be on the market 19].

The production of high-value pharmaceutical substances is the principal and most

promising application for transgenic pigs, goats. sheep and cattle [10]. Some wOrldwide companies are
investing great efforts in this promising technology. Due to its large-scale volume production. milk is the
preferred vehicle for expression of proteins in transgenic animals, although they can be produced in a
variety of biological fluids such as urine, saliva, blood and seminal fluid [11, 12].
Pronuclear DNA microinjection has long been the most reliable method to produce transgenic animals since
1985 when the first generation of transgenic farm animals were reported [l3, 14]. However, although
transgenic animals have been generated using this approach. it has many limitations: thus it is expensive
and laborious, has a low success rate (1-4%) and leads to random integration of the DNA into the genome [5,
15]. Different methodologies have been developed to improve the efficiency of transgenesis over the last
decade and a half. These include infection with retrovi ral vectors; nuclear transfer with modified somatic
cells and sperm mediated gene transfer.

Transfection with retroviral vectors is one of the best choices for creation of transgenic animals because of
their high gene transfer efficiency [16, 17]. although they have some limitations (size limit for the transgene
10 kb and possibility of gene silencing). Transgenic animals have been successfully generated by the
injection of virus into the periviteline space of oocytes (cattle, pigs, and monkeys), zygotes (cattle. mice,
and rats), and early preimplantation embryos (cattle, mice, rat) [16, 18]. However, the health risk is the
main barrier to the commercial use of this system. Another alternative for generating genetically altered
animals is to first make the modification in somatic cells and then use the modified cells as donors for
nuclear transfer (NT). Successful nuclear transfer (NT) of cultured cells, first demonstrated in cattle by
[19], has provided an alternative for obtaining genetically modified animals. Unfortunately the procedure i s
inefficient and frequently leads to animals that are abnormal. The cause of these abnormalities is probably
established during the first cell cycle after the NT.

Sperm Mediated Gene Transfer (SMGT) is based on the ability of sperm to bind, internalize, and transport
exogenous DNA into an oocyte during fertilisation [20, 21]. Rabbit sperm cells were reported to
spontaneously take up and transfer DNA into an oocyte during fertilisation resulting in the genetic
modification of the two cell-stage embryos [20]. In 1989, the birth of live transgenic mice was reported
after epidydimal sperm cells were used to transfer exogenous plasmid DNA into an oocyte during
fertilisation [21]. SMGT has been used more or less successfully in the production of transgenic embryos
and animals in a large number of species [22-26]. Although transgenic animals have been obtained using
SMGT, the repeatability of this technique is low, with only a few numbers of laboratories reporting clearly
positive results. In addition, inter-and intra-species success variability is still an unsolved problem
associated with this technology. Also the mechanism whereby the foreign DNA is bound and internalised
inside the sperm head, the mode of transmission upon fertilisation and the perseverance of the foreign DNA
in the resulting embryos, fetuses and offspring, are issues that have not yet been fully elucidated -although
the reader's attention is drawn to Chapter 12 where possible models are discussed.

Testis mediated gene transfer (TMGT) is a more recent system that introduces transgenes directly into the
reproductive tract of male animals, either as naked DNA or encapsulated in liposomes. The sperm take up
the transgenes in vivo (directly or through progression from sperm precursor cells) and transmit them via
natural mating or artificial insemination [26-28]. Injection into the rete testis appears to be more suitable for
in vivo gene transfer by electroporation than direct intratesticular injection [29].
In this chapter we review the state of art of SMGT in farm animals with a special interest in porcine and bovine
animals, with additional information related to other ruminants and horses. We evaluate the possible
applications of transgenic pigs and cattle and review the factors related to the success of the SGMT in these
species and offer our own experience based on studies analysing the main factors in porcine and bovine SMGT.
TRANSGENIC CATTLE

In bovines a number of different methods have been used to obtain transgenic animals. The pronuclear
injection approach was used for the first successful attempt to produce a transgenic bovine [30, 31];