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Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.

Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.

The use of prepuberal heifers as oocyte donors in breeding programs could decrease the generation interval in cattle and increase the genetic rate of gain. While large numbers of follicles develop in response to exogenous gonadotrophins the oocytes they contain lack developmental competence until the animals are 6 to 8 months old. In vitro fertilization rates are normal but rates of cleavage and subsequent development are low. The oocytes are smaller than those collected from adult cattle and the cortical granules fail to disperse evenly during maturation. Oocytes from prepuberal calves release less calcium in response to challenges with InsP3. The lower peak height and altered pattern of Ca2++ oscillations when compared to oocytes of mature cows suggests that cytoplasmic maturation is incomplete in heifers 175 days of age or less. However, the development of ultrasound guided methods for the recovery of oocytes from calves greater than five months old and their improved developmental capacity as demonstrated by development to blastocysts and birth of live calves indicates that the prepuberal calf will play a significant role in animal breeding programs of the future by serving as sire dams or by expanding the influence of genetically superior animals in producer herds.

Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.

Activation of the oocyte is the least efficient step in nuclear transplantation in the rabbit. We report the influence of age of oocytes, field strength, pulse duration and number, and shape of field on the rate of activation of mouse oocytes by electrical pulses. Regardless of oocyte age, activation rates were similar over a wide range of field strengths and pulse durations. Aged oocytes activated at a higher rate than recently ovulated oocytes (32 vs 3%), which lysed more frequently (13 vs 2%). Fragmentation rate was also higher among aged oocytes (42 vs 6%). The rate of activation increased with the number of pulses, from 9% with a single pulse, to 61% with six pulses. It also increased with the interval between pulses. Comparison of activation chamber geometries showed that the rate of activation was higher in a nonuniform field than in a uniform field and, for a particular field strength, varied from one electrode gap to another. These observations indicate that the rate of activation can be greatly increased by multiple electrical pulses. The activation rate also varied with consistent field strength in chambers with different electrode configurations.

Recent accomplishments in the cloning of mammals by nuclear transplantation have been driven by the enormous potential use of this technology for increasing genetic gain in animal agriculture, reducing the number of animals needed for experiments and investigating genetic and environmental interactions in animals. Nuclear transplantation entails transfer of individual nuclei from preimplantation embryos to unfertilized, mature oocytes. The mature oocyte is a uniquely suitable recipient because it has the ability to reprogram the transplanted nucleus, inducing it to develop as a newly fertilized embryo. Using the oocyte as a recipient for transplanted nuclei offspring have been produced in sheep, cows and rabbits. Before this technique can become of widespread commercial use, many problems remain to be solved to increase its efficiency. These include an economical source of recipient oocytes and refinements in methods of enucleating oocytes, fusion of the donor nucleus with the recipient oocyte, activation of the recipient oocyte and embryo culture procedures. If nuclear transplant embryos can serve as nuclear donors, then an unlimited number of genetically identical embryos could be produced, even with a relatively low rate of efficiency.

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.