A farmer or hatchery manager who wants to manage the genetic aspects of his population must know how inbreeding can be used to improve productivity and profits. Inbreeding is one of the three major traditional breeding programmes that breeders have used for centuries to improve animals and plants. While it is not as important as selection or crossbreeding, inbreeding is used to produce genetically improved livestock, plants, and laboratory animals. Inbreeding might be the most important breeding technique used in the production of laboratory animals, because genetically uniform lines of rats, mice, etc. are desired for biological and medical research.
Inbreeding will never be as important in animal husbandry as it is in agronomy, because many plants can be self-fertilized; it is easy to create matings by artificial pollination; and many individuals can be raised cheaply in a small plot. However, inbreeding has been used to create better, faster growing livestock, and new technologies have improved our ability to create and use highly inbred fish to improve a population or for research purposes. Inbreeding programmes should be far easier with fish than with livestock, because many species can be stripped, which enables fish culturists to create mating combinations livestock farmers can only imagine. Additionally, fish are highly fecund when compared to livestock, so different types of inbreeding programmes can be used.
In order to use an inbreeding programme to improve productivity and profits, a farmer or hatchery manager must understand how inbreeding works and what it does. The genetics of inbreeding was discussed in Chapter 2, and the techniques that are used to calculate individual inbreeding values were outlined in Chapter 3. This chapter will explain how inbreeding can be used to improve populations by itself, or how inbreeding can be combined with selection and crossbreeding to improve growth rate and other phenotypes. Finally, regular systems of inbreeding that can be used to produce inbred fish for breeding programmes are described.
Those who are not interested in learning how inbreeding programmes can be used to improve a population but only want to learn how to prevent unwanted inbreeding can skip this chapter and go to Chapter 7.
Although selection and crossbreeding are the breeding programmes that are usually considered when plans are made to improve a population genetically, inbreeding is a third option that can be used to produce good results. Inbreeding is generally shunned because it is a two-edged sword that can mortally wound a population. However, when used properly, inbreeding can be an effective and efficient breeding programme. In general, inbreeding programmes are used when you have superior animals. If you inbreed average animals, you produce average animals. But if you inbreed superior animals, you can create outstanding animals.
Some of the ways inbreeding programmes can be used are described in this section.
Inbreeding is often used when a new breed, strain, or variety is founded. In many cases it is inevitable and unavoidable. New breeds can be formed as the result of a fortuitous hybrid, or they can originate from a single individual with an unusual or desirable phenotype, as was the case with the Morgan horse, an American breed founded by a stallion named Justin Morgan. When breeds are new and small, inbreeding is inevitable. When breeds or strains are first created, there may be only a few males in the breed, and one is considered to be far superior to all others. When this happens, that male is bred to many females and to a good percentage of his daughters and granddaughters in order to produce a population of animals that resembles him. This is how a breed “type” (a particular body conformation) is created. A second round of inbreeding can occur if only one or two of the male's sons are used. When the size of the breed increases, inbreeding will be less important, but it is the breeding technique that is often used to develop a new breed or strain that breeds true for type.
Linebreeding is an inbreeding programme whereby an individual is mated to its descendants. Traditionally, a single male is bred to many females. Most linebreeding programmes that are used in livestock husbandry only linebreed males, because fecundity is low and gestation periods are long. However, in aquaculture the fecundity of fish should enable farmers and hatchery managers to use females as well as males in linebreeding programmes.
Linebreeding can be used when the breed is in its infancy to develop the breed, or it can also be used in an established breed or strain when an outstanding animal is discovered. Linebreeding is used to increase an outstanding individual's contribution to a population, especially when a farmer thinks that the animal is so superior that it is unlikely that he will ever find a better one.
Linebreeding is used by many livestock farmers, even when they say that they do not want inbred animals. Linebreeding is often not considered to be inbreeding, because the major goal is not to produce inbred animals but to increase an outstanding individual's contribution to the population and to succeeding generations. However, this will occur only because linebreeding is inbreeding.
Even if inbreeding reaches fairly high levels in a population, if the inbreeding is planned it can be used to produce outstanding individuals, and linebreeding is an inbreeding programme that is designed to accomplish that goal. Inbreeding depression is a measure of the population, not of an individual. Inbreeding depression is the difference between the mean of the inbred group and the mean of a control population with no inbreeding. Since inbreeding depression is a populational value, it represents the average value, which means an individual with a high level of inbreeding can be outstanding. If a breeder linebreeds a superior male only with superior females, this programme can quickly produce a superior population.
Linebreeding is the breeding programme that is needed if a farmer wants to keep the relationship of the next generation with a particular ancestor at a high level. The genetic contribution that a fish makes to a population is halved each generation: 50% in its offspring, 25% in its grandchildren, 12.5% in its great-grandchildren, etc. After seven generations, a fish contributes less than 1% of its descendants' genes. Linebreeding is the breeding programme that can be used to reverse this trend. For example, if a fish is mated with its grandchildren (the offspring would be both its great-grandchildren and its children), its contribution to those offspring would rise would rise from the normal value of 12.5% to 62.5%.
If the male that is being linebred mates only once or twice with his descendants, the linebreeding is considered to be mild linebreeding (Figure 19). If the male that is being linebred is repeatedly mated to his female descendants or is mated to them using a regular pattern, the linebreeding is considered to be intense linebreeding (Figure 20).
Intense linebreeding is a pre-biotechnology breeding programme that can be used to create a “near clone.” By repeatedly mating a male to his female descendants a farmer can create an “almost identical” genetic twin of the male. For example, in Figure 20, 93.75% of individual G's genes come from individual A.
This type of inbreeding programme is a variation on linebreeding. In this type of inbreeding programme, a farmer usually purchases an outstanding bull and uses it as a “herd bull”; i.e., it is the only male in the herd. This bull is mated to all females and many female descendents. This inbreeding programme is used to bring a herd of animals up to the herd bull's phenotypic standards.
Figure 19. An example of a linebreeding programme. This example can be considered to be mild linebreeding. Individual A is brought back and mated with his great-grandchild. Individual A can be a male or female, but linebreeding usually uses males. Consequently, individual A is the father and great-great-grandfather of individual I. This is used to increase the percentage of A's genes in his descendants; 56.25% of I's genes come from A; FI = 6.25%.
Figure 20. An example of a linebreeding programme. This can be considered to be intense linebreeding. Individual A is repeatedly mated to his daughters. In this example, individual A is the father, grandfather, great-grandfather, and great-great-grandfather of individual G. This breeding programme is used to increase the percentage of A's genes in his descendants; 93.75% of G's genes come from A; FG = 43.75%.
This breeding programme will create herds that are highly inbred, so some farmers use several herd bulls to reduce the level of inbreeding and thus minimize inbreeding depression. The inbreeding that will be produced by this inbreeding programme can be determined by the following equation:
where: number of males and number of females are the number that produce viable offspring. The formula shows that inbreeding is controlled by the sex that is least numerous, so the rate of inbreeding is governed by the number of herd bulls. For example, if 50 females are used, the inbreeding that will be produced per generation when 1, 2, 3, 4, and 5 herd bulls are used is:
|Number of herd bulls mated to 50 females||Inbreeding per generation|
Inbreeding can be used as an extreme form of progeny testing to expose detrimental recessive alleles and eliminate families that carry these undesired genetic bombs. Progeny testing is a selective breeding programme that is often used to accomplish this, but progeny testing is usually used to eliminate only one recessive allele. Progeny testing is a breeding programme where fish with the dominant phenotype are mated to a test fish (one that is homozygous recessive) to identify and cull the heterozygotes and to identify and save the homozygotes.
By mating relatives and developing highly inbred families, a farmer can unmask many detrimental recessive alleles simultaneously and cull families that carry many hidden genetic defects. Since most animals carry several to several dozen detrimental recessive alleles that are hidden in the heterozygous state, this type of breeding programme is a severe form of progeny testing, but families that exhibit no problems are genetically superior and defect-free. Because this type of breeding programme is so severe, many inbred families must be created in order to identify and save the few that will be defect-free. Although this is costly, it is a breeding programme that will produce outstanding animals, in terms of qualitative phenotypes, within a few generations.
Inbreeding can be used to improve the results of selection when the h2 for a trait is small. When h2 is 0.15, it is difficult to improve the population by individual selection, because most of the phenotypic differences that can be measured are not heritable. When this occurs, between-family selection is more efficient because this selective breeding programme magnifies the heritable differences (those due to VA) by minimizing environmental effects (VE).
When h2 is small, inbreeding can be used to create inbred families which will further magnify the heritable differences among the families. Inbreeding makes it easier to assess the heritable differences among families, by minimizing some of the non-heritable sources (VD, VI, and VE). Inbreeding does not change the absolute amount of VA; it changes relative amount, which improves the breeder's ability to identify and save families which are superior because ofVA. This makes between-family selection more efficient.
Once a breed is established, perhaps the most important use of inbreeding is to develop inbred lines that will be used in crossbreeding programmes to produce outstanding hybrids for grow-out. Crossbreeding and inbreeding are mating extremes along a continuum: inbreeding is the mating of animals that are more closely related than the average in a population, and crossbreeding is the mating of animals that are less related than the average in a population.
The genetics that controls phenotypic expression for both inbreeding and crossbreeding programmes is a function of heterozygosity, which means it is a function of VD. Inbreeding can be used to develop inbred lines that produce better hybrids-fish that exhibit more hybrid vigour. The creation of inbred lines improves the results of hybridization by “stretching” the genetic distance between lines; i.e., inbreeding can be used to create opposite types of homozygosity, and this will enable hybrids to be more heterozygous and hopefully to exhibit more hybrid vigour. Additionally, the development of inbred lines can be used to fix one or two qualitative traits in each line, so all F1 hybrids will have two or more desired qualitative traits.
There are three ways this type of breeding programme can be conducted: The first two types hybridize inbred lines. In one, inbred lines are created in two strains that were previously shown to produce outstanding F1 hybrids. The other option is to create inbred lines in a number of strains and then hybridize them in an attempt to discover a combination that works. The more efficient way is to create inbred lines in two strains that were previously shown to produce outstanding F1 hybrids.
This approach makes sense, because producing superior F1 hybrids is a hit-or-miss proposition. You cannot predict which mating combinations will nick and produce great hybrids and which mating combinations will produce duds. If you create inbred lines in two strains that have already been shown to produce outstanding F1 hybrids, it is likely that some of the inbred line combinations will also produce better F1 hybrids. This type of breeding programme is illustrated in Figure 21.
Inbreeding can also be used to create an inbred line that will be used in a topcrossing programme. Topcrossing is a crossbreeding programme in which an inbred line is hybridized with a randomly bred line to produce F1 hybrids. This type of breeding programme has been traditionally used by animal breeders to produce outstanding animals for judging contests.
Even though inbreeding can be used to improve a population, it does cause inbreeding depression. Fecundity is often adversely affected by inbreeding. When fecundity declines, it may not be possible to produce enough F1 hybrids for grow-out. This problem can be circumvented by developing four inbred lines and mating them to create di-hybrids.
The first step in this breeding programme is the creation of four inbred lines. The second step is the creation of two F1 hybrids. The third step is the mating of the F1 hybrids to produce a di-hybrid. The creation of the F1 hybrids reduces F to 0%, so fecundity will return to normal. The F1 hybrids are used to produce the seeds or animals that farmers will use for grow-out. This is how modern agribusinesses produce seed corn. If each inbred line is used to fix one or two qualitative phenotypes, the di-hybrids will exhibit heterosis, and all will also have several desired qualitative phenotypes. This type of breeding programme is illustrated in Figure 22.
The ability to use inbreeding in some of these breeding programmes depends on a farmer's or hatchery manager's ability to produce inbred lines quickly and efficiently. This can be accomplished only if breeders know how much inbreeding will be produced by a particular mating programme and the rate at which the inbreeding is created. The amount of inbreeding that will be produced by a given inbreeding programme depends on the relationship that exists between the fish that are mated. Consequently, it is important to know how much inbreeding is produced when relatives mate and produce offspring. This information can then be used to develop regular systems of inbreeding that will enable a farmer to produce predictable levels of inbreeding in a predictable time frame.
Figure 21. Breeding programme illustrating how an inbreeding programme can be combined with hybridization. Inbred lines of fish are created in two strains that have been previously shown to produce outstanding F1 hybrids. Once created, the inbred lines are hybridized to produce F1 hybrids. The use of inbred lines will improve hybrid vigour. This type of breeding programme is the way to maximize exploitation of VD. Once identified, selection can be conducted in the inbred lines to improve growth; this will exploit VA.
Figure 22. Breeding programme illustrating how inbreeding can be used to produce di-hybrids for grow-out. In this breeding programme, inbreeding is used to create inbred lines in four strains that have been previously shown to produce outstanding F1 hybrids. The F1 hybrids are then crossed to produce di-hybrids. This breeding programme is used when inbreeding depression decreases fecundity in the inbred lines to the point where it is impossible to produce enough F1 hybrids for grow-out. The F1 hybrids have F = 0%, which restores fecundity; the F1 hybrids are hybridized to produce the seed or animals that will be used for grow-out.
This section will show how much inbreeding can be produced when relatives are allowed to mate. In order to do this, it will be assumed that no previous inbreeding exists, so the amounts that are mentioned are those that are produced when inbreeding first occurs. Figure 23 shows a family tree with 41 individuals that could be mated to produce offspring with F ranging from 0% to 25%. The values presented in Figure 23 show that the matings of close relatives, such as parents with offspring and brothers with sisters, produce fish with high levels of inbreeding (25%) and that the inbreeding produced steadily decreases as the distance between relatives increases. For example, matings between third cousins, matings between fourth cousins, and matings between fifth cousins produce very little inbreeding (0.39%, 0.097% and 0.024%, respectively). Fifth cousins share so few genes that the inbreeding produced when they mate is insignificant; thus, on a practical level, many geneticists consider fifth cousins to be unrelated.
The inbreeding values listed in Figure 23 explain why first cousin marriages are forbidden in many cultures while second cousin marriages are sanctioned. First cousin marriages produce children with F = 6.25%, while second cousin marriages produce children with F = 1.56%.
One type of cousin marriage will produce a large amount of inbreeding. A double first cousin marriage (the offspring from the marriages of a pair of sibs that married a pair of sibs; first cousins are individuals that are not sibs but that share two grandparents in common; double first cousins are individuals that are not sibs but that share all four grandparents in common-individuals 22 and 23 in Figure 23 are double first cousins). Double first cousin matings produce as much inbreeding as half-sib, grandparent-grandchild, and aunt-nephew (or uncle-niece) matings-12.5%.
Figure 23 shows that some matings will produce F = 0%. It should be obvious that the mating of unrelated individuals such as 13 and 14 would produce offspring with no inbreeding, but other matings which would usually be considered to be between relatives would also produce offspring with no inbreeding. For example, if individuals 9 and 27 were mated, the offspring would have F = 0%, even though this would be considered to be an uncle-niece marriage. The reason why the 10 × 26 mating produces offspring with F = 12.5% while the 9 × 27 mating produces offspring with F = 0% is individual 9 is individual 27's uncle by marriage. Individuals 9 and 27 share no genes in common; they have no common ancestor and are not related, which is the requirement for the production of inbred offspring. The 9 × 27 mating might be incestuous in most societies, but it produces no inbreeding.
The relationships illustrated in Figure 23 and the amount of inbreeding produced by matings among those individuals can be used to develop regular systems of inbreeding in order to produce inbred lines of fish for breeding purposes. The reason why regular systems of inbreeding need to be used is simple: breeding programmes are expensive and take years, so random, haphazard breeding systems that produce unknown and unpredictable amounts of inbreeding are expensive lessons in poor planning and futility. All breeding programmes require meticulous planning, and inbreeding is no exception. If farmers or hatchery managers are going to use inbreeding to improve a population, they must know how to conduct regular systems of inbreeding. Regular systems of inbreeding enable a farmer or hatchery manager to know what matings must be made generation after generation, and this allows them to predict the amount of inbreeding that will be produced each generation.
There are a number of regular systems of inbreeding. Some are relatively simple, but others are so complex or produce so little inbreeding that their use is impractical. A major goal of an inbreeding programme is to produce F = 40–50% in a number of families and to do it fairly rapidly. The only liability of inbreeding programmes that quickly produce large levels of inbreeding is that inbreeding depression will reduce viability and fecundity, and this will cause some of the inbred lines to go extinct. Consequently, many inbred lines must created so that some will survive the ravages of inbreeding depression.
Figure 23 shows that there are six types of matings that produce large amounts of inbreeding. Some of these matings are difficult to make generation after generation (e.g., grandparent-grandchild), which limits their use in practical inbreeding programmes. Relatively simple regular systems of inbreeding that will produce F = 40–50% in four to five generations are: parent-offspring, full-sib, half-sib, and double first cousin.
Regular systems of inbreeding divide a population into a series of inbred families. This occurs because the only way a regular system of inbreeding can be maintained is by continually mating a specific combination of relatives, which means mating fish within a family. The size of the family is determined by the inbreeding programme: the closer the relationship between mates, the smaller family size can be.
|MATING||INDIVIDUALS IN FAMILY TREE||F PRODUCED BY MATING|
|Parent-Offspring||7 × 10||25.0%|
|Brother-Sister (full sibs)||10 × 15||25.0%|
|Half-brother-Half-sister (half sibs)||26 × 27||12.5%|
|Grandparent-Grandchild||2 × 10||12.5%|
|Aunt-Nephew (or Uncle-Niece)||10 × 26||12.5%|
|Double first cousins||22 × 23||12.5%|
|Great-grandparent-Grandchild||21 × 37||6.25%|
|First cousins||21 × 24||6.25%|
|Second cousins||29 × 30||1.56%|
|Third cousins||32 × 34||0.39%|
|Fourth cousins||37 × 39||0.097%|
|Fifth cousins||40 × 41||0.024%|
|Unrelated||13 × 14||0.0%|
Figure 23. A family tree with 41 individuals. Males are represented by squares and females by circles. This family tree is used to illustrate the amount of inbreeding that would be produced by matings between relatives. Possible mating combinations and the inbreeding that would be produced by these matings range from F = 0% to F = 25%. Individuals 1, 2, 3, and 4 have F = 0% and are not related, and they are not related to individuals 5 and 6. Individuals 5 and 6 have F = 0% and are not related.
A breeding programme of parent-offspring matings is an inbreeding programme that is traditionally used to quickly produce highly inbred animals. A single generation of parent-offspring mating will produce fish with F = 25%, and three generations will produce fish with F = 50%; eventually, F will approach 100%. A parent-offspring inbreeding programme is illustrated in Figure 24. Five generations of parent-offspring matings, along with the inbreeding that is produced, is illustrated. The inbreeding that is created by a parent-offspring inbreeding programme is contrasted to that which can be created by other regular systems of inbreeding in Table 3.
This type of inbreeding programme is simple and requires the fewest numbers of individuals in each inbred family. Only two individuals are needed in each generation-a parent (usually the younger parent) and an offspring. Consequently, this is the least expensive regular inbreeding programme. The major liability of this inbreeding programme is that it is often difficult to mate parents with their offspring generation after generation. A number of fish species spawn only once and then die. When this happens, the only way parent-offspring matings can occur is by using cryopreserved gametes.
Table 3. Percent inbreeding produced by regular systems of inbreeding: parent-offspring; full-sib; half-sib; double first cousin. There are two half-sib inbreeding programmes: a single male is mated to two half-sisters each generation (A) (Figure 26); a single male is mated to many half-sisters each generation (B) (Figure 27). Generation 1 is the first generation in which inbreeding is produced.
|Generation||Parent-offspring||Full-sib||Half-sib (A)||Half-sib (B)||Double first cousin|
After: Wright, S. 1921. Systems of mating. II. The effects of inbreeding on the genetic composition of a population. Genetics 6:124–143.
Full-sibs are brothers and sisters that share two parents. This regular system of inbreeding is as effective in producing inbreeding as parent-offspring matings, and it is equally simple and inexpensive. A breeding programme of full-sib matings is illustrated in Figure 25; five generations of full-sib matings along with the inbreeding that is produced is illustrated. As was the case with parent-offspring matings, full-sib matings produce F = 25% in the first generation and F = 50% after three generations; eventually, F will approach 100% (Table 3).
Figure 24. A regular inbreeding programme of parent-offspring matings. Five generations of parent-offspring matings and the inbreeding produced by this breeding programme are illustrated. The first generation of parent-offspring mating occurs in the F1 generation, and their offspring (F2 generation) is the first generation of inbred fish. Fish in the P1 generation have F = 0% and are not related. In subsequent generations, inbreeding values apply only to individuals which are produced in that generation; e.g., FC = 0%, not 25%.
Only two individuals are needed per inbred family per generation, but more can be used. Even though family size of parent-offspring and full-sib inbreeding programmes can be identical, a full-sib inbreeding programme is usually less expensive, because when parents are mated to offspring fish have to be cultured for two spawning seasons. When sibs are mated, broodstock can be turned over more rapidly. A major difference between regular parent-offspring and full-sib inbreeding programmes is the fact that it is far easier to make full-sib matings generation after generation. Because full-sib matings are rather easy to make and because this type of inbreeding programme quickly produces large levels of inbreeding, full-sib matings is probably the most commonly used regular system of inbreeding.
Figure 25. A regular inbreeding programme of full-sib (brother-sister) matings. Five generations of full-sib matings and the inbreeding produced by this breeding programme are illustrated. The first generation of full-sib mating occurs in the F1 generation, and their offspring (F2 generation) is the first generation of inbred fish. Fish in the P1 generation have F = 0% and are not related.
Half-sibs are brothers and sisters that share only a single parent in common. Regular programmes of half-sib matings are used when the same ultimate level of inbreeding is wanted, but a slower rate of increase is desired. A single generation of half-sib mating produces offspring with F = 12.5%, but the amount produced thereafter depends on the half-sib inbreeding programme that is used; F will reach 40–50% after four to five generations (Table 3).
There are several types of regular half-sib inbreeding programmes. The two simplest are illustrated in Figures 26 and 27. The breeding programme outlined in Figure 26 is one whereby a breeding set of three individuals is established to create an inbred family, and a single male is mated to two half-sisters each generation. There are two matings each generation: one produces the male and the other produces the two half-sisters.
The half-sib inbreeding programme illustrated in Figure 27 is similar to that illustrated in Figure 26, except a single male is mated to many half-sisters each generation. The inbreeding produced after the first generation in this programme is less than that produced by the smaller half-sib breeding family (Figure 26; Table 3); the additional females in this breeding programme moderates the accumulation of inbreeding. The inbreeding programme illustrated in Figure 27 shows that the size of the family decreases each generation, but this is only because the illustration shows that each mating produces a single offspring that is used in the next generation. To keep family size from decreasing, one mating each generation that does not produce the male brood fish must produce two half-sisters.
The cost and complexity of half-sib inbreeding programmes depend on the programme that is used. The one illustrated in Figure 26 is not much more expensive than a full-sib inbreeding programme. The half-sib inbreeding programme illustrated in Figure 27 is more expensive; the expense for this inbreeding programme is determined by the number of half-sisters that are maintained.
Half-sib mating programmes are used because large levels of inbreeding can be produced-but at a slower rate than is the case for parent-offspring or full-sib inbreeding programmes. This means regular half-sib inbreeding programmes take longer and are more expensive than full-sib and parent-offspring inbreeding programmes.
Double first cousins are first cousins that are twice as related as regular first cousins because the parents that produced them are a pair of full-sibs that mated with a pair of full-sibs (often a pair of brothers that mate with a pair of sisters). A regular inbreeding programme of double first cousins is illustrated in Figure 28. The inbreeding produced by mating double first cousins is similar to that produced by regular systems of half-sib matings (Table 3). The breeding programme illustrated in Figure 28 may look complicated, but in reality it is a fairly simple regular mating scheme. Each inbred family in a breeding programme of double first cousin matings requires a minimum of four individuals per generation. Consequently, the cost of raising fish each generation is about twice that of a programme of full-sib matings.
Although this inbreeding programme produces levels of inbreeding that are similar to those produced by half-sib matings, it is less efficient and more expensive. When double first cousins are mated, no inbreeding will be produced until F3-generation fish are produced, because the F2 generation is the first generation in which double first cousins can be mated (Figure 28).
Chromosomal set manipulation can be used to produce highly inbred fish in a relatively short period. Individual fish with F = 100% can be produced in a single generation, while inbred lines where all fish have F = 100% can be produced in two generations.
Chromosomal set manipulation to produce inbred fish can be done in one of two basic ways, but regardless of the technique used, the fish that are produced have only a single parent. The first technique is to prevent the first mitotic division that occurs when the zygote nucleus and zygote itself divides to become a two-celled embryo. To create inbred fish, this technique is done with haploid zygotes. This technique is called either “mitotic gynogenesis” or “mitotic androgenesis,” depending on the whether the haploid set of chromosomes of the zygote comes from the mother or from the father.
Figure 26. A regular inbreeding programme of half-sib matings. In this type of regular half-sib breeding programme, a single male each generation (marked with a star) is mated to two half-sisters. This is the simplest and least expensive type of half-sib inbreeding programme. Each inbred family is composed of three individuals per generation. There are two matings per generation: one produces the male and the other produces the two half-sisters. Five generations of half-sib matings and the inbreeding produced by this breeding programme are illustrated. The first generation of half-sib mating occurs in the F1 generation, and their offspring (F2 generation) is the first generation of inbred fish. Fish in the P1 generation have F = 0% and are not related.
Figure 27. A regular inbreeding programme of half-sib matings. In this type of regular half-sib breeding programme, a single male each generation (marked with a star) is mated to many half-sisters. Five generations of half-sib matings and the inbreeding produced by this breeding programme are illustrated. The first generation of half-sib mating occurs in the F1 generation, and their offspring (F2 generation) is the first generation of inbred fish. Fish in the P1 generation have F = 0% and are not related. This illustration shows that the size of the family decreases each generation. This is because each mating in the figure produces a single offspring that is used in the breeding programme. To keep family size from decreasing, one mating that does not produce the male must produce two half-sisters.
Figure 28. A regular inbreeding programme of double first cousin matings. Five generations of double first cousin matings and the inbreeding produced by the breeding programme are illustrated. The first generation of double first cousin matings occurs in the F2 generation, and their offspring (F3 generation) is the first generation of inbred fish. Fish in the P1 generation have F = 0% and are not related.
The second technique is to prevent equational division (second meiotic division) of the secondary oocyte (egg) after sperm penetration; this prevents the second polar body from leaving the egg. This technique is called “meiotic gynogenesis”, because chromosomal set manipulation is accomplished by disrupting a meiotic division; it produces fish called “meiotic gynogens”, because all chromosomes in the offspring come from the mother.
This technology requires highly skilled labour, and the methodologies have not been perfected. At present, these breeding programmes are important for some kinds of genetic research, but their practical use has not been quantified. Consequently, these breeding programmes should be done only by scientists who work at agribusinesses or research institutions that are capable of conducting sophisticated genetics experiments. The techniques and procedures needed to create the fish described in this section are species specific, so a detailed description for each important farmed species is beyond the scope of this manual.
Mitotic gynogenesis can be used to create mitotic gynogens (all genes come from the mother), fish that are 100% inbred. The technique that is used to accomplish this with species that have the XY sex-determining system (females are XX and males are XY; virtually all aquacultured species have this system of sex determination) is outlined in Figure 29.
The first step in this breeding programme is the production of first-generation mitotic gynogens. Ultraviolet radiation is used to destroy the DNA (the genes) in sperm. The irradiated sperm are then used to activate eggs. An irradiated sperm cannot fertilize an egg because its genes have been destroyed. The activation causes the egg to undergo the equational division (second meiotic division) and to extrude the second polar body. The egg now contains only a haploid egg nucleus; this produces a haploid zygote (the zygote contains only a single chromosome (homologue) from each chromosome pair, and each chromosome comes from the mother, which is why they are called “gynogens”).
When the haploid zygote undergoes first cleavage, a pressure or temperature shock is used to prevent the haploid zygote nucleus from dividing into two daughter nuclei. If the shock is timed perfectly, the haploid zygote nucleus has replicated its chromosomes so that each daughter nucleus will have a full and identical set of chromosomes, but the haploid zygote nucleus has not divided. By preventing first cleavage, the zygote remains a zygote, but the chromosome number of the zygote has doubled from the haploid state to the normal diploid state, which means that each chromosome occurs as a pair. Since mitosis (first cleavage is a mitotic cell division) produces two identical sets of chromosomes, each chromosome pair is composed of two identical chromosomes. Consequently, every gene comes from the mother, and every gene is homozygous; the mitotic diploid gynogen is 100% homozygous and 100% inbred.
This technique does not produce many viable fish. Since each fish is 100% homozygous, every detrimental recessive allele that reduces viability, produces an abnormality, or causes death will be expressed. However, mitotic gynogens that do survive are fish that are free from detrimental recessive alleles, which means they carry no recessive alleles that produce genetic defects.
If this is done with species that have the XY sex-determining system, each mitotic gynogen is a female. The only sex chromosome that exists in mitotic gynogens are those that were contributed by the mother, and females have only X sex chromosomes (all genetic material, including all sex chromosomes, in sperm were destroyed by UV irradiation). When the diploid chromosome number is restored by the shock, all fish go from X zygotes to XX zygotes, and all are females.
Each 100% homozygous mitotic gynogen is unique because there are thousands of genes. Consequently, each first-generation mitotic gynogen is a singular inbred line.
Figure 29. A schematic flow chart of the production of an inbred line of genetically identical 100% homozygous and 100% inbred fish by mitotic gynogenesis (all genes come from the mother) in species with the XY sex-determining system. In phase 1, first-generation mitotic gynogens are produced by preventing first cleavage (a mitotic nuclear division) of haploid zygotes. The end result is females that are 100% homozygous and 100% inbred. Each gynogen is genetically unique and is a singular inbred line. Phase 2 of the breeding programme is needed to produce a family of genetically identical, 100% homozygous, and 100% inbred fish that are capable of mating to perpetuate the inbred line. Second-generation gynogens can be produced either by a second round of mitotic gynogenesis or by meiotic gynogenesis (see Figure 31). Second-generation gynogens produced from each first-generation gynogen are divided into two lots, and one is sex-reversed to produce sex-reversed males. Within each family, sex-reversed males and their genetically identical sisters will be mated to produce an inbred line of genetically identical females that is 100% homozygous and 100% inbred.
If first-generation mitotic gynogens are to be used in a breeding programme to create inbred lines, a second phase of gynogenesis followed by sex reversal is needed in order to produce the lines of 100% inbred fish, in which all fish within each inbred line are genetically identical. Each first-generation mitotic gynogen will be used to create a unique line of 100% homozygous and 100% inbred fish by utilizing either of two possible types of gynogenesis. When the first-generation mitotic gynogens mature, their eggs are stripped, and either mitotic gynogenesis is repeated to produce second-generation gynogens or meiotic gynogenesis is used to create second generation gynogens. If meiotic gynogenesis is used, a shock is used to prevent the second polar body from leaving the activated but unfertilized eggs (meiotic gynogenesis will be described in more detail in a later section). Regardless of the technique used during phase 2 of this breeding programme, the second-generation gynogens are considered to be mitotic gynogens because that was the technique used during phase 1. Both techniques produce families (inbred lines) of identical fish that are 100% homozygous and 100% inbred but, in general, survival will be better if meiotic gynogenesis is used during phase 2.
The eggs and offspring from each first-generation mitotic gynogen must be isolated from all other second-generation eggs and offspring, because each first-generation mitotic gynogen is going to form a “cloned” line. The second round of gynogenesis produces hundreds of genetically identical copies of each first-generation mitotic gynogen (a form of cloning).
Half of the second-generation gynogens from each family are sex-reversed with anabolic androgens (steroid hormones) to produce XX sex-reversed males. The sex-reversed males are genetic females but phenotypic males. The fish that are not treated with hormones are raised normally. Within each family, the sex-reversed males and their sisters are genetically identical (genetically, they are all identical sisters); when they mate, they produce an inbred line of genetically identical fish that is 100% female, 100% homozygous, and 100% inbred. Sex-reversed males must be created every generation, because it is the only way males can be produced, and it is the only way each inbred line can be perpetuated without additional chromosomal manipulation.
If each inbred line is isolated from all other inbred lines, it will breed true and produce genetically identical 100% inbred fish. This breeding programme produces 100% inbred fish in one generation and 100% inbred fish that are capable of producing inbred lines of 100% inbred fish in two generations.
If this is done with species with the WZ sex-determining system (females are WZ and males are ZZ; the only major aquacultured species with this system of sex determination are: blue tilapia, Oreochromis aureus; Wami tilapia, O. urolepis; Japanese eel, Anguilla japonica), half the first-generation mitotic gynogens will be mitotic gynogenetic males (ZZ) and half will be mitotic gynogenetic superfemales (WW). Phase 2 of the breeding programme needed for mitotic gynogenetic WW superfemales is the same as that described previously in this section (Figure 29). ZZ mitotic gynogenetic males, on the other hand, must undergo mitotic androgenesis during phase 2 (Figure 30).
Mitotic androgenesis can be used to produce mitotic androgens (all genes come from the father), fish that are 100% inbred. The technique that is used to produce mitotic androgens for species with the WZ sex-determining system is outlined in Figure 30.
In phase 1, eggs whose DNA has been destroyed by ultraviolet radiation are fertilized by normal sperm. This produces a haploid zygote, and all chromosomes come from the sperm (from the father), so these zygotes are called “mitotic androgens”. When the zygote undergoes first cleavage, a pressure or temperature shock is used to prevent nuclear division, which produces a diploid androgenetic zygote. In species with the WZ sex-determining system, males are homogametic (ZZ), so all sperm contain a Z chromosome. Consequently, all haploid zygotes have a Z sex chromosome. The disruption of first cleavage changes Z zygotes into ZZ zygotes, and all are males. As was the case with mitotic gynogens, all mitotic androgens are 100% homozygous and 100% inbred, but in this case all fish are males. Each androgen is a singular inbred line.
Figure 30. A schematic flow chart of the production of an inbred line that is 100% homozygous and 100% inbred by mitotic androgenesis (all genes come from the father) for species with the WZ sex-determining system. In phase 1, first-generation mitotic androgens are produced by preventing first cleavage of haploid zygotes. All such fish are 100% homozygous and 100% inbred, and all are males. Each androgen is a singular inbred line. In phase 2, a second round of mitotic androgenesis is needed to produce a family of genetically identical 100% homozygous and 100% inbred fish that are capable of mating to perpetuate the inbred line. Second-generation androgens produced from each first-generation androgen are divided into two lots, and one is sex-reversed to produce sex-reversed females. Within each family, the sex-reversed females and their genetically identical untreated brothers will be mated to produce an inbred line of genetically identical males that is 100% homozygous and 100% inbred.
As was the case with mitotic gynogenesis, mitotic androgenesis does not produce many viable fish. Because each androgen is 100% homozygous, every detrimental recessive allele will be able to produce its phenotype. However, mitotic androgens that survive are free from detrimental recessive alleles.
Phase 2 of this breeding programme uses a second round of mitotic androgenesis, and half the offspring from each family are sex-reversed with anabolic estrogens to produce sex-reversed ZZ females. These females are genetic males but phenotypic females. Within each family, the sex-reversed females are mated to their genetically identical brothers to produce each inbred line; fish in each inbred line are genetically identical, 100% homozygous, and 100% inbred and all are males. As was the case with mitotic gynogens, each line is genetically unique.
Mitotic androgenesis can be done with species that have the XY sex-determining system. In this case, half the first-generation mitotic androgens will be XX androgenetic females and half will be YY androgenetic supermales. Second-generation androgens and inbred lines can be produced as follows: the phase 2 protocol needed for the YY androgenetic supermales is the same as that outlined in Figure 30; the phase 2 protocol needed for XX androgenetic females is that which is outlined in Figure 29.
Gynogenesis can be used to create another type of inbred fish-meiotic gynogens. This type of chromosomal manipulation is easier than mitotic gynogenesis, and meiotic gynogens have a higher survival rate than mitotic gynogens because they have less inbreeding. Meiotic gynogenesis is less useful in producing inbred lines because it is difficult to accurately predict the exact amount of inbreeding produced, and the inbreeding produced each generation is quite variable. Since regular systems of inbreeding are most useful when they produce reliable and predictable amounts of inbreeding, meiotic gynogenesis is less useful than mitotic gynogenesis for producing inbred lines. However, one to three generations of meiotic gynogenesis can be used to produce highly inbred fish.
The procedure needed to create meiotic gynogens for species that have the XY sex-determining system is outlined in Figure 31. Ultraviolet irradiated sperm are used to activate eggs. A pressure or temperature shock is applied shortly after activation to prevent the second polar body from leaving the egg during the equational meiotic division. The shock produces an activated egg that has a haploid egg nucleus and a haploid second polar body nucleus. These haploid nuclei fuse, and the egg “fertilizes” itself to produce a meiotic gynogenetic diploid zygote.
At first glance, it appears as if meiotic gynogenesis is a form of self-fertilization, which should theoretically produce fish with F = 50% in the first generation, F = 75% in the second generation, and F >99% in the seventh generation. However, this isn't a form of self-fertilization because the the genes in a haploid egg nucleus and the haploid second polar body nucleus are not truly random with respect to each other, as is the case with two randomly chosen haploid egg nuclei. A haploid egg nucleus and the haploid second polar body nucleus are created after the reduction division. Two randomly chosen haploid egg nuclei differ from each other in that independent assortment randomly distributed the maternal and paternal homologues of each chromosome pair. An egg haploid nucleus and the second polar body haploid nucleus have already undergone independent assortment, so both receive a replicated homologue from each pair of sister chromatids; consequently, they differ less from each other in that respect than two randomly chosen egg nuclei. When the egg haploid nucleus and the haploid second polar body nuclei fuse, the sister chromatids that were separated during the equational division are reunited. These sister chromatids tend to be homozygous with respect for genes near the centromere and heterozygous for genes further away due to crossing over. Because of this, the exact amount of inbreeding produced by one generation of meiotic gynogenesis is difficult to predict.
Figure 31. A schematic flow chart of meiotic gynogenesis. This type of gynogenesis differs from mitotic gynogenesis in that the egg “fertilizes” itself when a shock prevents the haploid second polar body from leaving the activated egg following the equational meiotic nuclear division. The haploid second polar body nucleus fuses with the egg haploid nucleus to produce a gynogenetic diploid zygote.
To further complicate matters, It is impossible to accurately predict the amount of inbreeding that will be produced by subsequent generations of meiotic gynogenesis, because the inbreeding will be a function of crossing over frequencies. Crossing over tends to occur at particular locations along each chromosome, so heterozygous loci tend to remain heterozygous for many generations. Because heterozygous loci will tend to remain heterozygous, full-sib mating will eventually produce greater levels of inbreeding than meiotic gynogenesis. Additionally, the inbreeding that is produced each generation will be different for each species and each population due to differences in crossing over frequencies.
The only way to quantify the inbreeding produced by meiotic gynogenesis is to examine the mothers and their offspring electrophoretically (a brief description of electrophoresis is presented in Chapter 8). Heterozygous loci in diploid meiotic gynogens that are produced from mothers that are heterozygous at those loci are heterozygous because of crossing over. The percentage of such loci that are heterozygous in the offspring can be used to determine crossing over frequency; once this is ascertained, it can be used to determine inbreeding for each meiotic gynogen. Studies with fish have shown that one generation of meiotic gynogenesis produced F = 55% to 79% (F = 79% is approximately that produced by seven generations of full-sib matings).
Although meiotic gynogenesis produces less inbreeding than mitotic gynogenesis, it can be used to quickly produce highly inbred fish. However, each meiotic gynogen will have a different level of inbreeding.
Most aquaculturists have been told that inbreeding causes problems and that it should be avoided at all costs. This is true only if inbreeding is undesired, because inbreeding is a powerful and valuable breeding technique that animal and plant breeders have used for years to produce superior brood stock and to produce superior plants and animals for grow-out. Any farmer or hatchery manager who wants to manage the genetic aspects of his population should know when and how to use inbreeding to improve productivity and profits, as well as when it should be avoided so that inbreeding depression does not ruin productivity and profits.
Inbreeding is used to create new breeds that breed true to form. Linebreeding can be used to bring a population up to the level exhibited by a superior animal and to increase a superior animal's contribution to his descendants. The use of herd bulls is a type of inbreeding that can be used to quickly improve a population. The creation of inbred families can improve the results of between-family selection when a breeder wants to improve a phenotype that has a low h2. Finally, the creation of inbred lines is a way to increase hybrid vigour in crossbreeding programmes and to produce outstanding F1 hybrids for grow-out.
A farmer can use inbred lines to improve selection and crossbreeding programmes only if he knows how to produce inbred lines efficiently. This means that he must know how to conduct regular systems of inbreeding so that he knows what matings must be made, generation after generation, in order to produce predictable levels of inbreeding. Several types of regular inbreeding programmes can be used to produce inbred lines. Parent-offspring and full-sib inbreeding programmes are the simplest and cheapest, and both quickly produce high levels of inbreeding. Full-sib inbreeding programmes are usually used because it produces large amounts of inbreeding, and it is rather easy to make this type of mating generation after generation. Half-sib and double first cousin inbreeding programmes are slightly more complicated and somewhat more expensive. Although they ultimately produce the same amount of inbreeding as parent-offspring and full-sib inbreeding programmes, they produce it at a slower rate.
Chromosomal manipulation can be used to quickly produce highly inbred lines of fish. One generation of mitotic gynogenesis or mitotic androgenesis will produce fish that are 100% homozygous and 100% inbred. A second generation of chromosome set manipulation is needed to produce 100% inbred fish that are capable of reproducing. One generation of meiotic gynogenesis will produce fish that have large, but unknown, levels of inbreeding; the amount of inbreeding produced by meiotic gynogenesis is variable and depends on crossing over frequencies. The use of chromosomal manipulation to produce inbred lines should be done only by scientists at large agribusinesses or at research stations.