexing

Hayashi et al.Nagashima et al.

1989 (contr. fr., hatch.bl.); 1995 (z.p.i.)

Embryo freeze

Kvasnickii1951ET

Polge et al.1970AI (frozen/thawed)

Rodin and Lipatow1936AI (fresh)

AuthorsYear of publicationTechnology

Factors affecting semen qualityand 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

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

Advantages of AI

•Genetic improvement e.g., widespread use ofoutstanding sires; improving accuracy of selection throughprogeny test; permitting crossbreeding; introduction of newgenetics

••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

Sex of offspring

•Genetic sex

•Nuclear transfer

•Parthenogenetic activation of ova

•Fusion of two oocytes

Differences between X and Y sperm

* DNA size

* Shape of sperm

* Weight

* Density

* Identity motility

* Surface charge/biochemistry

* Internal biochemistry

•age of semen

•repeat breeding

•co-twin with heifers

The degree of differencesmay be affected by

* Albumin separation

yielding 75-80% Y sperm

* Sephadex filtration

yielding 70-75% Y sperm

Valid laboratory methods to separateX and Y sperm

Methods used : transcervical insemination

surgical insemination

AI is facilitated withestrus synchronization programs

* Extending the luteal phase

* Shortening the luteal phase

The basic requirements forembryo transfer

• A source of embryos

• A reliable method of transferring the

embryos

• Suitably synchronized recipients

The advantage of embryo transfer

•Disease control

•Breeding improvement

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

The variable degrees of success in cryopreservationof embryos in different mammalian species

•Varied responses of certain stages of embryonicdevelopment to different biophysical and physiochemicalparameters

• The nature of and concentration protocol of thecryopreservation used

• The type of programmable freezer

•The thawing rate

•The dilution protocol of the concentration of thecryoprotectant after thawing

Embryos damaged caused by

•The formation of large intracellular ice crystals

•The increased intracellular concentration ofsolutes and accompanying changes

Freezing of the hatchingand hatched blastocysts

Removing cytoplasmiclipids from early embryos

Vitrification

Cryopreservation of pig embryos

Three approaches to the successful cryopreservation of pig embryos

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

Improvement of IVP of pig embryos•2-step protocol of IVM of pig oocytes

•Addition of glutathione is beneficial for the formationof male pronucleus

•Capacitated semen; epididymal semen after freezingand thawing

•Co-culture of oocytes with epithelial cells; addition ofoviductal fluid; preincubation of sperm with FF to reducethe high proportion of polyspermy

Addition of hyaluronic acid to increase monospermicfertilization in IVF

Generation of identical multiples

•Blastomeres from the 4- and 8-cell stage have potentialto grow into blastocysts and to undergo in vivo

developmentblastomere-derived blastocysts have lower ratio of ICM/TE than intactblastocysts

••Nuclear transfer produces larger numbers ofgenetically identical animals of a single genotype

methods: electrofusion or microinjectiondonor cells: blastomere, blastocyst-derived cells, fetal fibroblast

small fibroblasts with a smooth sufracerecipient oocytes: in vivo derived oocytesactivation: Ca2+ dependent and Ca2+ independent approaches

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

The methods to produce transgenic animals

•Retroviral gene transfer methods

•Pronuclear injection of foreign DNA into fertilizedova

•Injection of pluripotent or totipotent embryonic cells

•Sperm-mediated exogenous DNA transfer duringIVF

•Nuclear transfer with embryonic and adultsomatic cells

Embryos or oocytes are exposedin vitro to concentrated virus solutionsor incubated over a single layer ofvirus-producing cells.

The virus enters the perivitelline spacethrough a slit in the ZP

The injection of virusunder the ZP ofoocytes withsubsequentIVM and IVF.

The main advantage of retroviral-mediated genetransfer into animlas

•The technical ease

•the sequence of DNA transferred is limited by size

•the inserted gene is not always expressed in thesecond generation

•many founders are mosaic, with multiple insertionsites (breeding experiments required)

The disadvantage of retroviral-mediated genetransfer into animlas

Pronuclear injection of DNA into a fertilized ovum

The primary advantage of microinjection of cloned DNA

•The higher efficiency compared to other methods

•the method could not be used with embryos of laterdevelopmental stage (>4-cell stages)

•difficult to screen the insertion sites, multiple copieswith configuration of tandem head-to-tail matter or array

•livestock species have a much lower frequency ofintergatrion of foreign DNA into their chromsomes

The disadvantage of microinjection technique

The porcine ES cells (8-15 mm diameter) exhibit a highnuclear/cytoplasmic ratio and contain several prominent nucleioli.

The ES cells usually grow in colonies with colony diameters rangingfrom 0.08-1.5 mm.

Isolation of ES cells, transformation/transfection of ES cells andinjection of ES cells into blastocysts to produce chimeric animals

The main advantages of using ES cells forproduction 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 forincorporation of the foreign DNA before injectedex., tissue specific expression

•offspring arisen from the ES cells carry the transgene inan identical site in the genome

•allow large numbers of genetically identical animals tobe established

•efficiency of transgenic animal production may be increased

•it might be possible to replace existing livestock genes

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

Nuclear transfer with a karyoplast of a derived fromblastomere from a preimplantation embryo oran adult somatic cell