Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The expression of genes regulated by transcription factors at specific stages exerts important regulatory effects on the development process of germ cells. Male mice with overexpressed programmed death ligand 1 (PD-L1) (B7 homolog1) in the testis have infertility and abnormal sperm development, thereby exhibiting severe malformation and sloughing throughout spermatid maturation and collapsed and disorganized seminiferous epithelium structure. Furthermore, PD-L1 overexpression causes overexpression of cysteine-rich secretory protein 1 (CRISP1) in the epididymis and adversely affects or precludes sperm energization, sperm-pellucida binding and sperm-oocyte fusion. These findings suggest that CRISP1 and PD-L1 can interact with each other to induce male infertility and germ-cell dissociation.

Scientists recently attracted considerable public attention when they presented a featherless chicken tailored for production in hot climates. Although this chicken was actually the result of traditional breeding, it is an example of what might be achieved if targeted gene manipulation techniques become widely applied in agriculture. Through interfering directly with an animal's genome, scientists hope to be able to create animals with exactly the desired characteristics, such as lean meat or temperature tolerance. Industry and geneticists may be enthusiastic about the possibility of producing pork with polyunsaturated fatty acids or high-yielding dairy cows to be kept in tropical climates, but the European public often reacts with alarm at these prospects. A consistent pattern of the surveys conducted among members of the European public is that, of all of the potential biotechnology applications, those involving animals are the ones that people find the least acceptable. People fear a development of techniques that may get out of control, and they also have ethical concerns about humans’ right to ‘play God’ and about the welfare of the animals involved. All of these aspects seem to be relevant for an ethical discussion about animal biotechnology. Animal welfare scientists can play an important role by providing information for an animal welfare risk assessment at an early stage of research projects that involve the genetic modification of animals, and also by helping to develop guidelines for the housing and husbandry of animals with special needs. On the other hand, ethical problems remain that lie outside the area of science. In this paper we discuss the role of animal welfare science in aiding ethics decisions about animal biotechnology. We give a summary of the different ethical concerns expressed by ethicists and by the general public. Focusing on one of them, animal welfare, we give an introduction to the animal welfare implications of recent developments in reproductive and gene technologies. The importance of animal welfare aspects is discussed in relation to other ethical concerns about animal biotechnology.

This study was performed to improve production efficiency at the level of recipient pig and donor nuclei of transgenic cloned pigs used for xenotransplantation. To generate transgenic pigs, human endothelial protein C receptor (hEPCR) and human thrombomodulin (hTM) genes were introduced using the F2A expression vector into GalT–/–/hCD55+ porcine neonatal ear fibroblasts used as donor cells and cloned embryos were transferred to the sows and gilts. Cloned fetal kidney cells were also used as donor cells for recloning to increase production efficiency. Pregnancy and parturition rates after embryo transfer and preimplantation developmental competence were compared between cloned embryos derived from adult and fetal cells. Significantly higher parturition rates were shown in the group of sows (50.0 vs. 4.1%), natural oestrus (20.8 vs. 0%), and ovulated ovary (16.7 vs. 5.6%) compared with gilt, induced and non-ovulated, respectively (P < 0.05). When using gilts as recipients, final parturitions occurred in only the fetal cell groups and significantly higher blastocyst rates (15.1% vs. 21.3%) were seen (P < 0.05). Additionally, gene expression levels related to pluripotency were significantly higher in the fetal cell group (P < 0.05). In conclusion, sows can be recommended as recipients due to their higher efficiency in the generation of transgenic cloned pigs and cloned fetal cells also can be recommended as donor cells through correct nuclear reprogramming.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

The release of Bt cotton varieties genetically modified for increased tolerance to major cotton insect pests provided impetus for the reestablishment of a cotton industry in northern Australia. However, this stimulated concern that the addition of the gene might facilitate an increase in the potential for weediness of genetically modified cultivars in noncropping habitats. Bt and conventional cottonseeds were planted in 12 sites in northern Australia to test the hypothesis that there would be no increase in the ability of Bt cotton vs. conventional cotton to establish weedy, or invasive populations, defined as population growth over time greater than one, irrespective of location, habitat, seed type, or population density. Invasiveness was a factor of germination, survival, and recruitment. We examined whether the addition of the Bt gene would increase fitness of these parameters, and associated invasiveness. An irrigation drain was considered a high-risk habitat for cotton establishment, so an additional site was sown at that habitat to provide supplementary data to the original 12 sites. Location and habitat were the dominant factors influencing germination, survival, fecundity, and invasiveness. Bt and non-Bt cottonseeds did not differ in their ability to germinate, establish, and survive. After 2 yr, cotton plant survival was very low, and only 3 of 13 sites established fecund cotton populations. Measurements continued for an additional 2 yr at these sites. There was no increase in values for invasiveness for the Bt genotype treatments at any location or habitat after 2 yr or at two selected habitats after > 4 yr, demonstrating that the addition of the Bt gene will not confer increased fitness for weediness. Mean invasiveness values for each habitat, irrespective of genotype, were less than one, indicating that neither conventional nor Bt cotton would establish invasive cotton populations in northern Australian habitats.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

A study was carried out between 2008 and 2011 to investigate the potential risks of gene flow and its consequences in the crop–wild–weed S. bicolor complex in Ethiopia to aid efforts to conserve genetic diversity. Morphological measurements and genomic DNA samples were taken in situ from 30 wild and eight cultivated populations representing a total of 760 samples from five regions. Genetic diversity, gene flow, population structure and outcrossing rates of wild populations were computed using phenotypic measurements and/or polymorphic simple sequence repeat (SSR) markers. Moreover, morphological analyses of fitness of crop–wild hybrids were studied. High diversity was observed among the wild/weedy sorghum populations for phenotypic traits and SSRs. SSR diversity was high in both wild and cultivated populations, but the magnitude was greater in the former. Gene flow between the wild and the cultivated sorghum was observed to be higher than that within either pool. Wild sorghums exhibited variation in the multilocus outcrossing rate (range = 0.31–0.65) and fitness was not compromised in most wild × crop hybrids. The study indicated that crop-to-wild gene flow is possible in Ethiopia. Thus, genes from transgenic sorghum are expected to enter into the wild and non-transgenic cultivated populations and may spread and persist, if transgenic sorghum is deployed in Ethiopia and in other countries of Africa, which may pose risk of introduction of unwanted effects, which in turn may lead to loss of genetic diversity.

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals.

Stochastic Efficiency with Respect to a Function (SERF) is used to rank transgenic cotton technology groups and place an upper and lower bound on their value. Yield and production data from replicated plot experiments are used to build cumulative distribution functions of returns for nontransgenic, Roundup Ready, Bollgard, and stacked gene cotton cultivars. Analysis of Arkansas data indicated that the stacked gene and Roundup Ready technologies would be preferred by a large number of risk neutral and risk averse producers as long as the costs of the technology and seed are below the lower bounds calculated in this manuscript.

There is significant concern about potential ecological effects of introduced organisms, including non-indigenous species and those created by genetic modification. This paper presents an Ecopath with Ecosim modelling approach, designed to examine long-term trophic effects of growth hormone (GH) transgenic coho salmon should they ever escape to a coastal salmonid ecosystem, namely the Strait of Georgia in British Columbia (Canada). The model showed that the effects of introduced GH transgenic coho salmon varied with their biomass, diet, structure of the invaded ecosystem, and environmental conditions. Occasional escapes of non-reproductive salmon did not have a significant impact on the example ecosystem. However, effects of GH coho salmon varied with their diet when large numbers of these fish were present in the simulated ecosystem (for example, when they constituted 20% of total current aquaculture production in the area). Further, climate-driven changes in the biomass of low trophic levels (bottom-up effects) could have a greater impact on the ecosystem than the introduction of large numbers of GH coho salmon. A new version of Ecopath with Ecosim's Monte Carlo approach showed that the model predictions were robust to GH coho salmon's Ecopath parameters, but more sensitive to vulnerabilities of prey to GH coho salmon. Modelling ecosystem effects of genetically modified organisms provides a complementary approach for risk assessments when data from nature are not readily obtainable.

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

Oocyte genome cloning is a method by which haploid maternal embryos are obtained in such a way that parthenogenetic haploid blastomeres from these embryos can be considered as a clone of the original gamete. Our objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Furthermore, we generated biparental homogeneous transgene-expressing embryos using parthenogenetic haploid blastomeres that expressed a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation of oocytes in ionomycin and 6-dimethylaminopurine (DMAP) with a 3 h interval to permit their second polar body extrusion. The cleavage rate was 87.3%. To generate transgene-expressing blastomeres, activated oocytes were injected with pCX–EGFP–liposome complexes 3 h post ionomycin exposure, resulting in a cleavage rate of 84.4%. In the second experiment, haploid parthenogenetic blastomeres that were positive or negative for EGFP expression were used to reconstruct biparental embryos. Cleavage and blastocyst rates for the reconstructed embryos were 78.4% and 61.1% and 10.8% and 8.4%, using EGFP-positive or -negative blastomeres, respectively (P < 0.05). All of the reconstructed embryos showed EGFP expression, with 96.6% of them showing homogenic expression. Oct-4 expression in the reconstructed blastocysts displayed a similar pattern as IVF-blastocyst controls. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that this technique is efficient to generate homogeneous transgene-expressing embryos.

A total of 72 male weaned pigs were used in a 110-day study to investigate the effect of feeding genetically modified (GM) Bt MON810 maize on selected growth and health indicators. It was hypothesised that in pigs fed Bt maize, growth and health are not impacted compared with pigs fed isogenic maize-based diets. Following a 12-day basal period, pigs (10.7 ± 1.9 kg body weight (BW); ∼40 days old) were blocked by weight and ancestry and randomly assigned to treatments: (1) non-GM maize diet for 110 days (non-GM), (2) GM maize diet for 110 days (GM), (3) non-GM maize diet for 30 days followed by GM maize diet up to day 110 (non-GM/GM) and (4) GM maize diet for 30 days followed by non-GM maize diet up to day 110 (GM/non-GM). BW and daily feed intake were recorded on days 0, 30, 60 and 110 (n = 15). Body composition was determined by dual energy X-ray absorptiometry (n = 10) on day 80. Following slaughter on day 110, organs and intestines were weighed and sampled for histological analysis and urine was collected for biochemical analysis (n = 10). Serum biochemistry analysis was performed on days 0, 30, 60, 100 and 110. Growth performance and serum biochemistry were analysed as repeated measures with time and treatment as main factors. The slice option of SAS was used to determine treatment differences at individual time points. There was no effect of feeding GM maize on overall growth, body composition, organ and intestinal weight and histology or serum biochemistry on days 60 and 100 and on urine biochemistry on day 110. A treatment × time interaction was observed for serum urea (SU; P < 0.05), creatinine (SC; P < 0.05) and aspartate aminotransferase (AST; P < 0.05). On day 30, SU was lower for the non-GM/GM treatment compared with the non-GM, GM and GM/non-GM treatments (P < 0.05). On day 110, SC was higher for the non-GM/GM and GM/non-GM treatments compared with non-GM and GM treatments (P < 0.05). Overall, serum total protein was lower for the GM/non-GM treatment compared with the non-GM/GM treatment (P < 0.05). The magnitude of change observed in some serum biochemical parameters did not indicate organ dysfunction and the changes were not accompanied by histological lesions. Long-term feeding of GM maize to pigs did not adversely affect growth or the selected health indicators investigated.

The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous–hFVIII or exogenous–enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

The aim of our study was to compare the viability of sperm cells from transgenic (mWAP-hFVIII gene) or non-transgenic (normal) rabbit males as assessed by viability (SYBR-14/PI) and apoptosis (annexin V) tests. These results were evaluated using female conception rates following insemination with the respective sperm samples. No significant differences were found in concentration and motility between transgenic and non-transgenic spermatozoa. Spermatozoa from both transgenic (63.05 ± 20.05%) or non-transgenic (65.75 ± 22.15%) males, stained with SYBR-14 (green), were found to be morphologically normal. In both groups, the highest proportion of annexin V-positive sperm staining was found in the post-acrosomal part of the sperm head (8.66 and 27.53%). The percentage of sperm that stained with SYBR-14/PI or with annexin V/DAPI was correlated with liveborn in transgenic rabbits (R2 = 0.6118 and R2 = 0.2187, respectively) or non-transgenic rabbits (R2 = 0.671 and R2 = 0.3579, respectively). These data indicate that there was no difference in the viability of rabbit transgenic and non-transgenic spermatozoa when determined by both fluorescence assays.

The aim of this study was to compare the quality of rabbit transgenic embryos obtained upon microinjection of gene constructs containing different promoters and green fluorescent proteins (CMVIE–EGFP, PGK–EGFP and CMVIE–hrGFP). Developmental rate, total cell number in hatching blastocyst stage, number of apoptotic cells, diameter of embryos, transgene integration and transgenic mosaicism were investigated.

The rate of rabbit embryos microinjected with the different gene constructs developed up to morula stage was significantly lower (p < 0.05) than that of intact (non-microinjected) rabbit embryos (66–74vs. 98%). The highest efficiency of transgene integration (15%) was found when the CMVIE–EGFP (DrdI) gene construct was used, however a significantly higher transgenic mosaicism (60%) was found in rabbit embryos using this gene. The lowest cell number was counted in rabbit transgenic embryos with CMVIE–rhGFP linearized by ScaI (115.0 ± 8.20), the highest cell number (134.0 ± 35.00) was detected in rabbit transgenic embryos carrying PGK–EGFP (Not I) gene. The highest number of apoptotic cells (2.6 ± 0.33) was recorded in rabbit transgenic embryos with the integrated CMVIE–EGFP (ClaI) transgene.

Based on these results a more suitable gene marker for rabbit transgenic embryos production and selection is the CMVIE–EGFP (ClaI) gene construct. Prior to using microinjected embryos (for embryo transfer, vitrification or ESC isolation) it is necessary to pre-select microinjected embryos with evident transgenic mosaicism.

Transgenic fish in development for aquaculture could escape from farms and interbreed with wild relatives in the nearby environment. Predicting whether escapes would result in transgene introgression is a major challenge in assessing environmental risks of transgenic fish. Previous studies have simulated gene flow from transgenic fish using mathematical modeling of fitness traits to predict the relative selective value of transgenic genotypes. Here, we present the first study of gene flow over the full life cycle in openly-breeding populations of transgenic animals, along with measurement of fitness traits. We conducted two invasion experiments in which we released two lines of growth-enhanced transgenic fish (T67 and T400), Japanese medaka (Oryzias latipes), into populations of wild-type (W) medaka in structured mesocosms. After several generations, the frequency of transgenic fish varied across replicates in the first invasion experiment (6 months), but the frequency of transgenic fish decreased in the second experiment (19 months). We also measured selected fitness traits in transgenic and wild-type medaka because these traits could be used to predict the relative selective value of a genotype. We found that: T400 males were more fertile than W males; offspring of W females lived longer than those with transgenic mothers; and W and T67 females reached sexual maturity sooner than T400 females. In contrast with other research that reported larger transgenic males had a mating advantage, we found that W males obtained more matings with females than T males; genetic background effects may account for our differing results as we compared W and T fish derived from different strains. The decreasing frequency of transgenic fish in the second invasion experiment suggests that transgenic fish had a selective disadvantage in the experimental environment. Our finding of transgenic advantage of some fitness traits and wild-type advantage in others is consistent with our invasion experiment results.

This article addresses how people who handle transgenic animals in practice—laboratory workers and members of animal ethics committees—talk about and handle dilemmas with transgenic animals. It is shown how dilemmas associated with transgenic animals become back-grounded through rhetorical comparisons with ‘something else’. Through these comparisons, transgenic animals are framed as normal, ordinary and thereby unproblematic on the one hand, and as valuable treasures in which are embedded hopes and expectations of future medical treatments on the other. This tension builds up to a discourse on transgenic mice as ordinary treasures. Towards the end of the article we discuss how this discourse tends to exclude possibilities of discussing specific dilemmas of genetically modified animals. Instead the discourse is contributing to certain transgenic silences. This article is based on the project ‘Dilemmas with transgenic animals’, in which notions of culture and nature, risk and safety, innovation and organism, science and technology, are investigated in the scientific production, use and ethical evaluation of transgenic animals. The project builds on case studies in two different contexts: laboratories and animal ethics committees. The methods used are interviews and ethnographic fieldwork.

Human complement regulatory protein hCD46 may reduce the hyperacute rejection (HAR) in pig-to-human xenotransplantation. In this study, an hCD46 gene was introduced into porcine embryonic germ (EG) cells. Treatment of human serum did not affect the survival of hCD46-transgenic EG cells, whereas the treatment significantly reduced the survival of non-transgenic EG cells (p < 0.01). The transgenic EG cells presumably capable of alleviating HAR were transferred into enucleated oocytes. Among 235 reconstituted oocytes, 35 (14.9%) developed to the blastocyst stage. Analysis of individual embryos indicated that 80.0% (28/35) of embryos contained the transgene hCD46. The result of the present study demonstrates resistance of hCD46-transgenic EG cells against HAR, and the usefulness of the transgenic approach may be predicted by this cytolytic assessment prior to actual production of transgenic pigs. Subsequently performed EG cell nuclear transfer gave rise to hCD46-transgenic embryos. Further study on the transfer of these embryos to recipients may produce hCD46-transgenic pigs.

Impact assessments of virus resistance transgene introgression into wild, free-living populations are important for determining whether these transgenes present a risk to agriculture or the environment. Transgenic virus-resistant Cucurbita pepo ssp. ovifera var. ovifera L. (squash) cultivars have been commercialized, and may be cultivated in close proximity to cross-compatible wild, free-living relatives (C. pepo subsp. pepo vars. ozarkana and texana). Therefore, the potential impact of these virus resistance transgenes was studied by surveying the incidence and fluctuations of virus infection (as assayed by ELISA), virus symptoms (which may not be seen in an infected plant) and population size in forty-three free-living C. pepo populations in Illinois, Missouri, Arkansas, Mississippi, Louisiana, and Texas. Ten of these populations were studied over three consecutive seasons. Depending on the year, 61% to 78% percent of the populations had at least one individual infected by at CMV, ZYMV or WMV2, but the median incidence of infection within populations was 13%. The observed infection level in free-living populations was consistent with levels defined as “low” in field plot experiments conducted by others, leading to the conclusion that transgenic virus resistance should not provide a significant fitness advantage to the free-living populations examined. Viral symptoms were detected in only 2% of plants observed, indicating that severity of viral infection was low. CMV, ZYMV, and WMV2 were not the only viruses infecting these populations, further reducing the likelihood that resistance to these viruses would release populations from constraints imposed by virus diseases.