The overall aim of this project was to understand the mode of sex determination mechanism operates in chondrostean species. The following hypotheses were tested: i) white sturgeon has a genetic sex determination system, ii) one of the sexes in sturgeon is heterogametic, and iii) sex-specific DNA polymorphisms exist between the sexes.

Several experimental approaches derived from the fields of transmission, molecular, and cytogenetics were used to examine the nature of the sex determination process.

In the first series of experiments (Chapter 2) ploidy manipulation techniques were used  to produce gynogenetic and polyploid white sturgeon. A novel random amplified polymorphic DNA (RAPD)-based technique was developed to rapidly assess the overall success of treatments designed to induce gynogenesis, and measuring erythrocyte nuclei size with a Coulter Counter was found to be a rapid and accurate technique for ploidy analysis in sturgeon. Both sexes were observed in 23-24 month old gynogenetic progeny groups derived from four different females supporting the hypothesis that white sturgeon has a female heterogametic (ZW:ZZ) genetic sex determination system. The proportion of males in diploid (48%), gynogenetic (18%), and triploid (14%) progeny groups derived from the same female suggested that the sex-determining element on the W chromosome segregates independently of the centromere.

In the second series of experiments (Chapter 3) various molecular techniques were used in an attempt to isolate genetic markers for the identification of sex in white sturgeon. Subtractive hybridization, representational difference analysis and bulked segregant analysis were used, in conjunction with genetically unique groups of sturgeon, which were specifically developed  to increase the probability of isolating sex-specific DNA sequences. DNA sequences associated with sex in white sturgeon were not identified. These results suggest that either 1) there are no sex-specific sequences in sturgeon, or 2) the sex-specific DNA is comprised of unusual sequences which were not complementary to the 1200 random decamer primers or the recognition sequence of the restriction endonucleases used in this set of experiments, or 3) the sex-specific sequences comprise a very small portion of the genome.

Chapter 4 details a synaptonemal complex (SC) analysis of white sturgeon spermatocytes. Synaptonemal complex analyses have not been previously reported for any sturgeon species and this study was initiated to determine if it was possible to directly identify heteromorphic sex chromosomes in meiotic prophase SC spreads. No bivalent consistently exhibited asynaptic behavior or had axes of unequal length suggesting that heteromorphic sex chromosomes are not present in white sturgeon spermatocytes which concurs with the findings of Chapter 2. Pachytene nuclei had varying numbers of univalents, self-paired foldback elements with no obvious centromeric region (1-7), and other SC peculiarities. No consistent evidence of alignment or pairing involving more than two lateral elements was found indicating that the process of diploidization is now complete in this ancient polyploid. The SC number was 139 (± 3.4) with both between and within animal variation.

The final series of experiments (Chapter 5) involved mitotic analyses of metaphase chromosome spreads from four white sturgeon individuals of each sex. Conventional mitotic analyses provided no evidence for a heteromorphic sex chromosome pair or any sex-related chromosomal polymorphism. Likewise fluorescence in situ hybridization of labeled male and female sturgeon genomic DNA to metaphase chromosome spreads of each sex did not reveal a chromosome that was seen to be specifically hybridizing only to the genomic DNA of one sex. The average chromosome number was 271 (range 265-276) which concurred with the meiotic count found in Chapter 4. An  improved C-banding technique using propidium iodide and epifluorescence microscopy revealed between animal variation (2-7) in the number of entirely heterochromatic metacentric chromosomes. These heterochromatic chromosomes and the self-pairing elements observed in  Chapter 4 were approximately equal in number and relative size and they may represent accessory B chromosomes. In combination these results suggest that white sturgeon has a female heterogametic ZW:ZZ genetic sex determination system, and that the sex-determining element segregates independently ofthe centromere. No evidence of a heteromorphic sex chromosome pair was found in meiotic analyses of white sturgeon spermatocytes supporting the hypothesis of male homogamety in this species.  The inability to identify sex-specific DNA sequences despite evidence of a genetic sex determination system suggests that sex-specific DNA is rare, making up a very small portion of the genome. Mitotic analyses provided no evidence for a heteromorphic sex chromosome pair suggesting that the accumulation of sex chromosomal rearrangements is insufficient to be visible at the cytogenetic level. All of these results support the contention that white sturgeon sex chromosomes are at an early stage of differentiation.

DNA (deoxyribonucleic acid) is a molecule that is shaped like a double helix and made up of pairs of nucleotides. DNA transmits genetic information. DNA is packaged into chromosomes which are located within the nucleus of all cells. Every cell in the body contains all of the chromosomes that collectively make up the genome of that organism. DNA codes for amino acids which are linked together to make proteins. A gene is a stretch of DNA that specifies all of the amino acids that make up a single protein. Proteins are the building blocks of life. There are thousands of proteins in the body (encoded by thousands of genes). The interaction and structure of proteins determines the visible characteristics or phenotype of an organism, while the genotype refers to the genetic makeup.

The sequence of nucleotides that encode a gene can differ between individuals. These differences are called genetic variants. As a result of these nucleotide differences, genetic variants or alleles may differ in the amino acid sequence of the protein they encode, or they may regulate the production of different quantities of the encoded protein. These differences can have an effect on phenotype.

All individuals receive one copy (allele) of each gene from their mother, and one from their father. The DNA sequence of a gene inherited from each parent may be identical in which case the individual is said to be homozygous for that gene, or the sequence of a gene inherited from each parent may vary in which case the individual is said to be heterozygous. Genetic variants often differ from each other by the sequence of a single base pair. These differences are called single nucleotide polymorphisms SNPs (pronounced “snips”). Genotyping means using laboratory methods to determine the sequence of nucleotides in the DNA from an individual, usually at one particular gene or piece of a gene. SNPs are commonly the basis of genotyping tests. Genetic tests based on SNPs analyze DNA derived from an individual to determine the genetic variants that are present at one specific location (nucleotide pair) in the midst of the approximately 3 billion nucleotide pairs that make up the genome.

Historically we have not known which genes contribute to performance characteristics (traits), and so we have used performance records and EPDs (expected progeny differences) to infer the genetic merit of animals. This method has been very successful at improving certain traits. Research has shown that some genetic variants of specific genes are associated in a positive way with a given trait. It is therefore possible to genotype an animal using a DNA-based genotyping test and select individuals carrying the preferred genetic variant. Marker-assisted selection is the process of using the results of DNA testing to assist in the selection of individuals to become parents in the next generation. The word “assisted” implies that the selection is also influenced by other sources of information, such as animal’s observed performance and EPDs. The genotypic information provided by DNA testing should help to improve the accuracy of selection and increase the rate of genetic progress by identifying animals carrying desirable genetic variants for a given trait at an earlier age.

It is not known which specific genes contribute to an EPD – the genes are anonymous. Complex traits, including most of the economically relevant traits for cattle production (birth weight, weaning weight, growth, reproduction, milk production, carcass quality) are controlled by many genes, and they are also greatly affected by the environment (e.g. feed conditions). Although complex traits are influenced by a number of genes – each one of these genes is still inherited in the same way. An animal inherits one copy of each gene from its sire, and one copy of each gene from its dam. These copies may differ from each other, and these differences may have either a positive or negative effect on the trait that the gene controls or influences. When an animal has a positive EPD for a certain trait, what that is effectively saying is that based on its pedigree and phenotype, it has inherited a greater than average number of “good” genetic variants of each gene affecting that particular trait.

It is important to combine DNA results (which look at single genes) with other criteria, such as EPDs (which look at numerous genes) and the animal’s actual phenotype for the trait (if available), to ensure that selection is distributed over all the genes that contribute towards the trait of interest. Don’t ignore animals that have good EPDs for a given trait and yet are not carrying the favorable form of a gene for that trait. These animals are likely to be a source of good alleles for the many other genes that contribute towards that trait. Ideally the information from genetic tests should be integrated into a genetic evaluation system that weighs all the information about an animal. Combining information from both EPDs and genetic tests into a selection decision will be superior to selection on either EPDs or markers alone. The challenge will be to determine the weight that should be given to the marker information in this decision-making process. The magnitude of the effect of a genetic variant of a gene on the trait may vary among the different breeds, production systems and environments.

Animal biotechnology encompasses a broad range of techniques for the genetic improvement of domesticated animal species, although the term is increasingly associated with the more controversial technologies of cloning and genetic engineering. Despite the many potential applications of these two biotechnologies, no public or private entity has yet delivered a genetically engineered food-animal product to the global market, and the sale of milk or meat from cloned animals and their offspring is currently subject to a voluntary moratorium in the United States. The animal biotechnology industry faces a variety of scientific, regulatory, ethical and public acceptance issues. Effective and responsible communication among scientific, community, industry and government stakeholders will be required to reach a societal consensus on the acceptable uses of animal cloning and genetic engineering.

In this study, we report the generation of CD209 gene knockout sheep, utilizing electroporation-mediated CRISPR-Cas9 genome editing, as a model to test whether this might make cattle resistance against Bovine Leukemia Virus (BLV). This approach exploits the CD209 gene's role as a receptor for BLV, hypothesizing that its knockout would confer resistance to infection. Our methodology involves specific guide RNAs targeting the sheep CD209 gene, followed by electroporation into ovine zygotes to induce targeted gene disruptions. It was hypothesized that a gene knockout of CD209 would result in the inability of the virus to bind and enter the cell; therefore, creating disease resistance. The resultant lambs exhibited varied mosaicism and phenotypic outcomes associated with the gene edit, indicative of the CRISPR-Cas9 system's effectiveness and efficiency. This study not only demonstrates a novel application of gene editing in livestock but also underlines the potential of sheep as surrogate models in BLV research, due to their analogous immunological responses and shorter gestational periods compared to cattle. The successful application of this technology paves the way for future research in genetic engineering for livestock disease resistance, with significant implications for animal health management and agricultural productivity.

Genetic improvement of livestock is critical for animal production and wellbeing. Traditional breeding approaches however are slow and often result in unwanted genetic linkage drag. Genome-editing technologies offers an alternative that can quickly and precisely introduce useful traits into animals without linkage drag. Editing of mammalian livestock species has been achieved by somatic cell nuclear transfer of an edited cell or direct microinjection of zygotes. However, the efficiency of cloning in large livestock species is low and perinatal abnormalities are common, and introducing editing reagents through microinjection is a time-consuming task with a high technical barrier. Electroporation is a widely used technique for delivering gene-editing reagents into cells and poses as a possible high throughput approach to generate genome-edited animals through the treatment of early-stage embryos. Electroporators work by directing pulses of electrical current to create transient pores in the lipid bilayer of the plasma membrane, allowing the passage of reagents into cells. Upwards of 100 zygotes can be processed with the push of a button making electroporation a scalable and simple approach to producing genome edited livestock. Here, various electroporation parameters for generating gene-edited bovine, ovine, and caprine embryos were tested for producing targeted mutations.Targeted genetic knock-ins of over 1kb however have not been produced with electroporation alone as nucleic acids larger than 1kb are unable to pass the zona pellucida. To develop a high throughput approach to producing large template targeted knock-in livestock, another delivery system for large DNA repair templates must be employed. Non-pathogenic viruses such as rAAV can transport nucleic acid fragments of up to 4.9kb into cells. Here, a scalable approach was developed to transduce large DNA repair templates into bovine zygotes prior to electroporation for the production of 2.7kb knock-in blastocysts.

The reproductive biology of fish makes them particularly amenable to genetic manipulation. A genetically engineered or “transgenic” Atlantic salmon is currently undergoing federal regulatory review, and international research is being conducted on many other species. The innate ability of fish to escape confinement and potentially invade native ecosystems elevates the ecological concerns associated with their genetic modification. Escaped transgenic fish will not invariably result in deleterious effects on native populations, and careful risk assessment is required to determine the ecological risks unique to each transgene, species and receiving ecosystem combination. In response to public concerns about transgenic fish, California has developed stringent regulations for the importation, possession and raising of transgenic fish, and a California law prohibits their presence in waters of the Pacific Ocean regulated by the state.