ALAT Chapter 16 Heredity & Breeding PDF

ALAT Chapter 16 Heredity & Breeding • Genetics is the scientific study of heredity. Through genetics, we gain an understanding of why offspring resemble or differ from their parents. Some of these similarities and differences are due to heredity (inherited characteristics received from the biologi...

ALAT Chapter 16 Heredity & Breeding • Genetics is the scientific study of heredity. Through genetics, we gain an understanding of why offspring resemble or differ from their parents. Some of these similarities and differences are due to heredity (inherited characteristics received from the biological parents). Others are caused by either external (environmental) factors or mutations (a permanent change in the inherited or genetic factors). • The successful breeding of laboratory animals depends on both a basic understanding of genetics and the ability to choose the breeding system that best suits the needs of a particular research study. • Over the past several decades, advances in molecular biology have led to welldefined animal genetics and the development of genetically manipulated animals, such as transgenic mice. These genetically manipulated animals are used extensively as animal models to understand normal biological functions, disease states, and for the development of treatments. The Basis of Genetics: Genes & Chromosomes • Genetics: traits or physical characteristics of living organisms are passed from parents to offspring. • Genes: The biological units by which traits are inherited. • Trait: A physical characteristic, such as fur or eye color, the number of toes, or the length of the intestine. • Think of genes as sets of instructions that determine what traits can be expressed in the animal. • Genes are made up of deoxyribonucleic acid (DNA) molecules that are strung together to form long structures called chromosomes. • Chromosomes are long, paired structures found in the part of the cell called a nucleus. • The only types of cells in which the chromosomes are not paired are ova and sperm cells, which contain single or unpaired chromosomes. • When the female’s ovum is fertilized by the male’s sperm, the resulting fertilized ovum, or zygote, has paired chromosomes from the unpaired chromosomes it receives from each parent. • The number of chromosome pairs differs for various species, but all normal members of a given species have the same number of pairs. • Because chromosomes are paired, so are the genes they carry. In a pair of genes, one gene may be more easily expressed than the other. Thus, that gene’s set of instructions will determine which trait will be expressed in the animal. These genes are said to be dominant, and they contribute to the appearance or characteristics of the animal. • Recessive genes are those less easily expressed. • If an animal receives two dominant genes that carry instructions for black fur, the animal will have black fur. (Punnet square = BB) • If it receives a recessive gene that carries instructions for brown fur from one parent and a dominant gene that carries instructions for black fur from another parent, the animal will have black fur. (Punnet square = Bb) • Only those animals that receive a recessive brown fur gene from both parents will have brown fur. (Punnet square bb) BB BB Bb Bb bb bb BB BB Bb Bb bb bb • Genotype: The specific genetic makeup of an individual. Thousands of different genes are present in animals. When an animal is found to have a particular gene pair that codes for a specific fur color, for example, the animal is said to have a specific genotype. • Sometimes an animal’s genotype is detectable by looking at physical characteristics, such as the fur color. If the genotype does not present such visible characteristics, a laboratory test may be necessary to identify the genotype. Other times, however, an animal’s genotype is not detectable by these methods. In that case, scientists must extract the actual genes from some of the cells to be able to determine the genotype. • An animal’s genotype and its environment determine its physical and physiological characteristics. These characteristics are referred to as the phenotype and include the appearance of the animal, such as its size, height, or fur color and internal physiology, such as its immune function. Phenotyping is a process used to assess and document how the animal’s genotype is expressed. Phenotyping is commonly performed for genetically manipulated rodents, but can be used in any species. The procedures may include a wide range of assessments from behavioral characteristics to molecules at the cellular level. To summarize these terms, genotype refers to how the genes code the information for the body’s structure. Think of it like an architect’s plan for a building, in which the characteristics of the building are planned. In contrast, phenotype refers to how the body’s individual characteristics appear. If the genotype is the architect’s plan for a building, the phenotype is the building itself. • Note that phenotype does not always completely reflect the genotype as the construction of a building sometimes deviates from the plan. The phenotype can reflect more than the genotype. Environmental factors can influence the phenotype and shift it away from the plan coded by the genotype. Consider an animal as a fetus. Its parents provided all its genes, including those which will specify the animal’s future size in adulthood. However, poor nutrition and a heavy parasite infection of this animal as a juvenile will stunt its growth and reduce its size as an adult, thus affecting its phenotype. Mutations • Occasionally a gene is damaged or changed. When this happens, the gene will be expressed differently. Such a genetic alteration is called a mutation. • Mutations can occur spontaneously or be brought about by chemical or physical environmental factors. For example, exposure to radiation can cause a chemical change in the genes (a mutation) in some of an individual animal’s cells. • If the mutation occurs in the ovum or the sperm, it may be transmitted to the offspring. This mutation may show up as an observable change in behavior or appearance. Therefore, it is the responsibility of the technician to notice and report unusual behavior or appearance in animals whose phenotypes are different from others in the same group. Such animals could have a new mutation and potentially be models that will help scientists understand certain diseases or physiological systems. • Technicians should learn as much as possible about the particular traits and needs of the specific animal species under their care. For example, nude mice have a genetic mutation that prevents them from developing T lymphocytes, an important cell for the immune system. The nude mutation also prevents these mice from developing fur, hence the name “nude.” Impairment in a portion of the immune system is referred to as an immunodeficiency because the immune system is deficient in some functions and is unable to defend the body with a normal immune response. Immunodeficient animals are highly susceptible to infections and need to be maintained under barrier conditions. Genetically Engineered Animals • Techniques are now available that allow scientists to modify the genetic makeup of animals. Most genetically engineered animals are rodents, predominantly mice and, to a lesser extent, rats. However, many species, including pigs, zebrafish and others, have been genetically modified for research studies. These animals are useful for many aspects of biomedical research, such as examining specific disease pathways, normal aspects of physiological systems, and drug or vaccine testing. Genetic engineering means that the genes of these animals were originally manipulated in the laboratory and then transferred into an animal. A transgenic animal contains new genes that either are from another species or have been altered within the same species. The larger mouse in Figure 17.2 has been genetically • engineered to contain the gene for rat growth hormone. Overexpression of growth hormone caused the transgenic mouse to grow larger than its non-transgenic littermate. A “knockout animal” contains an altered gene that has been designed to “turn off” a specific gene, preventing the gene from being expressed. In many cases, this loss of gene expression may lead to observable changes in its appearance, behavior, and physiological or biochemical characteristics. In some strains of knockout mice, the changes may not be seen at all and they may appear to be normal. However, the animal may be immunocompromised or have other problems and may need special care. Knockout mice are used in many research areas, including cancer, obesity, neuroscience, diabetes, arthritis, substance abuse, anxiety and Parkinson’s disease studies.2 • A “knock-in” animal contains new DNA that has been designed to change a specific gene, generally by creating a mutation that is suspected of contributing to a disease. For example, a knock-in mouse may contain a mutation that causes a known cancer-causing gene to change its pattern of expression. In turn, this gene may become more or less aggressive at causing tumors. Another example of knock-in mice used in biomedical research is one that mimics the development of Huntington’s disease, a human neurodegenerative disorder characterized by motor, cognitive, and psychiatric alterations. If the gene that is added to the animal is a functional human gene, the mutant may be referred to as humanized. Reproduction • The first steps in the complex process of sexual reproduction are the production of sperm in the testes of the male and eggs (ova) in the ovaries of the female. Ovulation is the release of the egg from the ovary; it occurs at or near the estrus - the period of time in which the female is sexually receptive. The way in which a sperm and egg (ovum) come together for fertilization depends on the species. • In most mammals, fertilization occurs within the female’s reproductive tract; the resulting zygote becomes implanted and grows in the female’s uterus. • In other species, such as most amphibians, fertilization occurs outside the female’s body, as does the embryo’s development. • Males of most species of mammals produce sperm continuously and are ready to mate almost any time after reaching sexual maturity. Females, on the other hand, mate only during specific times. Under the influence of hormones, many female animals undergo estrous cycles, the equivalent of human menstrual cycles. • At the beginning of the estrous cycle, the female’s eggs undergo a maturation process that prepares them for fertilization. Once the eggs are mature, the female enters estrus (heat), during which she becomes receptive to mating with the male. The time between fertilization and birth is called the gestation period. The length of this period varies with the species. Knowledge of the normal gestation period for the species is important in the maintenance of laboratory animal colonies. The gestation period for many species is given in the text of the species-specific chapters in this manual and in the Appendix. When breeding animals, it is important to know the best time for mating to occur in order to increase the chance of a successful mating leading to a pregnancy and healthy offspring. It is possible to collect cells from the vagina and examine them under a microscope to determine when the female is entering the estrus stage of her cycle and is therefore ready for mating. • For non-mammalian laboratory animals, a thorough knowledge of reproductive strategy is even more important, as both zebrafish and Xenopus are used extensively for the harvesting of eggs and larvae. Personnel using these species should ensure they are familiar with the techniques and equipment available to ensure optimal spawning and reproductive behavior for these animals Likewise, the housing and husbandry of larvae is different from that used for adults, so it is important that SOPs regarding feeding and water quality are followed exactly. • As zebrafish are often kept in small, closed populations, breeding strategies are designed to maximize genetic diversity. To do so, researchers should: • eliminate sibling matings • maximize effective population size • periodically import individuals from outside populations • In the case of mutant and transgenic strains, genetic modifications can be maintained by incrossing or by outcrossing to wild-type animals. In either case, progeny should be tested to ensure transmission of the transgene or mutant phenotype. Breeding Colonies & Schemes The success of a colony management program depends, in large part, on choosing the breeding scheme that best meets the goal of the program. Different schemes are used for the breeding of laboratory rodents, depending on the research requirements. The most common schemes are outbreeding and inbreeding. Outbreeding • Outbreeding is a scheme in which only unrelated or distantly related animals of the same stock are mated. The result is a maximum number of genetic differences among the animals in the colony. This mating scheme usually produces more vigorous animals than those produced by inbreeding, and usually results in a larger litter size. • Commonly used outbred mouse stocks are CF1, ICR, and Swiss. • Some outbred rat stocks are Sprague Dawley, Long-Evans, and Wistar. Inbreeding • Inbreeding is a mating scheme used to produce animals with minimal genetic variation. The closely related animals that result from inbreeding share most genetic characteristics. • By definition, a strain is considered to be established after a minimum of 20 generations of brother-to-sister mating. All the animals of the strain are nearly identical, with almost 99% of the genes being the same in all animals of that strain. • Rodents are the most frequently inbred laboratory animals. • Inbreeding is occasionally used in non-rodent species, such as rabbits, to produce offspring that are as genetically similar as possible. • An accidental mating to an unrelated animal or an animal of a different strain contaminates the inbred line and destroys its usefulness. Thus, an escaped animal should never be returned to a cage housing other animals, even if you are certain of its home cage. Such an error may not be detected for some time, and may complicate or ruin the ability to produce valid research results. Other Breeding Schemes • Breeding schemes for other animals, such as dogs, cats, or swine, usually involve mating animals that show similar individual characteristics that are desirable to preserve in the offspring. • Line breeding: When such animals share one or a few common distant ancestors, such as a great-grandparent. • Cross breeding: Involves the mating of animals of different breeds, such as the mating of a German shepherd dog to a Labrador retriever dog. Mating Systems After determining the genetic characteristics needed for the animals to be bred for a particular project, the species-appropriate mating system is selected that will produce animals with the desired traits. For some species, more than one mating system may be appropriate. In this situation, management considerations determine which mating system should be used. Monogamous Mating • In the monogamous mating system, one male (the sire) and one female (the dam) are selected by the breeder and put together in a cage. • PROS: • Pairs may be left together long term, often for the duration of their breeding life. • This system simplifies recordkeeping • Once the proper pairing has been established, lends itself well to maintaining both outbred and inbred rodent colonies. Polygamous & Harem Mating In both polygamous and harem mating, one male is housed with two or more females. In polygamous mating, the females are removed as they become pregnant; in harem mating, the group is kept together. The harem mating system is often used for rodents, cats, and larger animals such as sheep and NHPs. • PROS: • These mating systems result in the largest number of offspring from the least number of breeder animals • The most economical methods of laboratory animal production. • Eliminates the need to move pregnant animals • CONS: • With harem breeding, won’t know who the dam is • Harem breeding complicates recordkeeping • With rodent harem breeding, space becomes an issue; It is important to ensure that the stocking density limits are not exceeded. The cage must be large enough to permit proper housing of adults and young until the offspring are old enough to be weaned (separated from their mother). Separate Housing • The male and female may be housed separately and brought together only for mating. This system reduces the number of breeding animals needed and permits accurate recordkeeping; however, labor costs for this system are high. • Separate housing is the system of choice if males are known to kill the young or if males and females are aggressive toward each other. • Rabbits and hamsters are typically housed separately for these reasons, and brought together only for mating. Maintenance of Breeding Animals It is important to select good breeding stock. The animals should be young adults that are healthy and nonaggressive. New breeding stock should not be introduced into the colony until the health status of the animals has been carefully checked. • Rodents and their pups communicate with each other by ultrasonic vocalizations, which humans cannot hear. • Larger animals may make sounds that are audible to humans as they care for their young. • Caging for breeding colonies should provide comfort, privacy, and enough space for the developing young while permitting routine observation. Most rodent species build nests for their young if provided with a supply of soft paper, shredded wood fiber, or cotton. • Lighting: A lighting system that provides 12 to 14 hours of light each day is best for most rodent breeding colonies because the longer light period helps females establish consistent estrous cycles. • The health and condition of breeding animals must be constantly monitored. Any signs of illness or disease should be reported immediately. Females should exhibit good care of the young and adequate milk production. When neonatal rodent pups are nursing well, a white spot should be visible on the left side of their abdomen. This is referred to as the “milk spot” and is where the milk is held in the pups’ stomach. • A rodent dam with good maternal instincts will keep her pups together in the nest area that she has created in the cage. She will “hover” over the nest to nurse the pups and to provide a source of warmth until the pups can stay warm on their own. If the dam relocates her nest, she will pick the pups up with her mouth, holding them by the scruff, and quickly transfer them. If the sire is present throughout the pregnancy and postnatal period, he may also help with nest care, keeping the pups warm while the dam eats, drinks, and grooms. • Dystocia (difficult birth) and vaginal prolapse are health problems that may occur in breeding females of many species. The dam may be at the end of her pregnancy and clearly pushing, but is not successful in giving birth to some or all of her young. A portion of a neonate may even be visible in the birth canal. • Dystocia and other reproductive problems, such as miscarriage, may be accompanied by vaginal prolapse. In vaginal prolapse, a portion of the vaginal tissue will be clearly visible and protruding from the vulva. In all cases of breeder animal health problems, the veterinary staff must be notified immediately. • Once a breeding colony is established, technicians must be observant in order to quickly identify any problem before it disrupts the production of animals. • Some animals may neglect, kill, or cannibalize their young if disturbed by frequent observations or unusual environmental changes. This is particularly true of both rodent and rabbit females with their first litter. • Minimal handling of newborn animals is a general rule. It is wise to delay cage changes and other husbandry practices that may not be immediately necessary for a few days following parturition to avoid the loss of the litter. • After 3 to 4 weeks of nursing, mouse and rat pups should be eating solid feed and can be weaned from their mother. • Certain highly specialized strains of mice, such as those produced by inbreeding or genetic engineering, tend to be small and often are not ready to be weaned for another week. • Approximate weaning times are included in the species-specific chapters in this manual. • Weaned animals are housed by sex in groups. These animal must be closely monitored during their first week to make sure that they are self-sufficient. Newly weaned pups placed in cages with automatic watering systems must be monitored at least daily for signs of dehydration. Sometimes supplements, such as packets of nutrient and water gel, are placed in the newly weaned animals’ cage to ensure they receive adequate water and nutrition. Breeding Records • Genetically modified or inbred animal strains are costly and time-consuming to generate and maintain. To ensure the pedigree of the animals, accurate and complete documentation is essential. Breeding records are required to monitor the reproductive efficiency of the colony, select the next generation of breeders, and troubleshoot problems in reproduction. Breeding records typically involve recording information on the animal’s cage card (Figure 17.4). The type of record system used varies between facilities. Some facilities keep information in a notebook, while others store them on a computer. Computer programs are commercially available for animal breeding records, and many are customized for transgenic animals. • Information included in breeding records includes, at a minimum, the following: • species, breed, and strain • animal identification numbers for both sire and dam • parentage or ancestry • date mated • date of parturition, number of offspring, and their sex • date the young were weaned • Additional pertinent information may be included, such as animal IDs of those used in experiments or results from tests performed on the neonates. Summary • In most research facilities, animal breeding is used for producing and maintaining genetically modified animals as tools for biomedical research. These animals are especially valuable because of the labor and resources invested to produce their genetic make-up. They often need special care because of diverse physiological impairments and a greater susceptibility to illness resulting from their genetic manipulations. Technicians should be especially alert to any change in these animals’ behavior and appearance, and rapidly communicate potential problems to the management and veterinary staff. Moreover, technicians working in a breeding colony are required to accurately document and interpret data on animal identities, matings, pedigrees, and other pertinent information. Technicians who have the knowledge and skills necessary for the proper care of these colonies are essential for the success of the breeding program.