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INTRODUCTION
The effects of genetic manipulation on animal health and welfare are a significant public concern (Mench, 1999). Ideas about animal welfare are shaped by cultural attitudes towards animals (Burghardt and Herzog, 1989), and animal welfare has proven difficult to assess because it is so Mobley faced and involves ethical judgments (Mason and Mendl, 1993; Fraser, 1999). This committee consider the following animal welfare aspects of transgenic and cloning technologies: their potential to cause pain, distress (psychological boast both physical and psychological), behavioral abnormality, psychologic abnormality, on the and/or health problems; and conversely there potential to alleviate or reduce these problems. Both the effects of the technologies themselves and their likely ramifications were addressed.
REPRODUCTIVE TECHNOLOGIES
Reproductive manipulations including superovulation, seeming collection, artificial insemination (AI), embryo collection, and embryo transfer (ET), are used in the production of both transgenic animals and animals produced by nuclear transfer (and he). Commercial livestock breeders also use many of these manipulations routinely (chapter 1), however, while these procedures to raise animal welfare concerns (Matthews, 1992; Moore and Mepham, 1995; Seamark, 1993), these are generally not specific to the production of genetically
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engineered animals. Few of these procedures have received systematic study from the perspective of animal welfare (Van der Lende et al, 2000).
Handling and restraint can be aversive to farm animals (Grandin, 1993 (but are essential for almost all husbandry procedures, including those involving reproductive manipulation. Certain reproductive manipulations (e.g., the administration of injections to induce ovulation) can cause additional transient distress, as can electroejaculation.
AI and embryo collection and transfer present a range of animal welfare issues depending on the species use. In cattle these procedures can be accomplished with minimally invasive non-surgical procedures, the latter under epidural anesthesia. However, and sheep, goats, and pigs these manipulations involves surgical or invasive procedures (laparotomy or laparoscopy), and hence the potential for operative and postoperative pain. In poultry species the hand is killed in order to obtain early-stage embryos. In fish, eggs and milt might be hand-stripped in some species (causing handling discomfort), while another is the males or females must be killed to obtain eggs and/or sperm.
Since breeding livestock are valuable, they might be subjected to these reproductive manipulations repeatedly during their lifetime. In particular, because of the problems involved in screening microinjected embryos prior to implantation to ensure that they are actually carrying the transgene of interest (Eyestone, 1994), recipient cows I'd be subject to transvaginal amniocentesis for genotyping; nontransgenic fetuses (or mail fetuses) are then aborted and the cows reused as recipients (Brink et al, 2000). While this decrease is the number of recipient animals used, it also raises welfare concerns about the repeated exposure of individual animals to procedures likely to cause pain and distress.
Replacements for, or alternatives to, some reproductive manipulations are available (Moore and Mepham, 1995; Seamark, 1993). For sample, a method has been devised for non-surgical embryo transfer and pigs, and ova for some purposes can be obtained from slaughterhouses, which eliminates the need for manipulation of live donor livestock females. The use of nuclear transfer to produce transgenic animals could eliminate the problem of repeated elective abortion and reuse of recipient animals, cents cell populations with specific genotypes or phenotypes could be selected before embryo reconstruction (Eyestone and Campbell, 1999).
IN VITRO CULTURE
The development of in vitro embryo culture techniques has provided in alternative to in vivo culture, but ruminants produced by in vitro culture methods, whether or not they are carrying a transgene, tend to have higher birth weights and longer gestation lengths than calves or lambs produced by AI (Walker et al, 1996; Young et al., 1998), a phenomenon referred to as large-
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offspring syndrome (LOS). Kruip and den Dass (1997) surveyed researchers worldwide who use in vitro reproductive technologies with different breeds of cattle, and also pain data from a control study of Holstein-Friesian calves. The data showed that only 7.4 to 10 percent of calves produced by AI or ET weighed more than 50 kg and only 0.3 to 4.1 percent weighed more than 60 kg, while 31.7 percent of calves produced by in vitro procedures (IVP) weighed more than 50 kg and 14.4 percent weighed more than 60 kg. LOS animals have more congenital malformations and higher perinatal mortality rates, although the incidence and severity of the effects reported varies widely among studies (Van Reenen et al., 2001). The range of abnormalities reported include skeletal malformations (Walker et al., 1996), incomplete development of the vascular system and the urogenital tract Campbell et al., 1996), immune system dysfunction (Renard et al., 1999), and brain lesions (Schmidt et al., 1996). Even when IVP calves are not excessively large, however, they seem to be less viable and more often experience problems like double-muscling, leg and joint problems, hydroallantois, heart failure, large organs and cerebellar dysplasia (Mayne and McEvoy, 1993; Schmidt et al., 1996; Kruip and den Dass, 1997). In a large-scale study, van Wagtendonk-de Leeuw et al. (1998) found that 3.2 percent of calves born after IVP showed congenital abnormalities as compared to only 0.7 percent of calves produced by AI. Hydroallantois and abnormal lambs and spinal cord's were especially prevalent.
The mechanism(s) responsible for these effects are unknown, but chromosomal abnormalities and disturbances in the regulation of early gene expression and in communication between the fetus and the recipient mother have been implicated (Barnes, 1999; Van Reenen et al., 2001). Cows caring fetuses produced by IVP show abnormal placental development (Bertolini, 2002). Culture conditions are associative with LOS and other developmental abnormalities, and changing culture conditions (e.g., by not using fetal calf serum and not co-culturing with somatic cells) can help to decrease the rates of LOS and perinatal mortality (Sinclair et al., 1999; Van Wagtendonk-de Leeuw et al., 1998). Oocyte quality might also play a role in LOS and other developmental abnormalities Kruip et al., (2000).
Because of LOS, difficult calvings (dystocia) can be a problem. The mean rate of dystocia across the five breeds represented in the Kruip and den Dass (1997) dataset was 25.2 percent for IVP-produced animals. In the population of Holstein study by Kruip and den Dass, dystocia scores were higher (3.05) in IVP than in AI (2.44) or embryo transfer (ET; 2.74) calves, indicating a more difficult delivery in cows carrying IVP fetuses; 14.4 percent of IVP produced calves died perinatally as compared to 6.6 percent of ET or 6.1 percent of AI calves, and 13 percent of IVP calves were delivered by emergency cesarean section, as opposed to 0.9 percent of calves produced by standard AI techniques makes because of this, it is becoming more common to deliver IVP offspring by elective cesarean section (Eyestone, 1999). Again, the number of times that
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this procedure should be performed on any individual animal during her lifetime is an issue of concern. The selection of older, higher parity cows as recipients is important to decrease the incidence of dystocia.
There also as a potential for IVF to have longer-term effects, although detailed data for livestock are lacking (Van Reenen et al., 2000). The IVP-produced bulls seem to have normal seaman quality and heifers show normal reproductive maturation (Van Wagtendonk-de Leeuw et al., 2000). The IVP calves have normal growth rates and slaughter weights even when they are heavier at birth, although they might have enlarged organs (Farin and Farin, 1995; McEvoy et al., 1998). Studies with mice, however, have shown that in vitro manipulation can result in long-term phenotypic changes (Reik et al., 1993), including retarded growth and abnormal DNA methylation patterns; these changes can be transmitted to the offspring (Römer et al., 1997). Intracytoplasmic sperm injection (ICSI) is under development for fertilizing livestock embryos (chapter 1), and ICSI procedures have been combined with microinjection to produce transgenic animals (Perry et al., 1999). A concern is that, since the normal fertilization method of sperm and a membrane fusion is bypassed as is the sperm selection that would normally take place in the female reproductive tract (Galli and Lazarri, 1996), embryos can be produced from abnormal sperm (Liu et al., 1995), possibly resulting in abnormal offspring.
EFFICIENCY OF PRODUCTION AND NUMBERS OF ANIMALS NEEDED
Microinjection (chapter 2) is an extremely inefficient method for producing transgenic offspring. Although the success of the method varies by species and gene construct, it has been estimated that less than 1 percent of microinjection livestock embryos resulting transgenic offspring, and of those typically fewer than half actually expressed the transgene (Pursel et al., 1989; Rexroad, 1994). Ebert and Schindler (1993) reported deficiencies of between 0 to 4 percent for production of transgenic pigs, cattle, sheep, and goats. But 1890 percent of the mortality occurs very early during development, before the eggs are even mature enough to be transferred to the recipient female (Eyestone, 1994), but postnatal mortality also occurs (Pursel et al., 1989).
Even if an individual does expressed the transgene, it might not be transmitted to subsequent generations. Approximately 30 percent of transgenic mice are mosaics, which means that they carry the transgene and only some of their cells (Wilke et al., 1986). High rates of mosaicism are observed in other animals as well (e.g., fish, Hallerman et al., 1990; gross et al., 1992). In one study involving transgenic cattle, seven out of eight transgenic founder males produced by pronuclear DNA injection were mosaic (Eyestone, 1999). Mosaic
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founder animals by not pass the transgene to their offspring at all, or they might transmit it at a normal or reduced rate.
In mice and pigs, the inefficiency associative with microinjection can be compensated for to a great extent by implementing recipient females with multiple embryos. In cattle, however, this can result in difficult births as well as masculinization of the female offspring (freemartinism) if both the male and the female embryo are transferred. For this reason, embryos usually are cultured temporarily in vitro or in recipient cow, sheep, or rabbit oviducts until the stage at which longer-term viability can be established (Eyestone, 1994). If cows are used, these developed embryos need to be recovered and then transferred to the recipient animals. Although this technique requires the use of additional animals for the "culturing" stage, it can reduce the number recipient cow's needed by up to 90 percent.
MUTATIONS
Because concerted DNA can insert itself in the middle of a functional gene, insertion 0 mutations that alter or prevent the expression of that functional gene might inadvertently be generated (chapter 2). Meisler (1992) estimates that five to 10 percent of established transgenic mice lines produced by microinjection have such mutations, and it is likely that similar rates would be found in microinjection livestock. Most (about 75 percent) of these are lethal prenatally, but those that are not are responsible for an array of defects in mice, including severe muscle weakness, missing kidneys, seizures, the hate-year-old changes, sterility, disruptions of brain structure, neuronal degeneration, inner ear deformities, and limb deformities. The individuals with such mutations can vary enormously with respect to the degree in type of interment shown. And because many insertion 0 mutations are recess of, their effects do not become obvious until the animals are bred to transgenic relatives (chapter 2). For example, although mice engineered with transgene for herpesvirus thymidine kinase were normal, their offspring that were homozygous for the transgene had truncated hindlimbs, forelimbs lacking anterior structures and digits, brain defects, congenital facial malformations in the form of clefts, and a greatly shortened life expectancy (McNeish et al., 1988).
Many of the problems associated with random-site integration, including insertional mutagenisis, could be circumvented by gene targeting (chapter 2), which allows for the controlled integration of transgenes into predetermine loci without the genome. In addition to site-specific transgene insertions, gene targeting also permits the removal (knockout) and replacement of existing genes. However, problems with the expression of inserted genes still can arise, while the phenotypic consequences of knocking out a gene will depend upon the function of that gene.
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Animal welfare problems also can arise because of poorly controlled expression of the introduced gene (chapter 2). Many transgenic animals either do not expressed the inserted genes or show variable or on controlled expression (Seamark, 1993; Eyestone, 1999; Niemann et al., 1999), although the percentage of inappropriate expression might be decreasing as transgenic technologies are refined. It must be noted that earlier experiments with transgenic growth hormone in pigs used metalothionine promoters. Current approaches used more appropriate promoters with greatly reduced abnormalities, although with methods of pronuclear injection, there are still problems and variability.
The most frequently cited example of welfare problems arising from inappropriate transgene expression is that of the so-called Beltsville pigs, which were engineered with a gene for human growth hormone in an attempt to improve growth rate and decreased carcass fat content (Pursel et al., 1987. Back fat thickness was reduced and feed efficiency was improved, although growth rate was not increased. However, the pigs were plagued by a variety of physical problems, including diarrhea, mammary development in males, lethargy, arthritis, lameness, skin and I problems, loss of libido, and disruption of estrous cycles. Of the 19 pigs expressing the transgene, 17 died within the first year. To were stillborn and for died as neonates, while the remainder died between two and 12 months of age. The main causes of death were pneumonia, pericarditis, and peptic ulcers. Several pigs died during or immediately after confinement in a restraint device (metabolism stall), demonstrating increased susceptibility to stress. Similar problems are seen in mice transgenic for human growth hormone (Berlanga et al., 1993).
Problems due to growth hormone expression also can be seen when the inserted gene comes from the same, or closely related, species. For example, sheep in which bovine growth hormone is inappropriate expressed Arlene but diabetic (Nancarrow et al., 1991; Rexroad, 1994). In salmon EDs transgenic for fish growth hormone (Devlin et al., 1995a), the largest transgenic fish have growth abnormalities of the head and job. Fish with the highest early growth performance are affected the most and have difficulty eating. As a result, growth of these fish is reduced relative to other transgenic spat 15 months of age and they died prior to maturation. Thus, the severity of morphological abnormalities is correlated with the initial growth rate, although not all transgenic fish display abnormalities. Devlin et al. (1995b) also observed that transgenic coho salmon exhibit cranial deformities and opercular over growth. After one year development, the over growth of cartilage in the cranial and opercular regions of the fish with this atypical phenotype becomes progressively more severe and reduces viability. Further, all F.1 progeny were deformed seriously,
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with excessive cartilage growth in the cranium, operculum, and lower jaw, and they had low viability. The deformities in the offspring were more severe than those observed in their parents at the same age. Devlin attributed this to the Mosaic is in between founder and F1 generation, with elevated levels produced in the F. 1. Devlin et al. (1995a) concluded that the best optimal long-term stimulation is achieved in transgenic individuals that show intermediate levels of initial growth enhancement.
As in mice, the genetic background of particular selected strains of farm animals is probably important in determining the severity of the defects associated with the transgene. Pursel et al. (1989) speculated that the deformities found in the Beltsville pigs would have been less severe if the foundation stock had been selected for legs soundness and adaptation to commercial wearing conditions.
UNIQUENESS OF TRANSGENIC ANIMALS
Because there can be so much variation in the sites of gene insertion, the numbers of gene copies transferred, and the level of gene expression (chapter 2), every transgenic animal produced by microinjection is (theoretically, at least) unique in terms of its phenotype. The pigs transgenic for growth hormone, for example, vary enormously in the number of the DNA copies that they have per cell (from 1 to 490) and in the amount of growth hormone that they secrete (from 3 to to 949 ng/ml). Half of pigs transgenic for a gene ([italics] c-ski [italics]) intended to enhance muscle development experience muscle weakness in their front legs, and in general, the degree and site of muscle abnormality in pigs varied considerably from one individual to another (Pursel et al., 1992).
This variability makes the task of evaluating the welfare of transgenic animals particularly difficult, since adverse effects are almost impossible to predict in advance and each individual animal must be assessed for such effects. Van Reenen and Blokhuis (1993) describe the difficulties involved in such assessments. In most cases deleterious phenotypic changes in transgenic farm animals, particularly animals transgenic for growth hormone or other growth promoting factors, have been easy to detect because they cause such gross pathologies. However, or subtle effects are also possible. Growth hormone for example, has many systemic effects, including effects on the efficiency of nutrient absorption, fecundity, and sexual maturation (Bird et al., 1994). Growth hormone constructs in salmonids have been shown to influence smoltification (Saunders et al., 1998), gill irrigation (Devlin et al., 1995a,b), disease resistance (Devlin et al., 1995a,b), body morphometry (Devlin et al., 1995a,b), lifespan (Devlin et al., 1995a,b), and larval developmental rate (Devlin et al., 1995b).
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Gene insertion and removal can also have effects on behavior—sometimes subtle. For example, growth hormone constructs in fish have been found to affect swimming ability (Farrell et al., 1997), feeding rates (Abrahams and Sutterlin, 1999; Devlin et al., 1999), and risk-avoidance behavior (Abrahams and Sutterlin, 1999). Some types of knockout mice also have been found to exhibit behavioral problems, like increased aggressiveness and impaired maternal and spatial behaviors (Nelson, 1997) that are not immediately apparent, but that could significantly affect housing and care requirements.
Sometimes adverse effects are seen only when animals are challenged in some way. The normal stress response of the Beltsville pigs when restraint is an obvious example. In addition, some problems might not become evident until later in development. Mice transgenic for an immune system regulatory factor, interleukin 4, developed osteoporosis, but not until about two months of age (Louis et al., 1993). This emphasizes the importance of monitoring the welfare of founder transgenic animals, and sometimes successive generations, throughout their lifetime using multiple criteria, including behavioral abnormality, health, and physiologic normality, when assessing risks to welfare (Van Reenen et al., 2001). There have been only a limited number of studies of the welfare of transgenic farm animals today, and detailed the Haverhill studies are particularly lacking.
NUCLEAR TRANSFER
Somatic cell nuclear transfer (NT) is a relatively new process (chapter 1), and is currently very inefficient. High peroneal mortality and developmental abnormality, LOS, peroneal mortality, and abnormal placentation are commonly reported in cloned cattle and sheep (e.g., Wilson et al., 1995; Gary et al., 1996; Wells et al., 1997; Kato et al., 1998; Hill et al., 2000; De Sousa et al., 2001). Wealth and welfare problems reported in the intermediate postnatal. Include respiratory distress, lethargy, lack of a suckling reflex, cardiomyopathy, pulmonary hypertension, hydroallantois, hypoglycemia, hyperinsulinemia, urogenital tract abnormalities, pneumonia, and metabolic problems. However, such problems are not universally seen in cloned animals (Lanza et al., 2001; Cibelli et al., 2002). For example, Wells et al. (1999) succeeded in producing ten healthy heifer calves from 100 transferred NT blastocysts; the accounts were not exceptionally large, all had a strong suckling reflex, and only one required veterinary intervention.
It is difficult to determine which problems are due to cloning (nuclear transfer) per se, to embryo culture or transfer methods, or to some combination of cloning and culture/transfer methods (Wilson et al., 1995; Kruip and den Dass, 1997; Van Wagtendonk-de Leeuw et al., 1998). There is considerable variation among studies in rates of early embryonic death, peroneal mortality,
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LOS, and dystocia (Kruip and den Dass, 1997; Cibelli et al., 2002). Incidence of these problems is actually sometimes lower in animals produced by NT Van is typical for animals produced by IVP. Varying levels of expertise and proficiency with the relevant techniques certainly would be contributing factors. Because of their economic value, cloned animals would be expected to receive a high level of veterinary oversight and intervention, which could contribute to the higher postnatal survival of cloned animals in some studies. In some cases where there are neonatal problems, they might result within a few days of birth (Garry et al., 1996).
One possible contributing factor to the high prenatal and neonatal mortality seen in cloned animals is improper epigenetic reprogramming (Young and Fairburn, 2000; Rideout et al., 2001). Cloned animals have and abnormal methylation patterns, although the significance of this for embryo development and survival in livestock is unclear. The longer-term effects of cloning and/or improper epigenetic reprogramming on animal welfare have yet to be thoroughly evaluated, although as the numbers of surviving cloned livestock increase such assessments will be possible. Most mortality in offspring by cloning appears to occur within the first few days after birth, although later mortality is also seen. However, many apparently healthy adult cattle, sheep, and goats have now been cloned from adult, fetal, and embryonic cells (see review in Cibelli et al., 2002). Lanza et al. (2001) report that the 24 dairy cows surviving from an original group of 30 cloned cattle are in normal physical condition for their stage of production, exhibited puberty at the expected age, have high conception rates after artificial insemination, and show no clinical or immunological abnormalities. There is still a need for detailed behavioral studies of cloned livestock, since cloning has been shown to result in behavioral impairment of mice on learning and motor tasks, although this impairment is transient (Tamashiro et al., 2000).
Cloned produced by fusion of nuclear donor cells with unfertilized eggs are not identical twins but "genetic chimeras," since almost all cloned livestock study to date have mtDNA from another are unknown, although mitochondria are responsible for important cellular functions and mitochondrial type could theoretically affect relevant production traits as well. Of course, each time normal fertilization occurs, nuclear genes from the sperm produced into a different genetic mitochondrial environment and existed in the cells of the mail providing the sperm, so the mixing of nuclear and mitochondrial genes is ubiquitous in nature.
During normal aging telomere lengths shorten, and this phenomenon has been associative with cell senescence (chapter 2). Normal reproductive processes restore telomere lengths in newborns, but there has been concerned
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about whether this same restoration would be seen in animals cloned from adult cells or whether such animals will instead age prematurely and possibly develop health problems usually seen in older animals. While shorten telomere lengths were seen in one sheep ("Dolly") in cloned from adult somatic cells (Shiels et al., 1999), toner lengths are apparently normal in cattle cloned from adult cells (Lanza et al., 2001; Betts et al., 2001).
BIOMEDICAL APPLICATIONS
In contrast to genetic manipulation of farm animals for production traits, transgenic manipulation for the production of human pharmaceuticals or transplant organs generally is not intended to cause changes that have as the logic effects on the animals themselves. Thus, although unexpected and undesirable phenotypic effects can still occur as result of gene insertion or cloning technology, there are generally fewer potential animal welfare concerns associated with the production of transgenic farm animals for biomedical purposes than for agricultural purposes (Van Reenen and Blokhuis, 1993).
Pharmaceuticals
Although there is a potential for producing pharmaceuticals in the eggs, blood, urine, or sperm of farm animals (Lubon, 1998; Sharma et al., 1994), the most common method is to produce transgenic cattle or goats that express the protein of interest in mammary tissue. The recombinant protein is then secreted in milk when the female lactates. This poses problems mainly when those proteins either are expressed in non-mammary tissues (so-called ectopic expression) or "leak" out of the mammary gland into the circulation (e.g., Lubon, 1998; Niemann et al., 1999). If the protein is biologically active in the species in which it is produced, it can cause pathologies and other severe systemic effects (e.g., Massoud et al., 1996). Rigorous regulation of the expression of the transgene is thus necessary to ensure that the animal welfare consequences of milk-borne pharmaceutical production are minimized, but such regulation is currently difficult to achieve. However, even when a pharmaceutical is confined to the mammary tissue, the expression of particular proteins has been associative with premature lactational shutdown and goats (Ebert and Schindler, 1993) and pigs (Shamay et al., 1992). In the pigs, there was evidence that the mammary tissue developed abnormally due to premature expression of the transgene, and that the condition of the mammary gland might have cause lactation to be painful. Similar concerns arise in the case of blood-borne proteins and neutraceuticals (see below) if the products are produced at levels higher than the animal's normal physiologic levels.
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Xenotransplantation
In an attempt to prevent hyperacute rejection of pig organs by humans (chapter 2), pigs have been made transgenic for the expression of human complement proteins, which are involved in regulation of the immune response (Cozzi and White, 1995; Tu et al., 1999; Cozzi et al., 1997; Byrne et al., 1997; Cowan et al., 2000). No phenotypic abnormalities have been reported in pigs as result of the expression of transgenes for these human proteins, although since the pigs are produced by microinjection there are the usual inefficiencies in terms of the numbers of embryos microinjected relative to the numbers of transgenic animals borne (Tu et al., 1999; Niemann and Kues, 2000).
He searches under way to produce pigs that, in addition to carrying complement transgenes, have both copies of the gene and coated the enzyme that produces the antigen associated with rejection knocked out. The animal welfare implications of this genetic manipulation are unknown; however, the knockout, which causes changes in cellular carbohydrate structure, potentially could have deleterious physiologic effects on the animals (Dove, 2000) and also render them susceptible to infection with human viruses (chapter 2).
An important animal welfare concerns related to xenotransplantation is the management and housing of pigs intended for use as organs worse animals. To minimize the potential for transmission of disease to human recipients, only specific pathogen free (SPF) the pigs are used. In SPF research animals are used in other contexts besides xenotransplantation, but there use raises several animal welfare issues. The SPF pigs are borne by hysterotomy or hysterectomy, and are then reared in isolators for 14 days before being placed in the source herd or and the xenotransplantation facility. The natural weaning age for pigs is about eight weeks (three to four weeks in commercial practice), and piglets subjected to extremely early weaning like this are known to develop abnormal behaviors (Weary et al., 1999). Older pigs intended for testing or organ donation might be housed in social isolation in unusually barren (i.e., easily sanitizable) environments. The pigs are extremely social animals that, when given the opportunity, will spend considerable time each day foraging, and that develop abnormal behaviors and confinement if not given the opportunity to root or build nests. In the United Kingdom, the Home Office Code of Practice (2000) for organ-source pigs, while recognizing the importance of maintaining biosecure facilities, nevertheless recommends that such pigs be housed in stable social groups and provided with environmental enrichment such as straw or other material suitable for a manipulation. The code requires justification if the animals' behavioral needs are to be compromised for a xenotransplantation protocol. There are no comparable standards for pigs intended for xenotransplantation in United States, and the lack of standardization of
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housing and care among U.S. facilities for these pigs is a source of concern. Although there are many forms of environmental enrichment available that are suitable for laboratory-housed pigs (Mench et al., 1998), appropriate methods for organ-source pigs require development and evaluation (Orlans, 2000).
Other Biomedical Applications
Farm animals might be genetically engineered for human biomedical applications other than xenotransplantation or the production of pharmaceuticals. Researches under way, for example, to produce it porcine model of cystic fibrosis, and there are already farm animals models for retinal degeneration (Petters et al., 1997) and neurodegenerative disease (Theuring et al., 1997). As genetic engineering techniques for farm animals improve, particularly such that single base coating changes are typical of many human genetic diseases can be introduced, and the production and use of farm animals models becomes more economically feasible, is likely that more models for disease research and toxicity testing will be developed. Discussion of the potential issues raised by these biomedical uses of farm animals is outside of the scope of this report. However, the welfare implications that depend upon specific features of the model understudy, including any unalleviated pain and suffering associated with the disease process itself as well as the need for specialized husbandry and veterinary care requirements (Dennis, 2000).
FARMING
If genetic technology becomes efficient and affordable, the primary farming applications of transgenesis and cloning likely will be to produce animals with increased growth, improved feed conversion, leaner meat, increased muscle mass, improved wool quality, improve disease resistance, increased reproductive potential, or that produce food of improved nutritional quality (neutraceuticals) or appeal.
Genetic engineering certainly has the potential to improve the welfare of farm animals. Decreasing mortality and morbidity by increasing resistance to diseases or parasites, or decreasing responses to ingestion of toxic plants, are obvious examples of welfare benefits, and an area in which some transgenic researches focused (Müller and Brem, 1994; Dodgson et al., 1999). It has also been pointed out that transgenic animals might receive a higher standard of care than nontransgenic animals because of their greater economic value (Morton et al., 1993). The cloning could be used as a strategy for breed preservation to maintain genes that are important for at a patient and resistance to disease, but
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could equally result in a further narrowing of the gene pool, with possibly deleterious effects on animal health (chapter 2).
Genetic engineering also could be used to deal with non-disease related welfare problems. It might be possible, for example, to engineer hands that produce only female offspring (Banner, 1995). This would eliminate the problems associated with surplus male checks, which are killed at the hatchery. The need for the so-called standard agricultural practices by castration and dehorning also could be reduced or eliminated by genetic engineering. Pigs are castrated to prevent boar taint in the meat, but this trait is strongly genetically linked and thus amenable to genetic manipulation. Similarly, horns on cattle, which are removed because they cause injuries to humans and other cattle, are the result of the single gene that could be knocked out by genetic manipulation without affecting other desirable performance traits; genetically polled (hornless) breeds of cattle are already available, produced by selective breeding.
The phenotypes of domesticated animals have already been changed significantly by selective breeding (chapter 1), including changes in certain aspects of behaviors such as temperament and sociability. The application of biotechnology to further adapt farm animals the modern intensive production systems, such as by making them more tolerant of crowding, less responsive, or less likely to display behaviors that are difficult to accommodate in intensive confinement while still maintaining economic efficiencies, might also be possible, are likely to generate the most controversy. Intensive farming systems that involve confinement, crowding and behavioral restriction are already been criticized for the negative effects on animal welfare (Fraser et al., 1997).
However, it is clear that serious welfare problems also can arise because of traditional breeding techniques. Broiler chickens are a case in point. Reading for increased growth has also led to serious physical disabilities, including skeletal and cardiovascular weakness. A large percentage of broilers have gait abnormalities (Kestin et al., 1992), and these might be painful and make it difficult for the birds to walk to the feeders and waterers. In addition, the parents of these birds must be severely feed restricted to prevent obesity, and this feed restriction is associative with extreme hunger and a variety of behavioral problems, including problems with mating behavior and hyperaggressiveness (Mench, 2002; Kjaer and Mench, in press). Traditional selection of pigs for increased leanness has led to increase excitability during handling (Grandin and Deesing, 1998), and selection for high reproductive rates (either by shortening the interval between births or increasing the number of offspring born) or increased lactation (chapter 1) has also led to welfare problems. In their report on [italics] The Use of Genetically Modified Animals [italics], the Royal Society (2001) concluded: "although genetic modification is capable of generating welfare problems...no qualitative distinction can be made between genetic modification using modern genetic modification technology and
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modification produced by artificial selection." Several ethical frameworks for evaluating the animal welfare implications of biotechnology is applied animals have been proposed in an attempt to resolve this difficulty. For example, Rollin (1995) has proposed the use of the "Principle of Conservation", which states that transgenic and cloning animals developed for agricultural uses should not be worse off than the founder animals or other livestock of the same species under similar housing and husbandry practices.
Improving disease resistance to pain and suffering is an application of transgenic technology that has clear animal welfare benefits. But it should be stressed that animal welfare is multifaceted, and this needs to be taken into account when assessing welfare impacts of the application of any technology—not just biotechnology. Important elements of animal welfare include freedom from disease, pain or distress, psychologic normality, and the opportunity to perform normal behaviors (Broom, 1993). While reducing diseases clearly beneficial, if this also permits animals to be more closely confined and thus decreases the opportunity for them to perform their normal behaviors, than the net effect on welfare could be negative.
The primary difference between traditional breeding and genetic engineering is that genetic engineering makes it possible to introduce genes that have not co-evolved over the evolutionary history of the target animal species or breed. Traditional methods of selection are subject to the checks and balances imposed by natural selection. Many related and apparently unrelated traits are genetically correlated, and selective breeding thus involves selecting for a whole phenotypes rather than he single gene product. Because most production and behavioral traits and livestock are polygenic and our understanding of livestock genomes is poor, few traits can reliably and predictably be engineered were introduced by manipulating only one gene (Moore and Mepham, 1995). For this reason, the production of the lien of transgenic will require generations of selective breeding after the introduction of gene constructs into the founder generation to ensure the animals display the desired phenotypes with few or no undesirable side effects. In making assessments about the production of genetically engineered animals for farming, costs and benefits need to be weighed carefully. When expression of growth hormone is appropriately regulated and transgenic pigs, for example, the increase is shown in growth and feed efficiency are modest, and similar to the increases that can be obtained by simply injecting pigs with porcine growth hormone (Pursel et al., 1989; Nottle et al., 1999). Pursel et al. (1989) suggest that centuries of selection for growth and body composition might limit the ability of the pig to respond to additional growth hormone. Indeed, it is possible that we already have pushed some farm animals to the limits of productivity that are possible by using selective breeding, and that further increases will only exacerbate the welfare problems that have already arisen during selection.
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The potential for reduction in genetic diversity in agricultural species also is posed by inappropriate application of certain biotechnologies (chapter 1). Transgenesis raises such concerns because each transgene integration event results in a genetically unique potential founder and only one founder is normally used to found a transgenic line. This can results in a profound genetic bottleneck unless genetic variability is restored to a production line by purposeful utilization of mating strategy involving backcrossing a transgenic line to a large number of distinct, presumably nontransgenic mates. The effects of cloning are more difficult to anticipate because competing processes are at issue. On the other hand, cloning by its nature produces identical copies of a particular individual, reducing genetic variability relative to what would have been transmitted by a conventional breeding. On the other hand, cloning makes it possible to save and utilize genetic variability that would not otherwise be available. For example, cloning could be employed to utilize the genetic resources from a steer that had proven to be a high performing individual. Cryopreserved cells could be utilized as donor material. Moreover, cloning is a tool that actually can be used to increase/maintain genetic variants in some situations quite independently of exploiting castrates (Seidel, Jr., 2001). The trade-off between the competing processes of loss in gain of genetic variants would be case-specific and is hard to quantify in the absence of simulation modeling with validation from field observations. Whatever the mechanism causing it, loss of genetic diversity could limit the potential for future genetic improvement of breeds by selective breeding or biotechnologic approaches. Further, disease could spread through susceptible populations more rapidly than through more genetically diverse populations.
Particularly serious concern that arises is susceptibility of species with low genetic diversity to infectious disease. Diversity of animal populations—particularly at major histocompatibility (MHC) loci—is a major factor preventing spread of disease, particularly viral disease (Xu et al., 1993; Shook et al., 1996; Kaufman and Lamont, 1996; Lewin et al., 1999). Different MHC types recognize different viral or bacterial epitopes encoded by pathogens for presentation to the immune system. In genetically diverse populations, pathogens can evade the immune response only if they adapt to each individual MHC type following transmission from one individual to another. The requirement for this evolutionary process provides a population of animals with significant protection against the spread of infection. Pathogens can more easily evade post immune response in genetically uniform populations (Yuhki and all Brian, 1990). The consequences of the failure of immunorecognition is illustrated by the deadly epidemics of diseases—such as measles—spread by initial contact between Europeans and isolated New World populations that lack adequate MHC diversity. Not only could enhance susceptibility create significant risk for spread of "new" infectious diseases in "monocultures" of
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cloned or highly inbred animal populations, it could also create new reservoirs for spread of zoonotic infections—like new strains of influenza—to humans.
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