LAT Chapter 8 Genetics & Breeding Colony Mgmt PDF

LAT Chapter 8 Genetics & Breeding Colony Management • Depending on the needs of the research program, a breeding colony may be maintained at your institution that provides animal strains or stocks to meet the requirements of a specific protocol. • Understanding genetics will help you maintain the...

LAT Chapter 8 Genetics & Breeding Colony Management • Depending on the needs of the research program, a breeding colony may be maintained at your institution that provides animal strains or stocks to meet the requirements of a specific protocol. • Understanding genetics will help you maintain the necessary quality control in the breeding colonies you may work with, at present or in the future. • Managing a breeding colony requires an understanding of anatomy, physiology, breeding schemes, progeny management, basic genetics, genotyping, phenotyping, and recordkeeping. • This chapter focuses on managing a rodent breeding colony, but the concepts and techniques are broadly applicable to all animals. Distinctions for other species are mentioned wherever appropriate. Glossary • Chromosomes: The genetic material of every nucleated cell is kept in coiled structures called chromosomes. Each diploid cell contains two copies of each chromosome, whereas the gametes (sex cells) have one copy only. In mammalian diploid cells, the sex chromosomes are unpaired in the male, which has one X and one Y chromosome. Females have a pair of X chromosomes. • Genes: Genes are segments along the strands of DNA that have specific sequences that code for specific proteins. There are two copies of each gene in the cell, one on each of the paired chromosomes. • Genome: All the genes in an animal are collectively referred to as its genome. • Alleles: Different versions of the same genes are called alleles. Individuals of the same species have the same genes, but they may have different versions of those genes resulting in different physical characteristics. This variation between genes is caused by a change in the type, number, or sequence of the individual nucleotides that make up the gene, their expression, and the genes’ interactions with each other. If both alleles are the same, the animal is homozygous. If the alleles differ, the animal is heterozygous. • Genotype: The genetic makeup of an organism with reference to a single trait, set of traits, or an entire complex of traits is called its genotype. Determining the genotype of an animal is vital to deciding whether an animal bred by the researcher can be used for a study or for breeding. If the animal does not have the desired genotype, it may be euthanized, enrolled in another study, or used for another type of study. Transgenic Animals Incredibly expensive, medically fragile, valuable animals • Transgenic animals have had DNA from a different source inserted into their genome, which may be inserted randomly or in a targeted manner. With a targeted mutation, DNA is inserted at specific locations on the chromosome. One type of targeted mutation is called a knockin, in which new genes are inserted. A human gene can be inserted to study a human disease, for example. In addition to adding genes, another type of targeted mutation blocks the function of a specific gene. Such animals can be referred to as knockouts since a gene has been “turned off” or “knocked out.” The function of this specific gene can then be determined through study of the outcome when the gene’s products are absent in thebody. • Knockins and knockouts are valuable animals used to study physiological processes in health and disease. • To create transgenic animals, scientists first create the DNA sequences they want to insert into the animal’s genome. Then pronuclear injection or homologous recombination is used to insert the new DNA into the developing cells. Transgenic Animals - Pronuclear Injection • Pronuclear injection is a technique performed on a fertilized egg in a single-cell stage. • A pronucleus is a structure containing the genetic material of the sperm or egg, prior to their fusion in the fertilized egg. • The egg may also be called an oocyte if it is still a single cell, or a zygote if it is a developing embryo. • Genes are injected with a tiny glass pipette directly into a pronucleus of the cell (Figure 8.2). With this technique, DNA is incorporated randomly into the chromosomes as they divide, and the embryo develops. The injected genes are not targeted to specific locations in the genome. Once the DNA is inserted into the developing fertilized egg, the embryo is surgically implanted into a surrogate mother, or in the case of non-mammalian species, left to develop into a larva. • Typically for pronuclear injections, only a small portion of the injected cells survive and develop into young. Of those that survive, only a small portion will have correctly incorporated the desired DNA. The offspring are identified by testing for the presence of copies of the injected genes in a small piece of tissue, usually a few millimeters collected from the tip of the tail. In general, pronuclear injections produce offspring in which every cell has the DNA because the DNA is injected prior to the first cell division of the embryo. Transgenic Animals - Gene Targeting by Homologous Recombination • Homologous recombination is a technique where the embryonic stem cells that line the inside of a developing blastocyst (a preimplantation stage of early embryos) are collected and used to produce embryonic stem (ES) cell lines. These special cells are pluripotent, meaning they are capable of becoming any of the different cells that make up the various body tissues. The DNA containing the mutated gene is inserted into these cells by a process called electroporation. A small electrical current is passed through a solution containing the genes and stem cells, which causes small pores to open in the cell membrane and allows the DNA to enter the cells. When the DNA is inside an ES cell nucleus, the cell may recognize portions of the nucleotide sequences (making up the mutated gene) that are the same as in their own genes. The cell integrates the mutated gene at the specific location corresponding to its own copy of that gene. This process is called homologous recombination. All the cells are then tested to determine which ones have incorporated the mutated gene. The cells containing the mutated gene are then injected into another developing blastocyst. • Injected blastocysts are surgically implanted into the uterus of a surrogate mother. If the cells containing the new gene are present, ordinarily they will have incorporated into some, but not all, of the animal’s tissues. An animal that has some cells containing the new gene and some without the new gene is called a chimera. Often chimeras are easy to recognize because they have more than one coat color, resulting in a mottled appearance. The most desirable chimera is one in which the new gene has been incorporated in the germ cells (egg and sperm). This is called germline transmission. A pure strain of animals carrying the desired DNA can be produced by breeding offspring with germ cell incorporation. Genotype Characterization • The genotype is the specific genetic makeup of an individual organism. Tissue testing will reveal if the animal carries a wild type gene, a different allele, or a mutant form of the gene they are seeking. • PCR (polymerase chain reaction): The most common molecular biology technique used to determine genotype. • To test an animal’s DNA using PCR, a sample of tissue is processed to extract the DNA from the cells. • In mice, this sample may be obtained from the tip of the tail or with less invasive sampling methods, such as plucking a hair shaft from the skin, taking a swab of cheek cells, using the tissue from ear punch performed for identification, or collecting fecal pellets. • The tissue is dissolved, and the mixture (which includes DNA) is placed into a tube with an enzyme and specially designed short DNA fragments that match the gene of interest. • The tube containing the sample mixture is placed into a PCR machine. The PCR machine takes the sample through many cycles of heating and cooling. • If the gene of interest is present in the tissue sample, multiple copies of the gene will be generated hundreds of thousands of times in a process known as amplification. The amplified gene is then visualized through a process called gel electrophoresis, • Pay attention to animal welfare and personal hygiene when collecting samples for PCR tests. Follow your institution’s or the lab's SOPs when collecting, labeling, and storing PCR samples. • SOPs should describe the amount of sample to collect and list any required anesthesia or analgesia. • It is important to prevent cross-contamination when collecting a sample for PCR. Your own skin cells, hair cells, and bodily fluids must not come into contact with the sample. • Gloves must be kept clean. • The scalpel blade or scissors used in the biopsy should be replaced or cleaned between animals. • Ethanol is often used for cleaning because it breaks down any protein that would interfere with the PCR and subsequent testing. • Store the sample according to the SOP, or it may not respond correctly during processing. For example, the storage temperature may be ambient, refrigerated, or frozen. The sample might need to be “dry” in the sample vial or be kept in a solution and chemically stabilized. Gel electrophoresis • In gel electrophoresis, the DNA sample amplified by PCR is loaded into a prepared agarose gel that is placed inside equipment called an electrophoresis gel box. • Many samples of amplified DNA from different animals can be loaded into the gel and processed simultaneously. Along with the samples from animal tissue, a sample of known DNA is also run at the same time so researchers can compare their samples to a known reference. • An electric current is passed through the gel, and the DNA separates into a long column according to the size of the fragment. • Researchers know the size of their gene of interest and can determine if an animal possesses the specific gene by where the bands accumulate in the gel. • From this process, it can be determined whether the gene of interest was amplified and therefore is present in the sample from the mouse. Records Once the genotype of an animal is known, the records must be updated to reflect this information. • Musts: • Record the information correctly. Animal identification is often done at the same time as sample collection, so the laboratory animal technician must be very careful to accurately label the sample tube with the correct animal number. • Only handle one animal at a time. If a sample vial is labeled with the incorrect animal, the entire study could be at risk. For example, if an animal were incorrectly identified as having a particular genotype and then bred, dozens or even hundreds of animals would be produced with the incorrect genotype. Phenotype Characterization • Phenotype: How genes express traits. The phenotype is the observable result of the genes and how they interact with the environment. For example, a mouse strain may have a short tail phenotype. As a laboratory animal technician, you may be involved in characterizing or determining phenotype in the animals with which you work. Think about the following categories regarding the genetically modified animals in your care. • Visible Phenotypes Examples include a mouse’s coat color or the shape of a fish’s fin. Are some offspring smaller or larger than others? Is there a color difference, such as a yellowish cast? Do some breeders produce larger or smaller numbers of offspring? Are there always one or two dead pups in a litter? • Behavioral Phenotypes Includes aggressive or timid behavior and excessive fear responses. Does the environment influence the reaction of the animal? Does it seem that the animal doesn’t hear or respond as you would expect from your experiences with the species? Do some animals explore more than others? Is the mother behaving appropriately toward her young? • Demonstrable Phenotypes Does the animal weave or stagger? Does the animal freeze at odd moments? Such observations may indicate a visual, neurological, or motor phenotype that could signal genetic anomalies. Some ways to test for such phenotypes include performance on a rotarod machine or in a Morris water maze. A rotarod tests the coordination or fatigue resistance in a rodent by the length of time the animal can hold onto a mechanically rotating rod.1 The Morris water maze is a test for behavioral phenotypes. An animal is placed in a large circular tub of water and must swim until it reaches a hidden platform found by memory. • Inducible Phenotypes Some transgenic animals have the gene of interest linked to another gene that can only be activated by the addition of an antibiotic. For these animals, adding the specific antibiotic (such as tetracycline or doxycycline) to the drinking water or their feed can trigger specific genes to turn on if present Properly label the cage when a treatment is given and follow SOPs for drug administration and observation. Remember that some treatments in water (such as doxycycline) are light-sensitive, meaning that the compound is degraded by exposure to light. • Physiological Phenotypes Examples of physiological phenotypes include the capability to mount an immune response or to secrete varying levels of enzymes in tissue. These capabilities may influence the survival of an animal or a line of animals. Your responsibility is to care for these animals by providing the appropriate environment to control and measure these differences. The phenotype may be measurable by histology or pathology. Genetic mutations may cause specific metabolites or other chemicals to be secreted. Histopathologists can apply various staining techniques to detect the presence of metabolites or changes in cellular structure, so they may be seen through a microscope. Pathologists may see changes in anatomy—something stunted, abnormal, or missing. Once again, the proper preservation, dissection, and handling of such samples are essential to the success of the research. Commercial companies provide services to run a battery of tests to characterize phenotype. Some research institutions have also developed their own unique tests. The combined results of these tests can suggest a genetic component to an observed phenotype. Such tests are used to target drug activity and to understand the role genes play in biological processes. Gene Linkage • Linked genes are located adjacent to one another on the same chromosome and tend to be inherited together. Thus, two or more linked but independent traits are almost always expressed together in the same genotypes and phenotypes. • A classic example of gene linkage is the linking of the recessive genes for albino (white) coat color and pink eyes on mouse chromosome. When mice inherit this allele for white fur, they will always have pink eyes. Strain & Stock Nomenclature • Nomenclature is used to characterize outbred stocks and inbred, hybrid, and transgenic strains of mice. • Most inbred strains are designated by capital letters or a combination of capital letters and numbers. • • Examples of inbred mouse strains include AKR, DBA, and C57BL. A few strains, such as 129, are designated by numbers only. • The substrain symbol shows the number of the line or an abbreviation of the name of the person or the laboratory that developed the substrain. The substrain symbol follows the strain name and is separated from it by a diagonal slash. For example, C57BL/6J indicates substrain 6 of a C57BL mouse bred by the Jackson Laboratory. An exception to this rule is BALB/c. The c in this name represents the allele for albino. • Outbred stocks, such as Swiss Webster, are usually designated by capital letters or a combination of capital letters and numbers. The abbreviation for the holder (producer) of an outbred stock precedes the stock name and is separated from it by a colon (:). For example, Tac:SW indicates the Swiss Webster (SW) mouse stock, bred by TaconicBiosciences. Mating Systems Pairing animals for mating depends on several factors, including species characteristics like genetics and space availability. For example, gerbils form a monogamous pair for life, and neither member of the pair will accept another mate. Other factors may be involved in determining the type of breeding method used, such as study requirements. Success of the breeding program requires a thorough knowledge of species variations, an understanding of the specific goals of the research project, and efficient and accurate recordkeeping. Monogamous Mating One female is bred with one male. The pair may be left together for the long term. • PROS: • Simplified recordkeeping - Once the breeding cage is set up, identification numbers and genetic information are recorded on the cage card. Litters are noted on the card after birth. • Reduces aggression • Takes advantage of post-partum estrus • The number of litters per female is maximized per unit of time. • Labor requirements are lower because animals are not removed from the cage. Polygamous & Harem Mating • These two mating systems are commonly used for mice and may be used for rabbits. • In both polygamous and harem mating, two or more females are bred with one male. • In polygamous mating, females are removed from the mating cage once they are determined to be pregnant. • In harem mating, the group is kept together. • Polygamous or harem matings are often used for mating transgenic or gene-targeted mice that have difficulty producing litters. By housing more than one female together, the female mice normally establish the same estrous cycle, thus maximizing the chances for pregnancy. • Recordkeeping is more difficult when using the harem system, because it is usually impossible to identify which offspring belong to which mother if they produce pups at the same time. • Of all mating schemes, the harem mating system demands the largest cage size, since cage size requirements are based on the number of animals, including pups. Intensive Breeding Keeping the male continuously with the female or females is referred to as intensive breeding. • PROS: • Simplified recordkeeping - Once the breeding cage is set up, identification numbers and genetic information are recorded on the cage card. Litters are noted on the card after birth. • Reduces aggression • Takes advantage of post-partum estrus • The number of litters per female is maximized per unit of time. • Labor requirements are lower because animals are not removed from the cage. • CONS: • There is a higher demand for space, including cages and equipment. • Intensive breeding usually requires more males in the colony, although fewer males can be used if one stud male is mated to multiple females as with harem or polygamous breeding. • The older litter must be weaned before the next litter is born to prevent overcrowding and the death of the newborns. Overcrowding is greater when two breeding females are in the same cage because they will often cycle simultaneously and produce their litters at the same time. Non-intensive Breeding • Non-intensive breeding is when the stud male and females are housed separately while the females are rearing their young. The female is not permitted to mate again until the young are weaned. • PROS: • It reduces the risk of fighting between aggressive females, which is typical of some strains. • The newborns cannot be killed by the male because he is removed prior to parturition. • There is greater control in timing litters; females will only become pregnant when placed in a cage with stud males. CONS:  Labor costs may be higher because animals need to be moved in and out of breeding cages.  The benefit of postpartum estrus is lost because no male is present at parturition.  Recordkeeping is labor-intensive because records must track which male has been with which females. In the non-intensive breeding method, if the male of the species marks territory, the animals are commonly mated in the male’s home cage. When a male mouse is placed in the female’s cage for breeding, for example, he will spend a significant amount of time marking his territory prior to mating with the female. To minimize the time for breeding, these mice are mated in a cage the male has already marked. Timed Mating A timed mating system is used to provide embryos or newborn rodents at a precise date needed for studies. • With the timed mating system, the female is introduced in the male’s cage, usually in the late afternoon before the technician leaves for the day. • Early the next morning, the technician checks for a vaginal plug to confirm that mating occurred during the previous night. The male's ejaculate coagulates to form a waxy plug in the vagina of the female; this may protrude from the vulva. When fresh, the plug is a cream color. • Technicians can check for plugs by gently inserting blunt-tipped forceps into the vagina and looking for whitish-yellowish material. These plugs may also be found on the cage floor a few hours after copulation has occurred. Because plugs may become lost in the bedding material, it is important to check females as early in the day as possible to detect breeding. • The morning when a plug is found is considered the first half-day of gestation. The presence of the plug only indicates that copulation took place; it does not indicate impregnation. • If a male is sterile but produces an ejaculate, a plug will be formed upon mating just as it would be for a fertile male. • It is also worth noting that the presence of only females in a cage over a period of time may depress the estrous cycle of all the females in that cage. • The addition of a male to the cage helps initiate estrus in group-housed females in about 3 days. This phenomenon is called the Whitten Effect. • Manipulating the onset of estrus is sometimes used by breeders to enhance timed matings. Breeding Schemes A breeding scheme refers to the plan for producing a colony with a desired genetic makeup. Following are breeding schemes that are used most often for the creation or maintenance of transgenic animal lines, particularly in rodent species. Outbreeding • Breeding animals that are unrelated or only distantly related is called outbreeding. • Outbred stocks are bred to maintain the genetic differences among the animals, thus making each animal genetically unique. This breeding scheme often produces more vigorous offspring and a larger litter size than inbreeding, and outbred females are often better mothers. Inbreeding Inbreeding uses sibling mating to produce animals with minimal genetic variation and, often, unique characteristics that meet specific research needs. • Regardless of the genetic outcome desired, establishing inbred colonies generally involves the propagation of three distinct colonies: the foundation colony, the expansion colony, and the production colony. The foundation colony consists of the original animals with the desired DNA, often referred to as founders. 1. Founders are bred to establish an expansion colony, which helps ensure that the desired genetic trait will not be lost if anything happens to the founders. 2. Breeding of the expansion colony generates the production colony, which are the animals used in research studies. Hybrid Breeding • Hybrid breeding is a selective system in which the parents come from different inbred strains. Hybrid breeding is frequently used to transfer a desired mutation from one to the other strain. The offspring are a mixture, or hybrid, of their parents. • If it is necessary to continue a particular hybrid breeding program, the two original strains must be crossed to produce each generation; mating hybrid offspring to each other or to the parents will not produce the same results. • Hybrid strains are designated with a shorthand combination of the two parental strains, noting the female parent first. For example, the hybrid C3D2F1 is an F1 (first generation) cross between a C3H/He female and a DBA/2 male. • All F1 hybrids are genotypically and phenotypically identical and are heterozygous at all genes. However, all F2 (second generation) hybrids are not genetically identical to each other; in fact, they are genetically heterogeneous. Therefore, it is important to keep excellent records and monitor breeding animals closely so that unplanned matings do not occur. Recombinant Inbreeding • Hybrid breeding followed by brother-to-sister matings or inbreeding of the F1 and subsequent offspring results in recombinant inbred strains. • Recombinant inbred strains can be helpful in determining inheritance of traits influenced by several genes or in determining the linkage of genes. Coisogenic Breeding Occasionally a spontaneous mutation occurs in an inbred strain. The animal with the mutation differs from other animals of that strain, usually by a single mutant gene, and is called a coisogenic animal. 1. If a coisogenic animal carrying the mutation is bred to other members of the same strain and the mutant gene is maintained, a new animal strain is created. Coisogenic animals are ideal for studying the effects of one gene when all the other genes remain identical. Congenic Breeding • Congenic strains can be constructed by selectively mating an animal carrying the mutation of interest to an inbred animal from a strain of choice. Of their offspring (the F1 generation), carriers of the mutation are mated with animals from the same inbred strain to produce the F2 generation. The same mating strategy is repeated in the F2 generation: carriers of the mutation are mated with animals from the same inbred strain, and so on. After 10 to 12 cycles of mating with the selected inbred strain, the mutant gene becomes fixed on the genetic background of the chosen inbred strain and will be expressed in subsequent generations. • The serial back-crossing removes the genome of the donor strain except for its mutated gene. Congenic strains are used in mapping a gene’s position on the chromosome (its locus) and determining how a single mutation affects phenotypic expression. Artificial Insemination • Artificial insemination entails manually placing semen into the reproductive tract of a female in heat. • It is used most often in large species, such as livestock, dogs, and rabbits. In large animals, a female in estrus may be used to induce a male to ejaculate into an artificial vagina for the purpose of sperm collection. In small animals such as rodents, sperm is collected directly from the ductus deferens and epididymis of a euthanized male. The female to be inseminated may be naturally cycling or may be artificially induced to ovulate with hormone injections. In Vitro Fertilization In vitro fertilization involves the union of eggs and sperm outside the body. These techniques require precise planning and timing to ensure that the surrogate mothers are synchronized with the egg or embryo donors. Much effort and expense are involved, and accurate records are essential. 1. To collect ova (oocytes) for in vitro fertilization, ovulation is induced in a female by injecting her with a combination of gonadotropins (reproductive hormones) in a treatment regimen that is appropriate for the species. a. 2. 3. Gonadotropin injections usually result in superovulation, which is the production of an increased number of eggs that mature and are released from the ovaries simultaneously. This technique is particularly useful when it is desirable to collect many eggs from the same female for in vitro fertilization. To collect ova from large animals, the females are anesthetized, and unfertilized eggs are aspirated directly from the ovaries or collected from the salpinges (fallopian tubes). In rodents, the female is euthanized, the reproductive tract is removed, and ova are collected from the salpinges. The eggs are fertilized in vitro with previously collected sperm. 1. 2. In rodents, the resulting zygotes may be injected or implanted into a pseudopregnant female’s uterus or salpinx, where they can continue normal development. (Females are made pseudopregnant by being bred with a sterile male.) In large species, the fertilized eggs are surgically transferred into the uterus (sometimes the salpinx) of the ovulated female. External Fertilization External fertilization in vertebrates is most common in amphibians and fish, where environmental factors and timing are the keys to success. • Working with fish or amphibians has the advantage that no surgery is required to obtain the embryos for study. Additionally, there are usually large numbers of embryos produced at the same time, which are easy to manipulate, develop rapidly, and exhibit high fecundity. 1. Females release eggs into the water simultaneously as males release sperm, and the gametes fuse and fertilization occurs. 2. Gonadotropins may be used to stimulate egg production. 3. Anesthetized males and females are squeezed or stroked gently but firmly on the body in the direction of the genital region, where gametes are collected. 4. The eggs and sperm can be mixed for a timed fertilization. Breeder Selection The selection of breeding animals is crucial to breed efficiently and obtain offspring with the desired genotype. Otherwise, unneeded animals are produced and resources such as caging, feed, and technician labor are wasted. Choosing the appropriate genotype of animals to mate, calculating the number of offspring of each genotype that will result, and preventing unplanned matings all fulfill the concepts of the 3Rs. The choice of which genotypes are needed for breeding is typically made by the research team, and you could be assigned the task of managing the breeding of a single line or an entire colony. For example, instead of being told to place male #154 and females #169 and #170 in a cage together, you may simply be asked to set up a breeding trio of a particular genotype, such as a wild-type male with homozygous females. Good recordkeeping plays an important part in the selection of mates, so detailed and accurate records will aid your selection of the correct animals for producing the desired progeny. Failure to correctly document the nomenclature or genetic information of animals can have far-reaching effects in later generations of offspring and potentially cause the loss of much research and grant funds. Dedication and keen observation skills will help make you an even more valuable part of the research team. Keep good records on breeding performance by noting which females are good mothers and which males are good studs. On the other hand, too purposeful a selection of animals could cause breeding problems. For example, always choosing the leanest females to minimize obesity in a mouse colony may eventually result in insufficient energy stores for lactation, and reduced lactation can cause the loss of pups. Overly selective breeding may also produce unexpected results in studies. Within any breeding colony, subtle changes can occur in anatomy and physiology, including at the cellular level. These changes can introduce unwanted variability into a study. So, by not thinking through the selection of breeders or by not verifying criteria for selection with the investigator, a technician can unduly change critical aspects of the research study. Other factors that can adversely affect breeding include the number of offspring, diet, age, if the female has successfully bred before, cage handling, environmental conditions (noise, vibration, temperature, nesting material, placement in the room or on the rack), frequency of cannibalism, behavior, and condition of the male (aggressive, too young or too old, “sterile” genes). Pay attention to all factors that undermine or support successful breeding and colony maintenance. With so many variables to consider, it is easy to overlook something that can affect breeding success. Be sure to alert the PI if you have a concern and involve your supervisor or veterinary staff when addressing these issues. Breeding Cage Management Laboratory animal technicians must know the basics of reproductive physiology to effectively manage breeding programs that meet the research program’s requirements. This knowledge is also essential for technicians to offer appropriate care to pregnant or lactating laboratory animals and their young. Basic Reproductive Physiology Estrous Cycle • Most female laboratory animals have estrous cycles. Those animals that have only one cycle per year are monoestrous, and those with repeated cycles throughout the year are polyestrous or seasonally polyestrous. Each estrous cycle has four stages: proestrus, estrus, metestrus, and diestrus. The duration of each stage is different in each species. • During proestrus, eggs (ova) develop in the follicles of the ovary, but a female cannot become pregnant during this stage. During this time, a female begins to react sexually toward males, but still refuses them, or in some species may display aggression. • Estrus: the time where the female is at maximum receptivity to copulation, and ovulation occurs. Physical and behavioral signs of estrus are commonly used to determine optimal breeding periods. In some species, the female stands near a male with her legs in a fixed position, her hindquarters elevated, and her tail to one side. This position is known as lordosis. • In the final two stages, metestrus and diestrus, the female ignores the male. • Anestrus is the long period between breeding seasons in some animals, such as dogs and cats. Basic Reproductive Physiology Copulation & Gestation • Proof of copulation: In some rodents, copulation can be verified by observing the presence of a vaginal plug. For most common laboratory animals, a more accurate method to verify that breeding has occurred is to take a vaginal swab and examine it microscopically for the presence of sperm. • Gestation period: the time from when an egg is fertilized to birth. The length of the gestation period is species-specific. • Pregnancy detection: is usually confirmed through visual inspection or gentle palpation of the abdominal area. Ultrasound and radiographs may also be used to confirm pregnancy. • Handling: Special care must be given to pregnant animals. Technicians must be aware that these animals are stressed by disturbances, so careful handling or restraint is necessary to prevent disrupting the pregnancy. Some species need to be handled differently; for example, when picking up a pregnant guinea pig, provide extra support to the hindquarters. • Feed: Nutritional requirements often differ during pregnancy; many species require more protein and calories. It is important to know any special requirements necessary during parturition, postparturition, and neonatal care. Basic Reproductive Physiology Parturition • Parturition is the birth of the young at the natural end of gestation. • To enhance a litter’s survival, it is best to leave the female undisturbed for 2 to 3 days before she delivers and perhaps as long as 4 to 5 days after parturition, particularly if the mother is very sensitive or of a delicate strain. • The cage should be handled minimally and not changed during this time. In general, the first few litters born have fewer pups than later litters if the female is not physiologically mature when impregnated. There is also a tendency for females to offer poorer maternal care to earlier litters, which may result in a higher mortality rate. As animals approach the end of their reproductive lives, litter size again tends to be smaller due to declining fertility. • Dystocia (difficult birth) is occasionally observed in laboratory animal species. The degree to which the technician can intervene and assist the dam depends on the species of animal with dystocia. • In general, for large animal species, more options exist for assisting in the delivery process should a problem arise. • In rodent species, the options for treatment may be limited to euthanasia of the distressed dam since the parturition process is extremely fast. • An institutional SOP should exist to guide the technician in responding to dystocia; in all cases, a facility veterinarian and the PI should be notified. • Some animals, such as mice, rats, and guinea pigs, have a postpartum estrus that occurs within 24 hours after giving birth. In continuous breeding pairs, females are usually fertilized during this period and remain pregnant while nursing their previous litter. Managing the Litter Maternal neglect and cannibalism, which can be a problem in many laboratory animals, are usually caused by inexperienced females, overcrowding, poor or varying environmental conditions, disturbance by staff, or stress caused by other laboratory animals in the area. To protect the litter, it is important to address and control these conditions when possible. Foster Care • A foster mother is a lactating female that rears the young in place of the biological mother. Young animals, particularly valuable ones, are given foster mothers under the following conditions: • The mother dies. • The mother shows no maternal instinct and does not care for the young. For example, the mother does not make a nest. • The mother has a physical or genetic abnormality that prevents her from caring for the young. • The mother does not produce enough milk. In mice and rats, if the neonates do not have a visible milk spot (milk seen in the stomach through the abdomen) within 8 hours, there is likely a problem with nursing. • In mice and rats, an early sign of impending cannibalism is observing the mother biting the pups. To avoid losing the litter, it may be wise to foster the pups as soon as possible. The earlier that fostering begins, the better the chance of raising healthy pups. An ideal foster mother will have had two or three successfully weaned litters. In fostering situations, there are two important conditions that must be met. First, the total litter size should remain about the same; at most, a foster mother may be able to produce enough milk for two more pups than she originally gave birth to. Some of the pups from the mother’s own litter may be culled when the foster pups are introduced. Secondly, the foster pups should be close in age to the mother’s own pups. For best results, select a mother whose current litter is within 48 hours of age of the pups to be fostered. The mother’s milk production is related to the age of the mother’s own pups. Follow these steps to introduce pups to the foster mother: • Wear clean gloves during the transfer to prevent contamination of the pups with human scent. • Move the foster mother or parents to a holding cage. • Promote acceptance by the foster mother by having her urinate on the gloves used for the transfer or on the foster pups. Scent can also be transferred by rubbing bedding from the foster cage onto the new pups. • Warm the new pups, then place them in the cage among the mother’s own pups and leave them alone for a few minutes before returning the foster mother (and father, if he was housed with the litter) to the cage. • Provide some distraction to the mother when introducing her to the foster pups. • Once the new pups are in the cage with the foster mother, they should be left undisturbed. Place the cage where the mice can be observed quietly from a distance without needing to remove the cage from the rack (such as at the end of a rack). If the foster mother accepts the pups, she should begin to nurse and clean them within 1 hour. If there is no such activity in the first 2 to 3 hours, another foster mother should be found. This short wait allows you enough time to rescue the pups and foster them again before they decline. The longest you can wait after fostering is 6 hours, else the pups will decline. In many circumstances, it is essential to clearly identify the foster pups so that they can be correctly separated from the original pups at weaning. It is very helpful if your fostermother’s pups are a different color than those fostered. The different coat colors help identify the foster pups when it is time to wean them. Both the birth mother’s cage card and the foster mother’s cage card should include information about the pups. Identification of pups should follow what is approved in the appropriate protocol. Culling • In some experimental or breeding management protocols, only a specific number of pups are allowed per litter. • For example, a mother from a specific strain can only support a certain number of pups, so excess pups would become malnourished or neglected by the mother. • In some facilities, it is common practice to limit the number of pups per breeding cage. • Sometimes pups from an oversized litter are transferred to an undersized litter (if genetic and pathogen status are the same), and sometimes the pups are euthanized. • If pups are euthanized, the AVMA Guidelines for the Euthanasia of Animals and institutional policies and guidelines must be followed. • Since rodent pups are resistant to CO2 asphyxiation, they are often briefly exposed to CO2 for anesthetic effect, then quickly decapitated using sharp scissors. Managing the Breeding Colony Environment Managing environmental parameters is vital to a successful breeding program. Housing breeding cages in a quiet area away from busy traffic areas of the facility can dramatically improve breeding success. Microenvironment Considerations • Larger cages may be needed to accommodate parturition and the numerous offspring of some laboratory animals. • Hanging wire cages should not be used for breeding programs for species such as rabbits, guinea pigs, hamsters, gerbils, rats, and mice unless a nesting box is added. Direct contact with the metal cage can cause chilling of newborns; also, smaller offspring, such as mice, may fall through the wire mesh of hanging cage floors. Macroenvironment Considerations • Consistency of the macroenvironment is essential to any successful breeding program. • Every attempt must be made to ensure that light cycles and intensity, temperature, and humidity are appropriate and maintained at consistent levels. • Noise and vibration should also be controlled, particularly ultrasonic noise. Many rodents communicate at ultrasonic frequencies, so the presence of environmental ultrasonic noise can have significant detrimental effects on behavior, mating, and animal health. • Variations in the macroenvironment can result in disruptions to the breeding colony, such as: • Failure to engage in normal mating behavior • Disruption of the estrous cycle • Abandonment of young • Cannibalism The Role of Pheromones • Pheromones are naturally occurring chemical compounds that are produced by all animals. Pheromones play an important role in animal mating behavior. These compounds are essential for attracting the opposite sex. • The role they play in mating can be either disruptive or supportive. • When pheromones from an unfamiliar male are detected, they may block pregnancy in a recently mated female. • Conversely, without the presence of pheromones, sexual activity such as courtship and mating may never take place. • Managing the exposure to pheromones when breeding animals is essential. • To avoid a disruptive exposure to pheromones, separate animals according to reproductive status, practice good exclusion techniques (such as PPE changes) and maintain proper ventilation in the macroenvironment. Weaning & Identification of Rodents • Outbred mice strains are more robust and are typically weaned on schedule, while some inbred strains may be smaller and thus need to remain with the dam a few days longer. • Rats are often weaned at 20 to 21 days. • Mice, hamsters, and gerbils are weaned between 21 and 28 days. • The right time to wean depends on the size of the pups. • If the pups cannot survive on their own, they should not be weaned. • Sometimes pups are weaned early because a second litter is due to be born. In such a case, a decision must be made on which litter is more valuable to save. If it is the first litter, the pups should not be weaned until they are ready, regardless of a new litter arriving. If the second litter is more valuable, the first litter can be weaned at 21 days of age. • To improve their chances of survival, feed pellets and packets of nutritive gel supplement may be placed on the cage floor for the first week or so, to help them obtain nutrients and moisture. Sexing • Males and females should be separated at weaning. • In many animals, including rats and mice, the sexes may be differentiated by the anogenital distance, which is the distance between the anus and the genital papilla (prepuce, or clitoris). • The anogenital distance is greater in males than in females. • Also, two visible lines of nipples develop in the female. • Sex determination of weanling animals is best done by comparing animals placed side by side. Weaning Cages • The Guide has guidelines for cage density for most laboratory animals based on the weight of individual animals, and the Animal Welfare Regulations specifies minimum standards for USDA-covered species. • When setting up a weaning cage, it is recommended to add some bedding and nesting material from the parental cage to provide the scent of familiar surroundings and ease the transition. Feed and water should be placed so that the pups can easily reach them, and some feed pellets can be placed on the floor of the cage. • The technician must consult institutional SOPs to determine the number of weaned animals allowed per cage. • Some institutions may allow more immature mice to be weaned together in one cage for a short period of time while they acclimate to life away from their mother. Retiring Breeders • It is important to know when to retire and replace breeders. For example, it is recommended to retire male and female mice after about 6 months, or after females have had 6 to 7 litters. If bred longer, the number of pups per litter decreases dramatically. • Males should be replaced when they have not produced a pregnancy in a receptive female after 3 to 6 weeks. • Consider replacing rodent breeders that have produced two consecutive litters in which the quality of the pups is not adequate. They may be producing pups that fail to thrive or are less productive than the rest of the colony. Recordkeeping • Accurate and appropriate records are very important for an efficient breeding program. Accuracy is vital when troubleshooting problems, for determining what needs to be accomplished daily, and to facilitate compliance with applicable policies, guidelines, and regulations. A well maintained recordkeeping system can be used to track: • Individual animals’ ancestors, siblings, and descendants; • Matings; • Litters born; • Offspring used in studies; • Selection of the next generation of breeders; and • Test results for genotype and phenotype. Cage Cards • Breeding colony records can be kept in several different forms, but the most essential record for rodent breeding programs is the cage card. Since the cage card generally stays with the animal throughout its life, it is critical that all pertinent breeding information is captured on the card prior to being entered into a computerized database or a centralized notebook. Cage cards can be an invaluable source of information; they include the animal’s genotype, date of birth, mating log, litter information, and weaning records. • There are some key strategies for organizing breeding records and cage cards: • Use a different and consistent color of cage card for each strain or each project. • Physically separate similar strains or similar coat colors. For example, put them on a different shelf, on a different rack, or in a different part of the room. • Always use correct nomenclature and identification on every cage card. • All cage cards for one cage should be kept together in the card holder until all animals in the cage are removed from production to ensure that all information about previous litters is available. Centralized Notebooks & Computer Databases • Notebooks and computer databases are helpful when managing transgenic rodent colonies. • If the cage card is considered the “picture,” then the compilation of all the information from the cage cards of a breeding colony can be considered the “story”. • Colony records contain overall information on ancestry. This information is used for analyzing and tracking genetic data, as well as for determining which mating pairs will produce progeny of specific genotypes. • Computerized database programs are commercially available and preferred by most laboratory animal facilities. However, when computers are not practical or available, handwritten records of mating logs, pedigrees, and breeding colony information serve the same purpose. • Do not transfer these handwritten records between rooms, as this can potentially transmit an unwanted pathogen through the colony. Colony Preservation & Animal Biosecurity • The Guide defines animal biosecurity as all measures to control known or unknown infections in laboratory animals. • Animal health is of primary importance in all laboratory animal species. • It is poor management to establish breeding colonies with foundation animals that carry disease. • Methods must be in place both to minimize the risk of infecting a colony and to deal with any disease that occurs within the colony. • SOPs are necessary for maintaining the biosecurity of animals during cage changing, transporting, shipping, receiving, quarantine, and routine tasks. • Institutions often have specific SOPs developed for breeding colonies that include more stringent entry requirements, enhanced use of PPE, and other bioexclusion methods. • These precautions typically include the sterilization of all equipment entering the colony area, specialized animal handling techniques, and enhanced health monitoring practices. • Once a healthy breeding colony is established, animals are continually monitored to ensure that the colony stays free of adventitious microorganisms. Despite these precautions, however, infectious diseases may occur in a breeding colony. • Strict adherence to appropriate husbandry practices is vital. Animal Import & Quarantine Programs • An institution’s animal procurement, quarantine, and health monitoring procedures are critical for the breeding program. • Some institutions have a separate, more stringent set of standards for receiving animals intended for breeding as opposed to those received for experimental colonies only. • Restrictions may be placed on the sources from which breeding animals can be obtained, the health status of the animals in the shipping facility, and the methods of shipping and receiving. • In most institutions, animals intended for use in breeding colonies will undergo a longer quarantine period and may be subject to multiple health screenings prior to being allowed into the breeding program. • Alternatively, methods of embryo rederivation and cryopreservation permit a faster admittance to the breeding colony. Rederivation Rederivation may be necessary to clear a colony of an infectious disease or to obtain an animal line from a facility where animals cannot be verified as specific pathogen-free. Embryo rederivation generally involves taking fertilized eggs or embryos from the animals that may have pathogens and then surgically implanting them for gestation into a “clean” female (one that has been tested and found free of pathogens). • Serological testing is performed on recipient females to verify their health status when the pups are weaned and before being used in a study. • Another method is cesarean rederivation. This method uses a combination of timed pregnancies, sterile surgical cesarean section delivery of the pups, and fostering methods. While cesarean rederivation is not considered as reliable as embryo rederivation for clearing a pathogen, it can save valuable strains of animals because most diseases do not transfer to the young in utero. Cryopreservation • One way to preserve a genetic line of some animals is to freeze (cryopreserve) embryos, eggs, sperm, or ovaries at very low temperatures. A special process is used when cryopreserving to minimize the formation of harmful ice crystals within the cells, which could result in cell death. • Cryopreservation can be used as a backup in the event of a natural disaster or a pathogen outbreak in the colony. The cost of producing genetically engineered animals is enormous. • Investigators may have many thousands of dollars and years of effort invested in a single genetic line. The loss of these animals due to disease or poor husbandry can be disastrous to the investigator and the research program. • Thus, cryopreservation should be an essential part of an animal management program. Summary Managing a successful breeding colony is not as simple as putting a male and female together and waiting for a litter of pups to arrive. Attention to all the details of colony management will enable you to generate the appropriate number of healthy animals while maintaining a cost-efficient facility. Knowing the basics of genotyping, phenotyping, and accurate recordkeeping will help ensure that the animals provided to the investigator have the genetic makeup needed for specific research protocols.