Animal Biotechnology Revised ABT Unit 1 Part 1 PDF

Animal Biotechnology Unit-I [part 1] Animal Biotechnology Animal biotechnology is the application of scientific and engineering principles to the processing or production of materials by animals to provide goods and services to human kind. Animal biotechnology includes livestock, poultry,...

Animal Biotechnology Unit-I [part 1] Animal Biotechnology Animal biotechnology is the application of scientific and engineering principles to the processing or production of materials by animals to provide goods and services to human kind. Animal biotechnology includes livestock, poultry, fish, insects, companion animals and laboratory animals. APPLICATIONS OF ANIMAL BIOTECHNOLOGY Transgenic technology Gene knockout technology Molecular genetics Embryo transfer technique In vitro embryo production Modern vaccines Molecular diagnostics Nutritional biotechnology  What is a Breed? A breed is a specific group of domestic animals having homogeneous phenotype, behavior, and other characteristics that distinguish it from other organisms of the same species. Therefore, breed is a stock of animals within a particular species with distinctive characteristics, which is produced by selective breeding. Hence, each breed consists of unique appearance and behavior when compared to another breed of the same species. Examples of Cattle & Sheep Breeds Angus Tharparkar Australian Charbray Red Sindhi Chianiana Sahiwal Ankole Gir Ongole Jersey Kangeyam Boer (Sheep) Vechur Thalichery (Sheep) Exotic breeds Indigenous breeds Examples of Dog & poultry breeds Kombai Aseel Rajapalayam Chittagong Kaikadi Kadaknath Gaddi kuta Busra Brahma German shepherd Java Golden Retriever Leghorn Boxer Styrian Great Dane Red cap Dog Breeds Poultry breeds What is a Species? In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species refers to the largest group of organisms in which any two individuals of the appropriate sexes or mating types can produce fertile offspring, typically by sexual reproduction. The exchange of genes between the individuals of the species or the gene flow is the major characteristic feature of a species. The gene flow does not occur among different species. Speciation refers to the emergence of new species from the existing species. It occurs due to physical, behavioral, and reproductive isolation of different populations of the same species. Animal Breeding Animal breeding is a branch of animal science that deals with the evaluation of the genetic value of livestock using different methods. Animals with superior breeding value in growth rate, egg, meat, milk, or wool production, and other desirable traits are selected. Animal breeding is performed to domestic animals in order to improve desirable qualities and to better suit human needs. Breeding stock It is a group of animals used for the purpose of planned breeding. In order to gain certain valuable traits in purebred animals. For example, when breeding cattle for milk, the “breeding stock should be sound, milch, having longer lactation period, higher productivity and reproductively efficient. Breeding animals is common in an agricultural setting, however, it is also a common practice for the purpose of selling animals meant as pets, such as cats, dogs, horses, and birds. Environmental factors must be considered and controlled in selecting breeding stock because it has greater effects on performance. Breeding and Variation There are two sources of variation namely, genetics and environment. So, breeding progress requires understanding these two. Continuous selective breeding leads to homozygosity in a population resulting a loss of variability. Differences in the animals’ environment, such as amount of feed, care, and even the weather, may have an impact on their growth, reproduction, and productivity. For example, only about 30 percent of the variation in milk production in dairy cattle can be attributed to genetic effects; the remainder of the variation is due to environmental effects. Genetic variation is necessary in order to make progress in breeding successive generations. Purebred Breeding It refers to the Mating animals of the same breed for maintaining such breed. Opposite to the practice of mating animals of different breeds, purebred breeding aims to establish and maintain stable traits that animals will pass to the next generation. Such animals can be recorded with a breed registry, the organization that maintains pedigrees Methods of breeding Generally, two ways of matings are prevalent: Natural mating: It refers to mating of animals by natural means. Artificial insemination (AI): It is the technique in which semen with living sperm is collected from the male and introduced into female reproductive organ. So, mating is done through artificial means. Inbreeding Breeding or matting of the related animals is known as inbreeding. Inbreeding is often described as “narrowing the genetic base” because the mating of related animals produces offspring that have more genes in common. Inbreeding is used to concentrate desirable traits. Inbreeding is generally detrimental in domestic animals. Types of Inbreeding Close breeding: Here animals are very closely related and can be traced back to more than one common ancestor. Examples: Sire to daughter, Son to dam, Brother to sister. Line breeding: Mating animals that are more distantly related which can be traced back to one common ancestor. Examples: Cousins Grandparents to grand offspring, Half-brother to half-sister. Disadvantage of Inbreeding Increased inbreeding is accompanied by Reduced fertility Slower growth rates Greater susceptibility to disease Higher mortality rates. Out breeding It is breeding of unrelated animals. The effect of out breeding is opposite of inbreeding because heterozygosity is increased here. It is of two types – Cross breeding: Crossbreeding involves the mating of animals from two breeds. Normally, breeds are chosen that have complementary traits that will enhance the offspring's economic value. Superior traits that results in the crossbred progeny from crossbreeding are called hybrid vigor or heterosis. So, it is done to take advantages of good qualities of two or more breeds. It is mainly used by commercial producers. Objectives of Cross Breeding Utilize the desired attributes of two or more breeds. Produce progeny better suited to target markets while maintaining environmental adaption. Improve productivity quicker in traits which are slow to change within a breed i.e. environmental adaption, fertility and carcass traits. Take advantage of the production improvements which arise from heterosis (hybrid vigor) when breeds are crossed. Advantage of Heterosis Heterosis is the production advantage that can be obtained from crossing breeds or strains, which are genetically diverse. The new combinations of genetic material can lead to production advantages over and above the average of the two parent breeds or strains. To be of economic advantage, the new production levels need to be above those of either parent strain or breed. Cross Breeding- 1.Two breed cross The two breed cross system produces first cross, or F1, progeny. In this system, the progeny resulting from the cross of two breeds are usually all sold for slaughter or to another commercial breeder. The system is most useful for situations in which females of a specific breed are well adapted to a given environment. These adapted females can be mated to a sire of another breed, resulting in heterosis for traits such as growth, improved carcase, feed conversion efficiency and vigour. Two breed cross occurs where breed A and breed B are two purebreds and the F1 progeny (AB) contains equal parts of the two breeds. 2.Backcross In a backcross system, Female F1crossbred progeny are mated to males of one of the parental breeds. This breeding system takes full advantage of heterosis for maternal traits such as fertility of the cow, milking and mothering ability. Continual backcrossing is the system used by producers to upgrade or change from one breed to another without having to buy purebred. The backcross is obtained where all the females from a two breed cross are mated to a purebred bull of either of the original breeds. 3. Three breed cross Three breed cross requires the input of three separate breeds. First cross females (F1 progeny) are joined with bulls of a third unrelated breed. This type of breeding takes advantage of both maternal and individual heterosis and of the complementarities of three breeds. The progeny is generally considered to produce the greatest lift in productivity, but it is influenced by the quality of the purebreds maintained to breed the F1 females. The three breed cross is obtained when all the females from a two breed cross are mated to a bull of a third, unrelated breed. All the three breed cross progeny are marketed. 4. Rotational cross Rotational crossbreeding (or) sequence breeding is when males of two or more breeds are mated to crossbred females. Over a number of years, each breed will have contributed its strengths and weaknesses equally. Levels of heterosis achieved in rotational crossbreeding depend on the number of breeds involved. Increased heterosis in rotational systems is a result of close to maximum heterosis being achieved in each cross with the purebred. Rotation breeding Starting at 50:50, and stabilises at 65:35 and giving 65 % heterosis from the last sire line used. 5. Composite breeding Development of a composite or synthetic breed results from the crossing of two or more existing breeds. The primary advantage of forming composite breeds is that after the initial crosses are made, management requirements are the same as for straight breeding. The initial choice of breeds must be based on those which have desirable traits for a particular environment and for the target market. The percentage of heterosis increases as more breeds contribute in the initial mating program. The heterosis will not be as high as that achieved with a rotational crossbreeding program. A simple approach to a composite breeding Grading up Grading up is the breeding of animals of two different breeds where the animals of an indigenous breed/genetic group is mated by an improved pure breed for several generations in order to get superior traits of the improved breed. Grading up is continuous use of purebred sires of the same breed in a grade herd. By fifth generation, the graded animals may reach almost purebred levels. Grading up Pure Thalichery Pure Boer X After 8th generation 100% Boer Grading up - Change in the genetic composition Number of generations Off-springs 1st Generation Percent replaced Percent non-descript 2nd “ 50 50 3rd “ 75 25 4th “ 87.5 12.5 5th “ 93.75 6.25 6th “ 96.87 3.13 7th “ 98.44 1.56 8th “ 99.22 0.78 off springs come closer to a 100% improved breed, as we go on breed  Chromosome Mapping & Identification of economically important genes in Farm animals  Quantitative trait loci (QTL) The loci controlling quantitative traits are called quantitative trait loci (or) QTL The term quantitative trait loci is first coined by Gelderman in 1975. It is the region of genome that is associated with an effect on a quantitative trait. The example of QTL is milk production, which is controlled by many genes. It can be a single gene or cluster of linked genes that affects the trait.  Salient features of QTL The traits are controlled by multiple genes, each segregates according to Mendel's Laws. The traits can also be affected by the environment to varying degrees. Many genes control any given trait and allelic variations are fully functional. Individual gene effect is small and genes involved can be dominant or co-dominant. The genes involved can be subject to epistasis or pleotrophic effect.  Aim of QTL mapping To identify the region of the genome that affects the trait of interest. To analyze the effect of QTL on the trait. To check the variation for the trait is caused by a specific region of a DNA. To test the gene action associated with QTL for Additive effect or Dominant effect. To identify the allele associated with favorable effect.  Genetic Marker Genetic marker is a general term used for any observable or assayable phenotype or the genetic basis for assessing of the detected phenotypic variability. Genetic markers are mainly classified based on physically evaluated traits (morphological and productive traits), based on gene product (biochemical markers) and based on DNA analysis (molecular markers). Molecular marker also known as DNA marker and is defined as a segment of DNA indicating mutations or variations, which can be employed to detect polymorphism (base deletion, insertion and substitution) between alleles of a gene for a particular sequence of DNA in a given population or gene pool.  Ideal DNA Markers High level of polymorphism Even distribution across the whole genome (not clustered in certain regions) Clear distinct allelic features Single copy and no pleiotropic effect Cost efficient marker development and genotyping Easy detection and automation High availability and suitability to be multiplexed Genome-specific in nature (especially with polyploidy)  Marker Assisted selection Marker-Assisted Selection (MAS) is used for indirect selection of superior breeding animals. MAS depend on identifying association between genetic marker and linked Quantitative traits loci (QTL). The association between marker and QTL depend on distance between marker and target traits. As soon as markers linked to QTL have been identified, they can be used in selection programme. This use of marker in selection is called Marker-Assisted Selection.  Objectives of MAS Testing for genetic defects e.g. BLAD. Testing for single gene trait e.g. coat colour. It is used to test multigenic trait or Quantitative trait e.g. Milk production. Identification traits using MAS Growth performance Milk production Maternal ability Carcass quantity and quality Fertility Reproductive efficiency Inherited genetic defects  Bovine Genome It contains 30 pairs of chromosomes. The Human and cattle genomes are 83% identical. Chromosome 1 – Birth weight Chromosome 2 – Rib eye area and weaning wt. Chromosome 4 – Tenderness Chromosome 5 – Rib bone & ovulation rate Chromosome 6 - Birth weight Chromosome 7 – Trypano tolerance Chromosome 8 – Back fat Chromosome 9 – Behavior Chromosome 12 - Birth weight Chromosome 14 - Carcass weight Chromosome 15 – Tenderness Chromosome 16 - Birth weight Chromosome 18 - Birth weight Chromosome 19 – Ovulation rate Chromosome 22 – Carcass weight Chromosome 23 - Weaning weight Chromosome 29 - Tenderness Applications of marker-assisted selection Disease resistance Product quality Improve longevity Feather pecking Stress resistance Desired behavior characteristics of animals  Classifications of Molecular Markers Non PCR-based or Hybridization based Molecular Markers. The most common example of this type of marker is Restriction Fragment Length Polymorphisms [RFLPs]. PCR-based DNA Markers these includes; Random Amplified Length Polymorphic DNAs [RAPDs], Simple Sequence Repeats or microsatellites [SSRs], Amplified Fragment Length Polymorphisms [AFLPs]. DNA Chip and Sequencing-based DNA Markers. Single nucleotide Polymorphisms [SNPs] is an example of these types of markers.  Restriction fragment length polymorphisms (RFLP) Restriction Fragment Length Polymorphisms (RFLPs) was the first form of DNA marker utilized to construct the first true genomic map. RFLP technology was first developed by Botstein and his co-workers since 1980. This hybridization based marker technology employed synthetic oligonucleotides as probes, which are labelled fluorescently to hybridize DNA This technology used the restriction enzymes that cleave the DNA at distinct site to observe the differences at the level of DNA structure. Differences are marked by using RFLP when the length of DNA segments are different, this imply that the RE (restriction enzymes) cleave the DNA at specific locations. The change or polymorphism that take place as result of mutation indicate creation or removal of the RE site and produce new RE site. The variations are determined by using hybridization probe. In RFLP analysis, the choice of the DNA probe is very crucial. Gel electrophoresis is needed for the identification of RFLPs to separate the DNA fragments of various lengths and to transfer the fragments into a nylon membrane. The radioactive labelled probe is applied to observe the segments of DNA exposed to an X-ray Film. This technique is normally employed in hybridization definition of nucleic acid, detection and diagnosis, description of polymorphisms on the gene construction of a genetic linkage map and recombinant DNA technology in livestock species. Randomly Amplified Polymorphic DNA (RAPD) Random Amplified Polymorphic DNA also called arbitrarily primed PCR. This technology utilizes an in-vitro amplification to randomly amplify the unknown loci of nuclear DNA with a matching pair of short oligo-primers, (8-10 base pairs) in length. Multiple primers in the range of (5 to 21) nucleotides are mostly used and has proven to be successful when detection is combined with polyacrylamide gel electrophoresis. The amplified products range from less than 10 to over a 100 depending upon the ratio and primer/template combination. RAPD technology had been used to estimates the genetic differences within or between the certain taxa of interest by evaluating the occurrence or lack of each product, which is directed by modification in the DNA sequence at each locus. RAPD technique offers a quick, simple, cheap and efficient technique for producing molecular information. Being highly polymorphic, only very small quantity of DNA is needed to be amplified by PCR technique in the absence of DNA sequence information. This is the major reason why RAPD technique has been used successfully in various phylogenetic and taxonomic researches.  Microsatellite/single sequence repeat (SSR) Microsatellites are two- to six-nucleotide repeats, interspersed throughout the genome. Microsatellites are highly polymorphic and abundant, often found in non-coding regions of genes. The most common dinucleotide motif in mammals is (CA)n, where n is the number of repeats. In avian species, the frequency of (CA) ≥10 is evaluated at once every 140 to 180 kb, and that of (CA) ≥14 is one every 350 to 450 kb. Microsatellites loci are also termed as short tandem repeats (STR's), simple sequence repeats (SSR's) and simple sequence tandem repeats (SSTR). The microsatellites and minisatellites altogether make up the variable number of tandem repeats (VNTRs). The mutation rate of microsatellites is thought to be high and there are often large numbers of alleles that vary in size at a single locus. The lengths of a specific microsatellite sequences tend to be highly variable among individuals. Slippage of DNA polymerase and mismatch repair during replication appear to be the mechanisms generating diversity of microsatellite length. Microsatellite length variation is easily detected by the polymerase chain reaction (PCR) using unique flanking primer sequences. Microsatellite derived markers represent a powerful way of mapping genes controlling economic traits. As soon as the simple repeat region is identified, by sequencing its immediate flanking regions, specific primers can be designed for PCR and for genotyping. Normally, the size of a microsatellite PCR product is obtained by electrophoresis in a denaturing polyacrylamide gel. One of the two primers employed in the PCR is often labeled with a fluorescent or radioactive tag. This technique of detection usually works better but suffers from an inherent weakness in determining the size of DNA accurately. More recently, there is an alternative to the gel-based approach to determine the size of DNA products; there are a series of technological advancement based on mass spectrometry. Microsatellite applications which includes, genetic characterization studies, analysis of population structure, estimation of genetic variability and inbreeding, determination of paternity, phylogenetic relationships among populations, disease diagnostics and forensic analysis.  Amplified Fragment Length Polymorphism (AFLP) AFLP method is a simple and inexpensive finger printing technique which provide more valuable information by producing multi-locus and consistent genomic fingerprints. The basic idea behind AFLP polymorphism was the insertion and deletion or substitution of nucleotides between and at restriction sites. The base substitutions are normally done at primer binding sites during PCR as in the case of RAPD. This technique is distinctive, as it enables the binding of adaptors of known sequences to DNA segments that are produced through the complete digestion of genomic DNA. This ensure easy separation of the generated DNA fragments following amplification the subset of entire fragments. Analysis of fragment using automated sequencing machine following gel electrophoresis. AFLP technique offer an effective, fast and cost-effective means for detecting a large number of polymorphic genetic markers which are very consistent and reproducible. The technique is considered as the most effective method for molecular epidemiological studies of pathogenic microorganisms and it is also used extensively in forensic science. The AFLP technology has been widely utilized in identification of genetic polymorphisms, evaluating and characterizing breed resources, measuring the correlation among breeds, constructing genetic maps and identifying genes in the main species farm animals. Single nucleotide polymorphisms Single nucleotide polymorphisms (SNPs) involve the substitution of one nucleotide for another, or the addition or deletion of one or a few nucleotides. It comprise more than 90% of all variances between the individuals; thus, they are the excellent genetic variation resource for population studies and genome mapping. SNPs type of marker are becoming highly attractive in molecular marker development due to their abundance in the genome of any organism (coding and non-coding regions). SNPs have an ability to identifying hidden polymorphism which is not commonly recognized by other genetic markers and techniques. There are four major reasons for the increasing interest in the use of SNPs as markers for genetic analysis. Firstly, they are prevalent and provide more potential markers near or in locus of interest Secondly, some SNPs are located in coding regions and directly affect protein function. These SNPs may be directly responsible for some of the variations among individuals in important traits. Thirdly, SNPs are more stably inherited than microsatellites, making them more suited as long term selection markers. Finally, SNPs are more suitable than microsatellites for high throughput genetic analysis, using DNA microarray technology. There are a number of methods to detect SNPs. The traditional gel-based approach uses standard molecular techniques, such as sequencing, PCR, restriction digests and various forms of gel elecrophoresis.  Microsatelli te Single locus marker RFLP STR Molecular Markers DNA Fingerprinting RAPD Multi-locus marker AFLP  DNA finger printing Identification of individuals is important in the domestic animals. For e.g., for the control of semen used in artificial insemination, the identification of cell lines and chimeras. DFP using in skin samples allows the diagnosis of zygosity even in species showing blood cell chimerism due to placental anastomoses. DFP also prove useful in investigating the phylogeny and genetic structuring of populations in particular cases. The control of inbred lines and determination of genetic relationships have already been realized by DNA fingerprints. In poultry, highly discriminating oligonucleotides (CAG)5, (CGAT)4 and (GT)8 have enough potential to differentiate between individual chickens and strains. Furthermore, the genetic makeup of inbred chickens can be evaluated by establishing fingerprints. Such data may be useful for selecting lines or individuals in cross breeding programmes.  List of genes associated with economically important traits Genes involved in Milk production Sl.No. Gene Gene name Function of gene Chromosome No. 1 ZNF232 Zinc finger protein 232 Regulating milk volume 5 Acetyl-CoA 2 ACACA Milk yield 19 carboxylase alpha Influence 3 PRL Prolactin protein 23 Lactation performance 4 CSN1S1 Casein alpha s1 Controlled total milk yield 6 ADAM metallopeptidase with Involved in the differentiation 5 ADAMTS20 5 thrombospondin type of mammary cells 1 motif 20 Genes involved in Composition of Milk Sl.No. Gene Gene name Function of gene Chromosome No. glycerol-3-phosphate biosynthesis of triglyceride 1 AGPAT6 27 acyltransferase 4 (milk fat) Peroxisome 2 PPARγ proliferator activated Control milk fat yield 22 receptor gamma 3 CSN3 casein kappa Regulating protein content 6 solute carrier family Associated with lactose 4 SLC27A6 7 27 member 6 content SREBP Play a central role in the 5 SCAP cleavage-activating regulation of milk fat 22 protein synthesis Genes involved in Reproductive and Fertility Trait Chromosome Sl.No. Gene Gene name Function of gene No. Influence the ovarian 1 CDC25C cell division cycle 25C 7 development 2 PSEN2 presenilin 2 Influence the litter size 16 Related to semen 3 YBOX2 Y-box Binding Protein 2 19 volume growth differentiation 4 GDF9 Related to follicular growth 7 factor 9 nuclear receptor Critically involve in 5 NR6A1 subfamily 6 group A 11 embryonic development member 1 Genes involved for Environmental adaptation Sl.No. Gene Gene name Function of gene Chromosome No. Related to high-altitude 1 LEPR Leptin receptor 3 and fitness traits Responsible for Sodium/Potassium/C 2 SLC24A4 hypoxia-related cellular 21 alcium Exchanger 4 responses Fibroblast growth Responsible for 3 FGF2 17 factor 2 thermotolerance ADP ribosylation Adaptation to different 4 ARFRP1 factor related protein 13 ecological environments 1 adaptation to oxidative 5 SR XN1 Sulfiredoxin 1 13 stress