Veterinary · Pre-clinical and foundation · also MSVE VPB-321, Animal Biotechnology
Molecular tools used in diagnosis, vaccines and reproduction: recombinant DNA, PCR, cell culture, monoclonal antibodies, cloning, transgenics and genomics, with their biosafety rules.
In the exams: ICAR AICE-JRF core group 08; ICAR AIEEA-PG 12 (Animal Biotechnology)
Veterinary and animal biotechnology is the use of molecular and cellular tools to diagnose, prevent and treat animal disease and to improve animal breeding. It covers recombinant DNA, the polymerase chain reaction and its quantitative form, molecular diagnostics, cell culture, monoclonal antibodies, recombinant and vectored vaccines, stem cells, cloning, artificial insemination, embryo transfer and in-vitro embryo production, transgenic and genome-edited animals, genomic selection and bioinformatics, and the biosafety and regulatory rules that govern all of them. In India the subject corresponds to the Minimum Standards of Veterinary Education course on veterinary biotechnology; elsewhere it is spread across molecular biology, microbiology, immunology, theriogenology and animal breeding courses.
For the practitioner the subject is not abstract. A clinician reads PCR reports and must know when a positive does not mean disease and when a negative is really an invalid test. A herd veterinarian advises on sexed semen, embryo transfer and genomic testing of heifers. A laboratory or vaccine scientist works with cell lines, hybridomas and expression systems under biosafety rules, and a regulator decides whether an edited animal, a clone or a new vaccine may be used.
The subject is examined heavily in India in the ICAR AIEEA (PG) Animal Biotechnology group, in core group 08 of the ICAR AICE-JRF/SRF (PhD) examination and in ASRB NET and scientist recruitment, where questions test definitions, landmark discoveries, principles of each technique and short calculations: qPCR efficiency and fold change, dilutions and concentrations, cell counts and the accuracy and reliability of breeding values. Licensing examinations elsewhere test the same material inside other subjects, chiefly the interpretation of molecular tests, vaccine types and reproductive technologies. This manual gives principles and interpretation, not bench protocols, and deliberately contains no methods for modifying pathogens. Regulations and product approvals differ by country and change often; they are described as they stood when this manual was reviewed and must be checked locally.
Deoxyribonucleic acid (DNA) is a double helix of two antiparallel strands held together by hydrogen bonds between complementary bases: adenine pairs with thymine by two hydrogen bonds and guanine with cytosine by three, which is why GC-rich DNA melts at a higher temperature. The central dogma describes the usual flow of information: DNA is replicated, transcribed into ribonucleic acid (RNA) by RNA polymerase, and messenger RNA (mRNA) is translated into protein on ribosomes. Retroviruses such as bovine leukaemia virus and feline immunodeficiency virus reverse part of this flow: their reverse transcriptase copies RNA into complementary DNA (cDNA), an enzyme that biotechnology borrowed for RT-PCR and for making cDNA libraries. A eukaryotic gene contains exons, which are kept in the mature mRNA, and introns, which are spliced out. Bacteria lack introns, which is why a mammalian gene expressed in Escherichia coli must be supplied as cDNA rather than as genomic DNA. Transcription is controlled by a promoter upstream of the gene and by enhancers that may act at a distance; translation begins at a start codon (AUG, methionine) and ends at one of three stop codons. These points matter in practice: a vaccine antigen made in bacteria will lack the glycosylation it carries in an animal cell, and a diagnostic primer designed across an exon-exon junction will amplify cDNA but not contaminating genomic DNA.
Recombinant DNA technology joins DNA from different sources and propagates it in a host. The first recombinant molecules were made by Paul Berg in 1972, and Stanley Cohen and Herbert Boyer showed in 1973 that a recombinant plasmid could be propagated in E. coli. Concern about safety led to the Asilomar conference of 1975, the origin of today's containment rules. The key tools are few. Type II restriction endonucleases cut DNA at short, usually palindromic, recognition sites; EcoRI, for example, cuts G^AATTC to leave four-base 5' overhangs called sticky ends, whereas other enzymes leave blunt ends. DNA ligase seals the sugar-phosphate backbone. Alkaline phosphatase removes 5' phosphates from a cut vector so it cannot re-close on itself. A cloning vector must carry an origin of replication, a selectable marker (usually an antibiotic-resistance gene) and a multiple cloning site. Plasmids carry inserts up to roughly 10 kb; bacteriophage lambda vectors and cosmids carry larger fragments; bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) carry hundreds of kilobases and were used to build physical maps of livestock genomes. Expression vectors add a strong promoter, a ribosome-binding site and often a purification tag so that the inserted gene is made into protein.
Bacteria are made competent by calcium chloride treatment followed by heat shock, or DNA is driven in by electroporation, a brief high-voltage pulse that opens transient pores. Only a small fraction of cells takes up DNA, so transformants are selected on antibiotic plates. Transformation efficiency is expressed as colonies per microgram of plasmid DNA. To tell recombinant from empty-vector colonies, many plasmids use blue-white screening: the insert interrupts the lacZ alpha fragment, so recombinant colonies cannot cleave the chromogenic substrate X-gal and stay white, while empty vectors give blue colonies when the lac promoter is induced with IPTG. Clones are confirmed by restriction mapping, colony PCR and sequencing. The blotting family completes the toolkit: Southern blotting detects a DNA sequence with a labelled probe, northern blotting detects RNA and western blotting detects protein with antibodies. Agarose gel electrophoresis separates DNA by size because the negatively charged phosphate backbone migrates towards the anode, with small fragments moving faster; a size ladder run alongside allows the product size to be read. Nucleic acid stains, some of them mutagens, must be handled and disposed of according to local rules. A genomic library contains fragments of the whole genome, whereas a cDNA library represents only the genes expressed in the tissue from which mRNA was taken.
The polymerase chain reaction (PCR), devised by Kary Mullis in 1983, copies a chosen stretch of DNA exponentially in a tube. A reaction contains template DNA, two oligonucleotide primers that flank the target on opposite strands, the four deoxynucleoside triphosphates (dNTPs), magnesium ions as the polymerase cofactor, buffer and a heat-stable DNA polymerase, originally from the thermophilic bacterium Thermus aquaticus (Taq). Each cycle has three steps: denaturation at about 94-95 degrees Celsius separates the strands; annealing, typically between about 50 and 65 degrees Celsius depending on primer melting temperature, lets primers bind; and extension at about 72 degrees Celsius, the optimum of Taq polymerase, synthesises new strands. In an ideal reaction the number of target copies doubles each cycle, so n cycles give 2 to the power n copies of each starting molecule. In reality efficiency falls below 100 percent and the reaction enters a plateau as primers and dNTPs are consumed and inhibitors accumulate, which is why the end-point band intensity of conventional PCR is a poor measure of starting quantity. Primer melting temperature can be estimated for short oligonucleotides by the Wallace rule, Tm = 2(A+T) + 4(G+C) degrees Celsius, a rough guide only. Taq lacks 3'-to-5' proofreading, so high-fidelity polymerases are used when the product is to be cloned or sequenced.
Reverse transcription PCR (RT-PCR) first copies RNA into cDNA and is the method for RNA viruses such as foot-and-mouth disease virus, avian influenza virus, Newcastle disease virus, peste des petits ruminants virus and canine distemper virus. Nested PCR uses a second primer pair inside the first product to raise sensitivity and specificity, but opening the tube between rounds greatly increases the risk of carry-over contamination. Multiplex PCR amplifies several targets at once with several primer pairs, for example to separate species or pathotypes in one tube, and needs careful primer design to avoid competition. Loop-mediated isothermal amplification (LAMP) uses a strand-displacing polymerase and four to six primers at a single temperature of about 60-65 degrees Celsius; it needs no thermal cycler and its product can be read by turbidity or colour, which makes it attractive for field and pen-side use, though primer design is harder and non-specific amplification must be controlled. Restriction fragment length polymorphism after PCR (PCR-RFLP) cuts the product with an enzyme whose site is created or destroyed by a mutation and was long the standard way to genotype single-gene defects in cattle and pigs.
Quantitative PCR (qPCR) measures fluorescence every cycle. Signal comes either from a dye that fluoresces when bound to double-stranded DNA, which detects any product including primer-dimers, or from a sequence-specific probe; the commonest probe is the hydrolysis (5' nuclease) probe, which carries a reporter and a quencher and releases fluorescence when the polymerase degrades it during extension. The threshold cycle (Ct, also called Cq) is the cycle at which fluorescence crosses a threshold set in the exponential phase. Ct is inversely related to the logarithm of the starting quantity: a low Ct means much target, a high Ct little. A standard curve of a ten-fold dilution series gives slope and efficiency: efficiency E = 10 to the power (-1/slope) minus 1. A slope of -3.32 corresponds to 100 percent efficiency, when each ten-fold dilution shifts Ct by 3.32 cycles; most laboratories accept roughly 90-110 percent. With dye chemistry, a melt curve after cycling checks that a single specific product was made. Relative expression is often calculated by the comparative 2 to the power -delta-delta-Ct method: delta-Ct is target Ct minus reference-gene Ct in each sample, delta-delta-Ct is the treated delta-Ct minus the calibrator delta-Ct, and fold change is 2 to the power -delta-delta-Ct. The method assumes near-equal, near-100 percent efficiencies for target and reference, and the reference gene must be shown stable under the experimental conditions. The MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments, 2009) list what a credible qPCR report must include. A Ct cut-off for calling a sample positive is set by each validated assay; it is not a universal number, and a high Ct may mean little target, degraded sample or late non-specific signal.
Molecular diagnostics detect nucleic acid of a pathogen or a host genetic variant. Their strengths are speed, high analytical sensitivity, the ability to detect organisms that are slow, dangerous or impossible to culture (Mycobacterium avium subspecies paratuberculosis, Leptospira, Anaplasma, Theileria, many viruses) and the ability to type strains. Their central limitation is that nucleic acid is not proof of a live, infectious organism or of disease: PCR can detect dead bacteria after treatment, residual vaccine virus after live vaccination, or latent herpesvirus DNA in a healthy carrier. Interpretation therefore always joins the result to the clinical picture, the sample and the timing. Sample choice is critical. Viraemia may be short, so a negative blood PCR days after fever has subsided does not exclude infection; for Leptospira, blood is most useful early and urine later in the course. Sample quality also matters: inhibitors in faeces, blood (haem), milk and tissues can give false negatives, so validated assays include an internal amplification control, and a failed internal control makes the result invalid rather than negative. Serology and PCR answer different questions: PCR says whether agent nucleic acid is present now; antibody tests say whether the animal has met the agent or a vaccine. A calf with persistent bovine viral diarrhoea virus infection is PCR positive and often antibody negative because it is immunotolerant, a classic pattern examiners ask about.
Because PCR copies a few molecules billions of times, amplicon carry-over is the main cause of false positives. Laboratories separate pre-amplification and post-amplification areas with one-way work flow, use filter tips, aliquot reagents and include controls in every run: a no-template (negative) control, a positive control, an extraction control and an internal amplification control. Some protocols use uracil-N-glycosylase with dUTP to destroy carry-over amplicons. Diagnostic performance is described in the same terms as any test. Diagnostic sensitivity is the proportion of infected animals that test positive; diagnostic specificity is the proportion of uninfected animals that test negative. These are distinct from analytical sensitivity (the limit of detection in copies per reaction) and analytical specificity (not cross-reacting with related organisms). Predictive values depend on prevalence: in a population where only 2 percent of animals are infected, even a test with 95 percent sensitivity and 98 percent specificity will give about as many false positives as true positives, so a positive result in a low-prevalence setting, such as surveillance in a country free of the disease, must be confirmed by a second test and investigation. The World Organisation for Animal Health (WOAH) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals sets out validation stages and the recommended tests for each listed disease, and assays used for international trade should be validated for that purpose.
Sanger (dideoxy chain-termination) sequencing reads a single PCR product of up to about 800-1,000 bases and remains the reference method for confirming a mutation or typing a virus gene. Next-generation (massively parallel) sequencing reads millions of fragments at once and underlies whole-genome sequencing of bacterial isolates for outbreak tracing and antimicrobial resistance genes, metagenomic detection of unknown agents and genotyping of livestock. Molecular typing places an isolate in a lineage or serotype: sequencing of the VP1 region is used for foot-and-mouth disease virus, and sequencing of the haemagglutinin cleavage site helps predict avian influenza pathogenicity, which is formally defined by the intravenous pathogenicity index or by that sequence. Inherited defects are diagnosed with allele-specific PCR, PCR-RFLP, probe assays or SNP arrays. Standard examples are bovine leukocyte adhesion deficiency (BLAD, a mutation in the integrin beta-2 gene, CD18) and deficiency of uridine monophosphate synthase (DUMPS) in Holsteins, complex vertebral malformation (SLC35A3) in Holsteins, porcine stress syndrome caused by a ryanodine receptor (RYR1) mutation, hyperkalaemic periodic paralysis (SCN4A) in Quarter Horse lines, and the ABCB1 (MDR1) deletion that makes collies and related breeds sensitive to ivermectin and some other drugs. Carrier testing of breeding sires lets breeders avoid carrier-by-carrier matings without discarding otherwise valuable genetics. Freemartinism in a heifer born co-twin to a bull is confirmed by detecting Y-chromosome sequences in her blood by PCR or by karyotyping for XX/XY chimerism.
Animal cell culture grows cells outside the body under controlled conditions and is the basis of virus isolation, many vaccines, toxicity testing and hybridoma work. A primary culture is taken directly from tissue, for example chick embryo fibroblasts or bovine kidney cells; it resembles the tissue closely but has a limited life. Subculturing gives a secondary culture and, if cells survive, a cell line. Finite cell lines, such as many diploid fibroblast strains, stop dividing after a limited number of population doublings (the Hayflick limit); continuous cell lines have become immortal, often with altered chromosome number, and grow indefinitely. Common veterinary lines include Vero (African green monkey kidney), MDBK (Madin-Darby bovine kidney), MDCK (Madin-Darby canine kidney), BHK-21 (baby hamster kidney, widely used to grow foot-and-mouth disease and rabies vaccine virus) and CRFK (Crandell-Rees feline kidney). Adherent cells grow attached to treated plastic and normal cells show contact inhibition, stopping when a monolayer is confluent; transformed cells lose it and pile up. Suspension cells grow free in medium and scale up in bioreactors. A basal medium such as minimum essential medium supplies salts, glucose, amino acids and vitamins; serum, classically fetal bovine serum, supplies growth factors and attachment proteins, and serum-free media are increasingly used to avoid variability and adventitious agents. Cultures are held at about 37 degrees Celsius, with a bicarbonate buffer balanced against about 5 percent carbon dioxide and phenol red as a pH indicator.
Adherent cells are detached for passage with trypsin, often with EDTA to chelate the calcium and magnesium that support cell adhesion. Cells are counted in a haemocytometer: with the standard chamber, the mean count per large square multiplied by the dilution factor and by 10,000 gives cells per millilitre. Trypan blue dye exclusion estimates viability because living cells with intact membranes exclude the dye while dead cells take it up and stain blue. All work is done aseptically in a biological safety cabinet. Contamination is the chief practical problem. Bacteria and fungi usually cloud the medium quickly; Mycoplasma is more dangerous because it passes through ordinary filters, does not cloud the medium and silently alters cell behaviour, so it is detected by PCR or DNA staining. Cross-contamination of one cell line by another has invalidated many published studies, which is why authentication is required. Pestiviruses such as bovine viral diarrhoea virus can contaminate fetal bovine serum and therefore cell lines and vaccines, so serum and cell banks are tested. Cells are preserved by slow freezing in medium containing a cryoprotectant, commonly about 10 percent dimethyl sulphoxide, and stored in liquid nitrogen at -196 degrees Celsius; thawing is rapid. Production uses a master cell bank and working cell banks tested for sterility, Mycoplasma, adventitious viruses and identity, so that every vaccine batch comes from characterised cells at a limited passage level.
Cell culture isolates viruses, which are recognised by cytopathic effect (CPE): rounding, syncytium formation (for example with paramyxoviruses), inclusion bodies or plaque formation. Non-cytopathic viruses, such as non-cytopathic strains of bovine viral diarrhoea virus, give no visible change and must be detected by immunostaining or PCR. Virus quantity is expressed as plaque-forming units or as the 50 percent tissue culture infective dose (TCID50), calculated by methods such as Reed-Muench or Spearman-Karber from a dilution series. Virus neutralisation tests in culture measure functional antibody and are reference serological tests for several diseases. Cell culture replaced embryonated eggs for many vaccines, though eggs remain important for avian influenza and Newcastle disease viruses. Beyond virology, cell and tissue cultures support in vitro toxicology and so help apply the 3Rs principle (replacement, reduction and refinement of animal use), organ culture, and the in vitro embryo work described in later chapters. Three-dimensional cultures, organoids and cultured meat grown from livestock muscle cells are active research areas; regulatory status of cultured meat differs sharply between countries and is changing.
A monoclonal antibody is a single antibody species, of one specificity and one isotype, produced by a clone of cells derived from one B lymphocyte. Georges Kohler and Cesar Milstein described the hybridoma method in 1975 and shared the 1984 Nobel Prize for it. A mouse (or rat) is immunised with the antigen; its spleen B cells, which make antibody but die in culture, are fused with myeloma cells, which are immortal but chosen not to secrete their own immunoglobulin. Fusion is induced with polyethylene glycol or by electrofusion. Selection relies on HAT medium, containing hypoxanthine, aminopterin and thymidine. Aminopterin blocks the de novo pathway of nucleotide synthesis, so cells must use the salvage pathway, which needs the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The myeloma partner is HGPRT-deficient and dies in HAT; unfused spleen cells die naturally within days; only hybrids, which gain HGPRT from the B cell and immortality from the myeloma, survive. Supernatants are screened, usually by ELISA, for the wanted antibody; positive wells are cloned by limiting dilution so that each culture derives from a single cell, and the clone is expanded in culture and cryopreserved. Growing hybridomas as ascites in mice is discouraged or banned in many countries on welfare grounds, and in vitro bioreactor production is preferred.
Mouse antibodies given repeatedly to another species provoke an anti-mouse antibody response, which shortens their action and can cause hypersensitivity. Recombinant DNA methods solve this by chimerisation (mouse variable regions on host constant regions), humanisation or, in veterinary medicine, caninisation and felinisation, in which only the antigen-binding loops are grafted onto a species framework. Antibody fragments (Fab, single-chain variable fragments) and camelid single-domain antibodies (nanobodies, from the heavy-chain-only antibodies of camels and llamas) are smaller and more stable. Phage display selects antibody fragments from large libraries without immunising an animal. Diagnostic uses are the largest: monoclonals give competitive and blocking ELISAs their specificity, type viruses and bacteria, identify lymphocyte subsets by flow cytometry and underpin lateral-flow pen-side tests. Therapeutic monoclonal antibodies have entered companion-animal practice; licensed products include caninised and felinised antibodies against nerve growth factor for osteoarthritis pain and a caninised antibody against interleukin-31 for pruritus in atopic dermatitis. Availability and approved indications differ by country. Polyclonal antisera remain useful where recognition of many epitopes is an advantage, for example in some antitoxins.
Conventional vaccines are either live attenuated or inactivated whole organisms. Live vaccines give strong cell-mediated and humoral immunity with few doses but can revert to virulence, spread to unintended hosts, and be unsafe in pregnant or immunosuppressed animals; inactivated vaccines are safe from reversion but usually need adjuvant and boosters and give weaker cellular immunity. Biotechnology offers vaccines built from only the protective parts of a pathogen or delivered by a harmless carrier. This chapter covers principles; laboratory methods for modifying pathogens are outside its scope and are regulated as described in the biosafety chapter. A sub-unit vaccine contains one or a few purified antigens. They may be purified from the organism or, more often now, produced as recombinant proteins in bacteria, yeast, insect cells infected with a baculovirus vector, or mammalian cells. Hepatitis B surface antigen made in yeast, licensed for humans in 1986, was the first recombinant vaccine. In veterinary practice, the capsid protein encoded by ORF2 of porcine circovirus type 2, expressed in the baculovirus-insect cell system, assembles into virus-like particles (VLPs) that form widely used vaccines. VLPs mimic the virus surface in repeated arrays that stimulate B cells strongly but contain no genome and cannot replicate. A recombinant tick gut antigen, Bm86, is the basis of anti-tick vaccines for cattle, which reduce tick numbers and reproduction rather than preventing attachment. Sub-unit vaccines are safe and can be designed to allow DIVA, but they are usually less immunogenic and need adjuvants.
Vectored vaccines put a gene for a protective antigen into a harmless live carrier that expresses it inside the host, so they combine the safety of a sub-unit with the immune stimulation of a live vaccine. Examples include canarypox-vectored vaccines for canine distemper, feline leukaemia and equine influenza; herpesvirus of turkeys (HVT) vectors carrying Newcastle disease, infectious bursal disease or avian influenza genes, given in ovo or at hatch; fowlpox vectors in poultry; and a vaccinia virus expressing the rabies glycoprotein used in oral baits that helped eliminate fox rabies from much of Western Europe. Canarypox does not replicate productively in mammals, which is part of its safety. Gene-deleted marker vaccines lack a non-essential gene, and the classic example is the glycoprotein E (gE)-deleted Aujeszky's disease (pseudorabies) vaccine: vaccinated pigs have no antibodies to gE, so a gE ELISA separates infected from vaccinated animals. This DIVA principle (differentiating infected from vaccinated animals) also underlies gE-deleted infectious bovine rhinotracheitis vaccines and non-structural-protein tests for purified foot-and-mouth disease vaccines, and lets a country vaccinate while still proving freedom. DNA vaccines inject a plasmid encoding the antigen, which host cells express; examples licensed in animals include a West Nile virus vaccine for horses licensed in the USA and a canine melanoma vaccine encoding human tyrosinase. Messenger RNA vaccines in lipid nanoparticles are under active development for animals. Reverse vaccinology screens a sequenced genome for surface-exposed proteins as candidate antigens. Adjuvants such as aluminium salts, oil emulsions and saponins shape the response; aluminium salts favour antibody, while some emulsions and saponins also drive cell-mediated immunity. Injection-site reactions, and sarcomas at injection sites in cats, are recognised adverse events.
A stem cell can both renew itself and give rise to differentiated cells. Potency describes how many cell types it can form. The zygote and early blastomeres are totipotent, able to form the whole conceptus including extra-embryonic membranes. Embryonic stem (ES) cells from the inner cell mass of the blastocyst are pluripotent, able to form all three germ layers; they were derived from the mouse in 1981 and are proved by teratoma formation and, in mice, by contributing to chimeras including the germ line. Establishing true germ-line-competent ES cells from livestock was historically difficult, and stable bovine and porcine pluripotent lines have only been reported relatively recently. Induced pluripotent stem cells (iPSCs) were produced in 2006 by Kazutoshi Takahashi and Shinya Yamanaka, who reprogrammed mouse fibroblasts with four transcription factors: Oct4, Sox2, Klf4 and c-Myc. Yamanaka shared the 2012 Nobel Prize with John Gurdon, whose frog nuclear-transfer work had shown that a differentiated nucleus can be reprogrammed. iPSCs have since been made from dogs, horses, pigs and cattle. Adult (somatic) stem cells are multipotent; they include haematopoietic stem cells, spermatogonial stem cells and mesenchymal stromal cells.
Mesenchymal stem or stromal cells (MSCs) are isolated from bone marrow, adipose tissue, umbilical cord blood and tissue, and other sites. Minimal criteria widely cited from the International Society for Cellular Therapy (2006, for human cells) are adherence to plastic, expression of CD73, CD90 and CD105, absence of haematopoietic markers such as CD45 and CD34, and ability to differentiate into bone, cartilage and fat in vitro; markers differ somewhat between species, so veterinary laboratories validate their own panels. Much of their effect is now attributed to secreted factors that modulate inflammation rather than to engraftment and differentiation. Veterinary use began in horses, where autologous bone-marrow or adipose-derived cells are injected into superficial digital flexor tendon lesions, and extends to canine osteoarthritis. Evidence from controlled trials is limited and mixed, and products range from minimally manipulated autologous preparations to licensed allogeneic cell products; the regulatory framework differs widely by country, with some regulators treating cell therapies as veterinary medicinal products. Risks include contamination, transmission of infection with allogeneic cells, ectopic tissue formation and, with pluripotent cells, tumour formation. Spermatogonial stem cell transplantation, in which donor germ-line stem cells colonise the testis of a sterile recipient, is a research route to spreading valuable male genetics. Platelet-rich plasma is often used alongside cell therapy but is not a stem cell product.
Cloning produces animals genetically identical in their nuclear DNA. Embryo splitting (bisection) divides a morula or blastocyst into two halves that are transferred separately, giving identical twins; it is used commercially in cattle but gives at most a few copies. Blastomere nuclear transfer uses cells of an early embryo as nuclear donors. Somatic cell nuclear transfer (SCNT) transfers the nucleus of an adult or fetal body cell into an enucleated oocyte, usually one matured in vitro. The reconstructed embryo is activated by electrical or chemical stimuli, cultured to the blastocyst stage and transferred to a synchronised recipient. Dolly the sheep, born at the Roslin Institute in Scotland in 1996, was the first mammal cloned from an adult somatic cell: the nucleus came from a mammary gland cell of a Finn Dorset ewe and was placed in an enucleated oocyte from a Scottish Blackface ewe, which is why Dolly was white-faced. Cattle, goats, pigs, mice, cats, horses, dogs, camels and water buffalo have since been cloned; India's National Dairy Research Institute, Karnal, produced cloned buffalo calves from 2009 using a simplified hand-guided method that does not need a micromanipulator. Handmade cloning of this kind removes the zona pellucida and bisects oocytes manually.
SCNT remains inefficient: typically only a few percent of reconstructed embryos transferred give a live, healthy offspring, although rates vary with species, donor cell and laboratory. Losses occur throughout pregnancy, and cloned calves and lambs may show large offspring syndrome, with oversize, placental abnormalities (fewer, enlarged placentomes and hydrallantois), respiratory and metabolic problems at birth. The cause is incomplete epigenetic reprogramming: the donor nucleus carries the DNA methylation and histone marks of a differentiated cell, which the oocyte must erase and reset, and errors affect imprinted genes that control fetal and placental growth. Clones that survive the perinatal period generally appear healthy, and their offspring produced by normal breeding do not show the syndrome, because the germ line resets the epigenetic marks. A clone shares its nuclear genome with the donor but has the mitochondrial DNA of the oocyte, and it may differ in phenotype because of environment and epigenetics, so a cloned animal is not a guaranteed copy of the donor's appearance or performance. Uses include multiplying elite breeding animals, preserving rare or endangered genotypes, restoring valuable animals from cryopreserved cells, and above all producing genetically modified animals, because a cell line can be edited and checked in culture before it is used as a nuclear donor. Regulators in several countries have assessed food from clones and their offspring; approaches differ, with some requiring no special controls and others restricting or seeking to label such products, and animal welfare concerns about the method weigh heavily in some jurisdictions.
Artificial insemination (AI) is the first and still the most widely used reproductive biotechnology. Lazzaro Spallanzani inseminated a bitch in the eighteenth century, Ilya Ivanov developed practical methods in Russia in the early twentieth century, and the discovery that glycerol protects sperm during freezing (Polge, Smith and Parkes, 1949) made frozen semen, stored in straws in liquid nitrogen at -196 degrees Celsius, the basis of dairy breeding worldwide. AI multiplies a proven sire's genes across thousands of herds, prevents venereal diseases such as bovine genital campylobacteriosis and trichomonosis when semen comes from tested bulls, and makes progeny testing possible. Its success depends on heat detection and timing; in cattle the traditional morning-evening guide is to inseminate cows seen in oestrus in the morning that evening, and those seen in the evening the next morning, while fixed-time AI after hormonal synchronisation removes heat detection altogether. Sexed semen is produced by flow cytometric sorting. X-chromosome-bearing sperm carry slightly more DNA than Y-bearing sperm, a difference of about 4 percent in cattle; after staining with a DNA-binding fluorescent dye, the sorter measures each cell's fluorescence and deflects X- and Y-bearing sperm into separate streams. Accuracy of about 90 percent for the chosen sex is typical in cattle. Because sorting is slow and damages some sperm, straws contain far fewer sperm than conventional ones, and conception rates are generally lower than with conventional semen, so sexed semen is usually recommended for well-managed heifers. Its main value is producing replacement heifers in dairy herds and avoiding unwanted male calves, a significant welfare and economic issue where male dairy calves have little value or cannot be slaughtered.
Embryo transfer (ET) lets a genetically superior female produce many more offspring than she could carry herself. In multiple ovulation and embryo transfer (MOET), the donor is superovulated, most commonly with follicle-stimulating hormone given twice daily over about four days because of its short half-life, or with a single injection of equine chorionic gonadotrophin (eCG, formerly PMSG), whose long half-life can over-stimulate. Prostaglandin F2-alpha is given during the treatment to regress the corpus luteum, and the donor is inseminated at oestrus, often twice. Embryos are recovered non-surgically in cattle about day 6-8 after oestrus, usually day 7, by flushing the uterus through a balloon catheter; small ruminants and pigs often require surgical or laparoscopic collection. Embryos are evaluated under a stereomicroscope and graded by the International Embryo Technology Society (IETS) system for stage of development and quality. Response to superovulation is highly variable between donors and treatments, and the number of transferable embryos per flush varies widely. Recipients are synchronised so that their oestrus falls within about a day of the donor's, and a single embryo is placed in the uterine horn on the side of the corpus luteum. Embryos can be frozen by slow freezing (with ethylene glycol permitting direct transfer after thawing) or by vitrification. Embryos from tested donors, handled according to IETS and WOAH washing protocols with intact zona pellucida, carry a much lower risk of transmitting many diseases than live animals, which is why embryos are used to move genetics across borders. MOET nucleus breeding schemes, proposed in the early 1980s, increase selection intensity on females and shorten the generation interval.
In-vitro embryo production (IVP) has overtaken conventional MOET in cattle in several countries, notably Brazil. Oocytes are collected from live donors by transvaginal ultrasound-guided follicle aspiration (ovum pick-up, OPU), which can be repeated regularly, including in pregnant cows and prepubertal heifers, or from abattoir ovaries. The procedure has three laboratory steps. In-vitro maturation (IVM) takes about 22-24 hours in cattle, during which the oocyte resumes meiosis to metaphase II. In-vitro fertilisation (IVF) follows, with frozen-thawed sperm capacitated, often with heparin, since sperm must be capacitated before they can fertilise. In-vitro culture (IVC) then continues for about 7-8 days to the blastocyst stage, under reduced oxygen in many laboratories. Blastocyst rates are commonly quoted in the range of roughly a third of matured oocytes in cattle, varying widely by laboratory and donor. A single expensive dose of sexed semen can fertilise oocytes from several donors, so IVP makes good use of sexed semen. In-vitro-produced embryos are more sensitive to cryopreservation than in vivo embryos and have been associated with larger calves and altered gene expression, a milder form of the problems seen after cloning. Intracytoplasmic sperm injection (ICSI) injects one sperm into the oocyte and is used routinely in horses, where conventional equine IVF has been unreliable. Buffalo, sheep, goats and camels are also served by OPU-IVP, generally with lower efficiency than cattle. OPU-IVP combined with genomic testing of embryos or young females is the fastest current route to genetic progress in dairy cattle.
A transgenic animal carries foreign DNA integrated into its genome and passes it to offspring; a genome-edited animal carries a targeted change to its own DNA, which may or may not include foreign sequence. Pronuclear microinjection, in which a DNA construct is injected into a pronucleus of a fertilised egg, produced the first transgenic mice around 1980, the growth-hormone mice of Palmiter and Brinster in 1982, and the first transgenic pigs, sheep and rabbits in 1985. It is inefficient in livestock, with only a small percentage of injected eggs giving transgenic offspring; integration is random, copy number uncontrolled, expression depends on the surrounding chromatin (position effect), and founders may be mosaic because integration happened after the first cell division. Retroviral and lentiviral vectors integrate efficiently but carry limited DNA. In mice, gene targeting in ES cells by homologous recombination produced knockouts. In livestock, the route was instead to modify cultured somatic cells and then use them as SCNT donors, so that every cloned offspring carries the change. Programmable nucleases transformed the field: zinc-finger nucleases, TALENs and above all CRISPR-Cas9, in which a guide RNA directs the Cas9 nuclease to cut a chosen sequence. The cell repairs the break by error-prone non-homologous end joining, often knocking the gene out, or by homology-directed repair using a supplied template to introduce a precise change. Editing reagents can be injected directly into zygotes, avoiding cloning, though mosaicism is common. Off-target cuts and unintended on-target outcomes must be checked by sequencing.
Early aims were faster growth and disease resistance; the outcome was more varied. Pigs carrying growth hormone transgenes in the 1980s showed severe health problems, an early lesson in animal welfare. Transgenic animals as bioreactors secrete human proteins in milk: recombinant human antithrombin from goat milk was approved in the European Union in 2006 and the USA in 2009. A growth-hormone transgenic Atlantic salmon, which reaches market size faster, was approved as food by the United States Food and Drug Administration in 2015. Pigs lacking the alpha-gal epitope were approved in the USA in 2020 for food and potential medical use, and multi-gene edited pigs are central to xenotransplantation research. Edited or naturally occurring variants have been introduced for traits already present in some breeds, such as polledness (hornlessness) moved from beef into dairy cattle to avoid dehorning; a later regulatory analysis found that some of these animals also carried unintended bacterial plasmid DNA integrated at the edited site, an example of why whole-genome checks matter. Knocking out or editing CD163, the receptor used by porcine reproductive and respiratory syndrome virus, gives pigs that resist infection in experimental challenges. Myostatin knockouts reproduce double muscling seen naturally in some cattle breeds. Mice and pigs engineered to model human or animal diseases are research tools. Concerns include welfare of founders and surrogates, unintended effects, escape of fertile animals such as fish into the environment, consumer acceptance and trade, and whether small edits indistinguishable from natural mutations should be regulated like transgenes, a question countries answer differently.
The first livestock reference genomes were the chicken (2004) and cattle (2009) assemblies, followed by pig, sheep, goat, horse, dog, buffalo and others, and they are continually improved. The cattle genome is about 2.7 billion base pairs across 29 pairs of autosomes plus X and Y (2n = 60). Genetic markers are variable DNA sites whose inheritance can be followed. Microsatellites (short tandem repeats) are highly polymorphic and were the standard for parentage testing and early linkage mapping; single nucleotide polymorphisms (SNPs) are far more numerous, suit automation on arrays carrying tens to hundreds of thousands of SNPs, and have largely replaced them, including for parentage. Genome-wide association studies (GWAS) test each SNP for association with a trait and rely on linkage disequilibrium, the non-random association of alleles at nearby loci, between markers and causal variants. Classic major genes found this way or by candidate-gene work include DGAT1 (the K232A variant on bovine chromosome 14 with a large effect on milk fat yield and content), myostatin loss-of-function mutations causing double muscling (for example an 11-base-pair deletion in Belgian Blue cattle), the Booroola fecundity gene FecB (a BMPR1B mutation that raises ovulation rate, carried by Garole sheep of India), callipyge muscle hypertrophy in sheep, and RYR1 in pigs.
Marker-assisted selection (MAS) uses a few markers linked to, or inside, genes of large effect. It works well for single genes, such as eliminating carriers of recessive defects, fixing polledness, or introgressing FecB, but most economic traits are polygenic, with many genes of small effect, and MAS explained too little of their variance to change breeding much. Genomic selection, proposed by Meuwissen, Hayes and Goddard in 2001, instead estimates the effects of all markers across the genome simultaneously in a reference population of animals that have both genotypes and reliable phenotypes or daughter-proven breeding values. Young animals with genotypes only then receive a genomic estimated breeding value (GEBV), the sum of their marker effects. Common methods are genomic best linear unbiased prediction (GBLUP), which replaces pedigree relationships with a genomic relationship matrix, Bayesian methods that allow some markers to have larger effects, and single-step GBLUP, which combines genotyped and non-genotyped animals in one evaluation. The accuracy of a breeding value is the correlation between estimated and true breeding value; reliability is accuracy squared. Genomic accuracy rises with reference population size and trait heritability and falls with the effective number of independent chromosome segments, so small breeds and indigenous populations benefit from pooling reference populations across countries. Genetic gain per year follows the breeder's equation: gain equals selection intensity times accuracy times additive genetic standard deviation, divided by generation interval. Genomic selection in dairy cattle gives young bulls accuracies below those of fully progeny-tested bulls but cuts the generation interval from about five to seven years to about two to three, so annual gain has risen markedly; analyses of US Holsteins found annual gains for yield traits rose by roughly 50 to 100 percent after its introduction. Low-heritability traits such as fertility and health gained most.
Bioinformatics stores, retrieves and analyses biological sequence data. The International Nucleotide Sequence Database Collaboration links GenBank (National Center for Biotechnology Information, USA), the European Nucleotide Archive (EMBL-EBI) and the DNA Data Bank of Japan, which exchange data daily; protein information is held in UniProt and structures in the Protein Data Bank. Genome browsers such as Ensembl display annotated genomes. Sequences are stored as FASTA (sequence only) or FASTQ (sequence plus a quality score for each base). A Phred quality score Q equals -10 times log10 of the probability that a base call is wrong, so Q20 means a 1 in 100 error and Q30 a 1 in 1,000 error. The Basic Local Alignment Search Tool (BLAST) compares a query with a database and reports the best matches with an E-value, the number of hits of that score expected by chance; the smaller the E-value, the more significant the match. Pairwise and multiple sequence alignment underpin primer design and phylogenetic trees, which place an outbreak virus among known lineages. Read depth or coverage equals number of reads times read length divided by genome size. Whole-genome sequencing of bacterial isolates, compared by SNP distances or core-genome typing, now links cases in food-borne and antimicrobial-resistance investigations, and metagenomics identifies microbial communities in the rumen, gut or an unexplained disease outbreak. Bioinformatic results are only as good as the reference databases, which contain errors and uneven coverage of animal pathogens and livestock breeds.
Biosafety protects people, animals and the environment from biological agents and genetically modified organisms; biosecurity in the laboratory sense protects agents from loss, theft or misuse. Agents are placed in risk groups 1 to 4 according to their ability to cause disease, transmissibility and the availability of prevention and treatment, and work is done at a matching containment or biosafety level (BSL-1 to BSL-4). The World Health Organization Laboratory Biosafety Manual (fourth edition, 2020) moved from rigid levels to a risk-based approach in which each procedure is assessed. Animal pathogens are also classified for their risk to animal health; foot-and-mouth disease virus, for example, is not a significant human pathogen but requires high containment because escape would be economically devastating. WOAH's standard on managing biological risk in the veterinary laboratory, in its Terrestrial Manual, applies the same risk-assessment logic. Biological safety cabinets are the core primary barrier: a class I cabinet protects operator and environment but not the product; class II, the usual choice for cell culture and diagnostic work, protects operator, product and environment with HEPA-filtered downflow and exhaust; class III is a sealed glove box for the highest risks. Good microbiological practice, personal protective equipment, validated decontamination by autoclaving or chemical disinfection, and waste segregation complete the system. Research that could be misused to cause harm, termed dual-use research of concern, is subject to additional oversight in many countries.
Internationally, the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, adopted in 2000 and in force since 2003, governs the transboundary movement of living modified organisms with advance informed agreement procedures; the Nagoya Protocol (in force 2014) governs access to genetic resources and sharing of benefits. National systems differ. In India, the Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms, Genetically Engineered Organisms or Cells (1989), under the Environment (Protection) Act 1986, set up a tiered structure: an Institutional Biosafety Committee (IBSC) at each organisation, the Review Committee on Genetic Manipulation (RCGM) under the Department of Biotechnology, and the Genetic Engineering Appraisal Committee (GEAC) under the Ministry of Environment, Forest and Climate Change for large-scale and environmental release, with State and District committees for monitoring. In the USA, the Coordinated Framework for Regulation of Biotechnology (1986) divides responsibility; the Food and Drug Administration regulates intentional genomic alterations in animals, and the Department of Agriculture's Center for Veterinary Biologics licenses veterinary vaccines under the Virus-Serum-Toxin Act. The European Union regulates contained use and deliberate release of GMOs by separate directives and authorises veterinary medicines under Regulation (EU) 2019/6. Vaccines for international use should meet WOAH Terrestrial Manual standards, including purity, safety, potency and freedom from extraneous agents. Animal experimentation is overseen by ethics committees applying the 3Rs of Russell and Burch (1959), in India under the committee now called CCSEA. Rules on genome-edited animals, clones and cultured meat are changing quickly; always check the current national rules.
Learn each technique as a principle you can explain in three sentences and a calculation you can do without a calculator's help: PCR doubling, efficiency from slope, delta-delta-Ct, C1V1 = C2V2, haemocytometer counts, reliability as accuracy squared and the breeder's equation. Know the landmark names and dates examiners use (Mullis, Kohler and Milstein, Dolly, Takahashi and Yamanaka, Meuwissen and colleagues) and the Indian biosafety committees. Practise reading a qPCR result with its controls and saying whether it is positive, negative or invalid. Link every technique to one animal example: PCV2 VLP vaccine, gE-deleted Aujeszky's vaccine, BLAD carrier testing, sexed semen in heifers.
After five years in mixed or dairy practice, biotechnology is mostly interpretation and advice. You decide whether a PCR-positive Leptospira or Ehrlichia result explains the clinical signs, request repeat samples when an internal control fails, and explain to a farmer why a PI calf is antigen positive and antibody negative. You test collie-breed dogs for ABCB1 before high-dose macrocyclic lactones, advise on sexed semen for heifers, schedule synchronisation for embryo transfer recipients and interpret genomic reports on heifers to decide which to breed from. You choose vaccines knowing whether they are live, sub-unit, vectored or DIVA-compatible, especially where eradication programmes depend on serology.
With twenty-five years' experience you set policy and run programmes: an AI and embryo transfer centre, a breed genomic reference population, a state diagnostic laboratory or a vaccine plant. Decisions include which PCR assays to validate and accredit for trade, how to confirm positives in a low-prevalence surveillance programme, whether to adopt OPU-IVP and sexed semen at scale, how to protect indigenous breeds' genetic diversity while using genomic selection, and how to meet biosafety committee and WOAH Terrestrial Manual requirements for containment, cell banks and vaccine quality control. You also explain edited animals, clones and cultured products to farmers, consumers and ministers in plain language.
At the research frontier the questions are open: improving genomic prediction accuracy for small and crossbred populations and low-heritability health traits; using whole-genome sequence and functional annotation to find causal variants; reliable pluripotent cell lines and in-vitro gametogenesis in livestock; reducing epigenetic errors in cloning and in-vitro embryo production; genome editing for disease resistance (CD163 and PRRS, for example) with rigorous off-target and on-target analysis; mRNA, vectored and structure-designed vaccines with DIVA properties; metagenomic and sequencing-based surveillance; and science-based, internationally coherent regulation of edited animals. A senior scientist must also judge dual-use risks and animal welfare in study design.
This manual teaches principles and interpretation, not laboratory protocols, and it deliberately omits any method for modifying pathogens. It does not give reagent recipes, validated Ct cut-offs for particular assays, or country-by-country product approvals, which change often; regulatory statements (India, USA, EU) are summaries that must be checked against current law. Aquatic animal biotechnology, proteomics and metabolomics, RNA interference, microbiome engineering and detailed statistical methods of genomic evaluation (Bayesian models, genotype imputation) are only mentioned or not covered. Figures quoted for efficiencies (sexing accuracy, blastocyst rates, cloning success) are typical textbook ranges and vary widely by species, laboratory and year.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions. Every numerical answer was computed by script and the working is shown in the explanation.
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