Application of new biotechnologies for improvements in swine nutrition and pork production

Abstract

Meeting the increasing demands for high-quality pork protein requires not only improved diets but also biotechnology-based breeding to generate swine with desired production traits. Biotechnology can be classified as the cloning of animals with identical genetic composition or genetic engineering (via recombinant DNA technology and gene editing) to produce genetically modified animals or microorganisms. Cloning helps to conserve species and breeds, particularly those with excellent biological and economical traits. Recombinant DNA technology combines genetic materials from multiple sources into single cells to generate proteins. Gene (genome) editing involves the deletion, insertion or silencing of genes to produce: (a) genetically modified pigs with important production traits; or (b) microorganisms without an ability to resist antimicrobial substances. Current gene-editing tools include the use of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats-associated nuclease-9 (CRISPR/Cas9) as editors. ZFN, TALEN, or CRISPR/Cas9 components are delivered into target cells through transfection (lipid-based agents, electroporation, nucleofection, or microinjection) or bacteriophages, depending on cell type and plasmid. Compared to the ZFN and TALEN, CRISPR/Cas9 offers greater ease of design and greater flexibility in genetic engineering, but has a higher frequency of off-target effects. To date, genetically modified pigs have been generated to express bovine growth hormone, bacterial phytase, fungal carbohydrases, plant and C. elagan fatty acid desaturases, and uncoupling protein-1; and to lack myostatin, α-1,3-galactosyltransferase, or CD163 (a cellular receptor for the "blue ear disease" virus). Biotechnology holds promise in improving the efficiency of swine production and developing alternatives to antibiotics in the future.

Keywords: Biotechnology; Disease; Growth; Health; Meat production; Swine.

Fig. 1

Role of genes in the growth, development, lactation, reproduction, and health of swine. The domesticated pig has 19 pairs of chromosomes (a total of 38 chromosomes), with one set of chromosomes from each parent. A chromosome contains segments of the DNA molecule that are called genes. A trait is controlled by two variant forms of a gene (called an allele) located at the same position (genetic locus) in the pair of chromosomes, with one allele inherited from each parent. Expression of genes through the transcription and translation processes to produce proteins is affected by environmental factors (including nutrition, ambient temperature and disease) in a cell-specific manner

Fig. 2

Scheme of pig cloning from embryonic or adult donor cells. An unfertilized, enucleated oocyte from an adult female pig is fused with the nucleus from an embryonic or adult donor cell via electric pulse to form a new cell. This new cell undergoes division in a test tube (culture medium) into an early stage embryo, which is implanted into the uterus of a sow. Ultimately, the sow gives birth to piglets that have the same genetic makeup as the animal that donated the embryonic or somatic cell

Fig. 3

Recombinant DNA technology. With the action of restriction enzymes, a segment of DNA (insert) is isolated from a pig DNA and a bacterial DNA plasmid is cleaved. Catalyzed by a ligase, a DNA insert joins the open plasmid to create a recombinant DNA plasmid, which is then introduced into E. coli to produce a protein or polypeptide of interest. The plasmid and bacteria replicate rapidly to generate a large amount of the protein or polypeptide

Fig. 4

Production of transgenic animals via the injection of the recombinant DNA into the pronucleus of a fertilized ovum (Method I) or the injection of transformed embryonic stem cells that contain the recombinant DNA into a blastocyst (Method II). In the first and most common method used for livestock species, an ovum is surgically collected shortly after its fertilization, and a recombinant DNA (e.g., the plasmid DNA of interest) is then microinjected through a very fine needle into the pronucleus of the fertilized ovum. The transformed ovum is developed into a blastocyst in vitro and the embryo is then transferred into a surrogate mother for development to term. In the second method, established embryonic stem cells (prepared from a preimplantation embryo) expressing the gene of interest in their genome are transfected with a recombinant DNA. Stably transformed ES cells are selected and then injected into the inner cell mass of a recipient blastocyst. After a short period of culture, the embryo that contains the recombinant gene in its genome is transferred into a surrogate mother for development to term. In both methods, surrogate mothers produce transgenic offspring, but the origin of the blastocyst differs

Fig. 5

Gene (genome) editing of animals using the ZFN, TALEN or CRISPR/Cas9 technique. a designer nuclease (ZFN, TALEN or CRISPR/Cas9) cleaves a DNA molecule to generate a double strand break (DSB) at a desired genomic locus. Thereafter, one of two endogenous repair mechanisms may repair the DSB DNA: non-homologous end joining (NHEJ) and the homology-directed repair (HDR). In the NHEJ pathway, the two ends of the DSB DNA are brought together and ligated without a homologous template for repair, which often inserts or deletes nucleotides (indels) to cause gene disruption (knockout). The HDR pathway requires the provision of an exogenous DNA template along with a site-specific genome editing nuclease to repair the DSB DNA, thereby causing the knock-in of a desired sequence of DNA into the genome of an embryo or animal cells. Because of its more precise targeting of genes, CRISPR/Cas9 is gaining momentum in life sciences as the preferred editor of gene editing of livestock species

Fig. 6

Mechanisms responsible for the development of antimicrobial resistance in bacteria. Bacteria naturally acquire new genes (including antimicrobial-resistant genes) to survive in a new environment or host. The antimicrobial-resistant genes produce enzymes (e.g., extended-spectrum β-lactamase in E. coli) to destroy or inactivate antibiotics. For example, penicillin-resistant bacteria synthesize β-lactamase, which breaks down the β-lactam ring of penicillin to an inactive degradation product. Through this mechanism, the bacteria cannot be killed by penicillin, leading to antimicrobial resistance in infected animals and humans. The sign (X) denotes an inability to kill bacteria

Fig. 7

Utilization of the CRISPR system as a new alternative to antibiotics. The CRISPR-Cas system has an ability to selectively target specific DNA sequences and, therefore, can easily distinguish between pathogenic or commensal bacterial species. Bacteriophages can be utilized to deliver the CRISPR-Cas cargo into bacteria through receiving either a designed DNA that encodes a guide RNA and Cas9 or a guide RNA and Cas3 to cut bacterial DNA molecules at multiple sites, causing self-destruction of the bacteria. Alternatively, the CRISPR-Cas9 system can be utilized to knock out genes responsible for antimicrobial resistance and re-sensitize the multidrug resistant bacteria, so that they will be killed by antibiotics. Finally, a CRISPR-Cas9 system can be constructed as a CRISPR interference (CRISPRi) plasmid vector that carries a DNA sequence for inactivated Cas9 and a guide RNA to silence the expression of membrane-bound virulent proteins and antibiotic-resistant genes