Danish milk consumers are critical of advanced breeding methods in dairy production, but only 1 in 5 is unwilling to drink milk from dairy cows bred with semen derived from such methods.

Assisted reproductive technologies and genetic technologies can accelerate progress in breeding programs in dairy farming, but it is unclear how consumers will react to the use of these technologies. Using representative questionnaire data on Danish citizens (n = 2,036) this cross-sectional study examined consumer attitudes to the application of advanced technologies in dairy cattle breeding. Attitudes were examined in 2 ways. First, we prompted about general attitudes to assisted reproductive technologies and genetic technologies in dairy cow breeding. Here we found that most of the participants were critical of cow impregnation involving hormone therapy and the insertion of cloned fetuses. Second, we used a vignette experiment to study whether acceptance of and willingness to drink milk varies with the type of technique that farmers use for their breeding work, as well as the traits being bred for. We included 5 breeding methods with differing degrees of technological complexity. Participants were randomly assigned to receive tailored information about 1 of the 5 breeding methods. The information specified that dairy farmers' own use of advanced technologies is limited to using semen in artificial insemination on the farm. The potentially concerning technologies are here not applied at farm level but are represented in the semen used in artificial insemination because they were used by breeders on earlier generations of cows and bulls to develop semen with higher genetic merit. There was much less concern about this indirect use of the technologies. Only 1 in 5 participants thought the most advanced method we prompted about (use of semen from breeding methods involving genetic engineering and cloning) was unacceptable. Unwillingness to drink milk from cows produced through such a breeding method was also modest (18%) and not much higher than the unwillingness to drink milk from a cow produced by natural fertilization (10%). A likely reason for the unexpectedly low level of unwillingness to drink milk is that people regard the genetic engineering as distant from the final product. We also found that high-frequency organic milk consumers were more critical of advanced breeding methods. Thus, 28% within this group were unwilling to drink milk from cows impregnated with semen derived from earlier generations of cows and bulls bred using gene editing and cloning. Further, this share rose if the high-frequency organic consumers were very averse to the manipulation of nature. The organic sector may need to cater to this subgroup (e.g., by ensuring the traceability of the semen that organic farmers use to artificially inseminate their cows).

Danish dairy farmers' acceptance of and willingness to use semen from bulls produced by means of in vitro embryo production and genomic selection.

A novel technology combining in vitro production and genomic embryo selection is currently under development in dairy cattle breeding. Adoption of this technology will probably accelerate genetic progress toward the main breeding goals of economic interest, as well as allow selection for traits of societal concern such as decreased methane emissions and improved animal welfare. However, dairy farmers, and especially organic farmers, could find the technology morally questionable and reject its use. This cross-sectional study surveyed Danish dairy farmers' general acceptance of the combined technology and their reported likelihood of using semen produced with it. Drawing on diffusion theory, a questionnaire was developed to examine the way farmers discover and communicate about new technological breeding options, and to measure the factors which predict acceptance and likelihood of adopting the technology. The questionnaire was sent to a randomly selected sample of organic and conventional dairy farmers in Denmark, and 85 organic and 71 conventional farmers (41% response rate) completed it. Seventy-six percent of farmers reported that they would be likely to use semen from bulls derived from the technology. A majority (61%) also found the technology acceptable, but many (33%) were unsure or undecided. Most farmers saw the technology as beneficial, but ethical reservations were aired by around a fifth of the farmers. There were no differences between organic and conventional farmers in likelihood of using, perceived utility, and ethical reservations about the technology. Self-reported idealistic organic farmers showed lower acceptance of the technology, but reported similar likelihood of using semen produced by it. Young farmers (20-39 yr) exhibited higher acceptance of the technology. Larger producers (in terms of number of cows) were more likely to report that they will use and accept the technology. We conclude that it is likely that semen from the technology combining in vitro production and genomic selection would be widely used by both organic and conventional farmers provided that costs can be kept low, and that there are advantages in terms of achieving breeding goals. Structural developments, growth in size of dairy farms, acceptance by young farmers, and the fact that economic incentives (and even ethical arguments) seem to favor the technology all point to this conclusion.

Eating out in four Nordic countries: National patterns and social stratification.

Using a survey conducted in four Nordic countries (Denmark, Finland, Norway and Sweden) in 2012, we investigated whether eating out in cafés and restaurants in these countries is best characterized as a workday pattern activity or a leisure activity; and, whether frequent eating at cafés and restaurants is related to socio-demographic factors and factors relevant to the organization of daily life. We found that eating out is not a fundamental part of everyday eating. It is something which takes place occasionally. This may be taken to suggest that eating out in the Nordic countries is primarily a leisure activity. However, while this is an accurate portrayal of Denmark and Norway, eating out in Finland and Sweden is somewhat more common and linked to work-life. This difference probably reflects contrasting historical-institutional paths: in Finland and Sweden a food culture emphasizing cooked lunches and dedicated public policies supporting the provisioning of lunches outside the home may have promoted eating out. Multivariate analysis revealed that eating out declines with age. An urbanization effect exists, as residence in a capital city increases the propensity to eat out. There were socio-economic differences in all countries. We hypothesized that education and status would not significantly explain eating out activity after people's gastronomic interest had been controlled for. However, education was statistically significant in Sweden and Norway, and surprisingly status was negatively associated with eating out in Sweden. As expected, eating out was positively associated with income, and it was more frequent among the higher salariat and the self-employed (although not in Norway).

Neutering increases the risk of obesity in male dogs but not in bitches - A cross-sectional study of dog- and owner-related risk factors for obesity in Danish companion dogs.

Knowledge of risk factors for canine obesity is an important pre-requisite of effective preventative strategies. This study aimed to investigate risk factors for canine obesity in adult companion dogs across Zealand, Denmark. Client-owned dogs (>2 years of age and without chronic illness) were recruited and examined at eight companion animal veterinary practices in areas with varying socio-economic characteristics. The body condition score (BCS) of the dogs was examined by two investigators based on a 9-point scoring scheme. Dog owners answered a questionnaire that had prompts regarding: 1) dog characteristics, including neuter status, 2) owner characteristics, 3) feeding and exercise practices and 4) the owners' attachment to the dog. The effect of these factors on BCS and the risk of being heavy/obese (BCS scores 7-9) were analysed in two separate analyses. A total of 268 dogs were included in the analysis, of which 20.5% were found to be heavy/obese. The average BCS was 5.46. In terms of dog characteristics, neutering dramatically increased both BCS and the risk of being heavy/obese in male dogs but not in bitches. BCS and the risk of being heavy/obese increased in senior bitches and decreased in senior male dogs. The risk of being heavy/obese was higher in dogs with overweight and obese owners. Regarding feeding and exercise practices, providing only one meal per day increased BCS and risk of being heavy/obese. Treats during relaxation increased the risk of dogs being heavy/obese. It also increased the dogs' BCS, but only if the owners were overweight or obese. An increased duration of daily walking increased the risk of the dog being heavy/obese, but only if the owner was overweight or obese. Allowing the dog to run free in the garden/property decreased the risk of the dog being heavy/obese. The owners' attachment to the dog was not associated with the dogs' BCS or dogs' being heavy/obese. An important and novel finding was that neutering increased the risk of being overweight or obese for male dogs while bitches were at risk irrespective of neuter status. Furthermore, a complex interaction between owners' weight status, feeding practices and the risk of dogs being overweight or obese was found, which stresses the need to consider companion animal obesity from a One Health perspective in future prospective studies. Finally, this study was unable to confirm that canine obesity is a product of owners being too attached to their dogs.

Why do people buy dogs with potential welfare problems related to extreme conformation and inherited disease? A representative study of Danish owners of four small dog breeds.

A number of dog breeds suffer from welfare problems due to extreme phenotypes and high levels of inherited diseases but the popularity of such breeds is not declining. Using a survey of owners of two popular breeds with extreme physical features (French Bulldog and Chihuahua), one with a high load of inherited diseases not directly related to conformation (Cavalier King Charles Spaniel), and one representing the same size range but without extreme conformation and with the same level of disease as the overall dog population (Cairn Terrier), we investigated this seeming paradox. We examined planning and motivational factors behind acquisition of the dogs, and whether levels of experienced health and behavior problems were associated with the quality of the owner-dog relationship and the intention to re-procure a dog of the same breed. Owners of each of the four breeds (750/breed) were randomly drawn from a nationwide Danish dog registry and invited to participate. Of these, 911 responded, giving a final sample of 846. There were clear differences between owners of the four breeds with respect to degree of planning prior to purchase, with owners of Chihuahuas exhibiting less. Motivations behind choice of dog were also different. Health and other breed attributes were more important to owners of Cairn Terriers, whereas the dog's personality was reported to be more important for owners of French Bulldogs and Cavalier King Charles Spaniels but less important for Chihuahua owners. Higher levels of health and behavior problems were positively associated with a closer owner-dog relationship for owners of Cavalier King Charles Spaniels and Chihuahuas but, for owners of French Bulldogs, high levels of problems were negatively associated with an intention to procure the same breed again. In light of these findings, it appears less paradoxical that people continue to buy dogs with welfare problems.

Veterinarian's dilemma: a study of how Danish small animal practitioners handle financially limited clients.

This study examined the extent to which Danish veterinary practices encounter financially limited clients and how different factors relating to the animal, the client and the veterinarian affect decisions to provide treatment for these clients. 300 small animal practices were invited to participate in an online survey. 195 participated, giving a response rate of 65 per cent. The results show that Danish small animal veterinary practices encounter clients with limited finances regularly: 33.8 per cent of them 3-4 times, 24.6 per cent 5-10 times and 19.5 per cent 1-2 times a month. Only around 9 per cent reported having a written practice policy on handling financially limited clients. Factors affecting decisions to treat include the severity and type of the animal's condition, the medical care needed and the client's expressed emotions. The propensity to treat is significantly higher in female veterinarians and in situations involving unborn animals. The overall conclusion is that small animal veterinary practices often provide treatment to clients who are not able to pay-far beyond what is legally required. This can be considered a major economic and psychological challenge for the practising veterinarians.

Eating practices and diet quality: a population study of four Nordic countries.

BACKGROUND/OBJECTIVES: Daily practices related to eating are embedded in the social and cultural contexts of everyday life. How are such factors associated with diet quality relative to motivational factors? And, are associations universal or context-specific? We analyze the relationship between diet quality and the following practices: social company while eating, the regularity and duration of eating and the activity of watching TV while eating. SUBJECTS/METHODS: A cross-sectional, questionnaire-based internet survey was conducted in April 2012 with stratified random samples of the populations (aged 15-80 years) in Denmark, Finland, Norway and Sweden (N=7531, completion rate 9-13%). The questionnaire elicited detailed accounts of one day of eating focusing on social and practical aspects of eating events. The validated Dietary Quality Score was the dependent variable. This measure is based on eight food frequency questions focusing on fats, vegetables, fruits and fish in the diet. RESULTS: Eating activities were associated with diet quality even when motivation to eat healthily and sociodemographic factors were controlled for. The number of daily eating events and eating main meals was positively correlated with diet quality in all countries. Beyond that, activities that were significantly associated with diet quality varied with country. When measured separately, the association between each activity and diet quality was weaker than motivation to eat healthily, but in combinations that are found in parts of the populations, the association was substantial. CONCLUSIONS: Daily practices related to eating are correlated with diet quality. Practices that are important are in part universal but also country-specific. Efforts to promote healthy eating should address not only cognitive factors but also everyday contexts of eating that facilitate or hamper healthy practices.

In a class of their own: the Danish public considers obesity less deserving of treatment compared with smoking-related diseases.

BACKGROUND/OBJECTIVES: This study examined public support for publicly funded treatment of obesity (weight-loss surgery and medical treatment) and two pulmonary diseases (chronic obstructive pulmonary disease (COPD) and lung cancer) in Denmark. It also investigated whether beliefs about the causes of lifestyle-related diseases (external environment, genetic disposition and lack of willpower) and agreement that 'people lack responsibility for their life and welfare' influenced support. SUBJECTS/METHODS: This was a questionnaire study in which a sample of 1003 Danes (age 18-65 years) drawn from an Internet database were surveyed. RESULTS: Approximately one in three supported publicly funded weight-loss surgery (30%) and medical treatment of obesity (34.4%). A large majority supported treatment for lung cancer (86.1%), and a clear majority also supported treatment for COPD, whether it was framed as 'smoker's lung' (61.9%) or COPD (71.2%). The belief that lifestyle-related diseases are caused by the external environment or genetic disposition did not systematically influence support. Agreement that 'people lack responsibility for their life and welfare' reduced support significantly for all treatment types. However, in contrast with pulmonary diseases, support for publicly funded obesity treatments decreased considerably when beliefs about individual failure (that is that people lack 'willpower' and 'individual responsibility') were detected. CONCLUSIONS: Support for publicly funded COPD and lung cancer treatment is considerably higher than that for obesity treatment. This could encourage institutional discrimination through policies that involve charging patients for the treatment of obesity but not for the treatment of other lifestyle-related diseases.

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.

Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primed in situ labelling and chromosome painting (PRINS-painting).

In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primed in situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting).

Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells.

A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.

Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria.

Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.

Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest that the MCAD mutations can be modulated by chaperones, a phenomenon that may influence the manifestation of the MCAD disease.

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.