Farmers' Attitudes in Connection with the Potential for Rodent Prevention in Livestock Farming in a Municipality in North Rhine-Westphalia, Germany.

Rodents in livestock farming constitute a threat to the one health approach. In the present observational case study, livestock farmers worked together with a pest controller within a pilot project. The aim of the study was to assess determinants associated with rodent-prevention potential. The study started in March 2019 on 24 livestock farms in a municipality in North Rhine-Westphalia/Germany. At the beginning of the project a survey on the determinants expected to be related to prevention potential was conducted. To determine the potential for rodent prevention, an expert person, who was not involved in the project before, assessed the on-site conditions of the farms after 1.5 years of project duration in 2020. The potential for rodent prevention was good for about half of the farms. There were significant differences in the willingness to make changes at the project's start between farms with a high and a low potential for rodent prevention after 1.5 years. There is a general need for action to provide farmers with more practical information on rodent control. This is aggravated by the insufficient advisory services offered to farmers. This study confirms the importance of implementing preventive measures in the control of rodents to ensure that anticoagulant rodenticides are handled responsibly to reduce the impact on non-target species.

Pilot study on nasal microbiota dynamics and MRSA carriage of a pig cohort housed on straw bedding.

Methicillin-resistant Staphylococcus aureus (MRSA) can be transmitted between pigs and humans on farms. Hence, the reduction of MRSA carriage in pigs could decrease the risk of zoonotic transmission. Recently, straw bedding has been found to significantly reduce MRSA carriage in pigs. The mechanisms behind this effect remain unclear but changes in the nasal microbiome may play a role. In this exploratory study, the nasal microbiota of pigs kept on straw was examined using V1/V2 16S rRNA gene sequencing. Nasal swabs were collected from 13 pigs at six different time points during the course of a full fattening cycle resulting in 74 porcine samples. In addition, straw samples were collected at each time point. Eleven out of 13 pigs were MRSA positive at housing-in. We found a strong temporal pattern in the microbial communities. Both microbial diversity and abundance of Staphylococcus species peaked in week 5 after introduction to the straw stable decreased in week 10, when all pigs turned MRSA-negative, and increased again toward the end of the fattening period. These findings show that the introduction of pigs into a new environment has a huge impact on their nasal microbiota, which might lead to unfavorable conditions for MRSA. Moreover, other Staphylococcus species may play a role in eliminating MRSA carriage. We designed a follow-up study including two different husbandry systems to further assess these effects.

Comparative Investigation of Coincident Single Nucleotide Polymorphisms Underlying Avian Influenza Viruses in Chickens and Ducks.

Avian influenza is a severe viral infection that has the potential to cause human pandemics. In particular, chickens are susceptible to many highly pathogenic strains of the virus, resulting in significant losses. In contrast, ducks have been reported to exhibit rapid and effective innate immune responses to most avian influenza virus (AIV) infections. To explore the distinct genetic programs that potentially distinguish the susceptibility/resistance of both species to AIV, the investigation of coincident SNPs (coSNPs) and their differing causal effects on gene functions in both species is important to gain novel insight into the varying immune-related responses of chickens and ducks. By conducting a pairwise genome alignment between these species, we identified coSNPs and their respective effect on AIV-related differentially expressed genes (DEGs) in this study. The examination of these genes (e.g., CD74, RUBCN, and SHTN1 for chickens and ABCA3, MAP2K6, and VIPR2 for ducks) reveals their high relevance to AIV. Further analysis of these genes provides promising effector molecules (such as IκBα, STAT1/STAT3, GSK-3ß, or p53) and related key signaling pathways (such as NF-κB, JAK/STAT, or Wnt) to elucidate the complex mechanisms of immune responses to AIV infections in both chickens and ducks.

Pet Rats as the Likely Reservoir for Human Seoul Orthohantavirus Infection.

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient's rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007-2020) as well as 594 wild rats from the Netherlands (2013-2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

Prevalence of LA-MRSA in pigsties: analysis of factors influencing the (De)colonization process.

Professional pig husbandry is often associated with a more or less high load of LA-MRSA. Possible risk factors for LA-MRSA colonization in pig herds have already been identified in studies suggesting that housing conditions may affect LA-MRSA prevalence. In Europe, pigs are kept under variety of conditions. The aim of this study is to identify husbandry and housing condition factors that affect colonization with LA-MRSA. 78 pig farms were selected and assigned to three categories according to housing conditions: conventional, alternative and organic. Animal and surface samples were taken and examined for the presence of LA-MRSA at beginning and end of one fattening period per farm. Altogether, a significant (p < 0.05) decrease in colonization with LA-MRSA from beginning to end of the fattening periods in pigs and surfaces can be observed. Alternative farms showed a higher dynamic in the colonization. In organic farms, almost no colonization was found. Influencing housing condition factors that determine LA-MRSA status at the end of the fattening period are the number of pigs in the building, LA-MRSA status at the beginning of fattening period, material of the floor (straw bedding), strictness of black-white separation and antibiotic treatment during the fattening period. For pig farming in general, knowledge and measures to reduce the colonization with LA-MRSA would be important.

Development and validation of a triplex real-time qPCR for sensitive detection and quantification of major rat bite fever pathogen Streptobacillus moniliformis.

Streptobacillus (S.) moniliformis is the most important pathogen causing rat bite fever (RBF) worldwide. This zoonotic pathogen is understudied mainly due to difficulties in culturing S. moniliformis as a fastidious microorganism. Therefore, advances in molecular detection techniques are highly needed, especially with regard to the widespread availability of real-time quantitative (q) PCR in laboratories. In this study, we aimed to develop a qPCR for the identification of Streptobacillus species and quantification of S. moniliformis in clinical samples, especially those derived from tissue samples of animal origin. We optimized a previously described PCR protocol in order to develop a qPCR, which can detect different Streptobacillus species with high specificity and is simultaneously able to quantitate S. moniliformis in different clinical matrices. The qPCR exhibited a limit of detection (LOD) of 21 copies/reaction representing ~4-5 streptobacilli, while the limit of quantification (LOQ) was 2.1 × 103 copies/reaction. It was also more sensitive than conventional PCR by two orders of magnitude and proved to have a substantial agreement (Kappa 0.74) compared to it with a superior detection rate in 374 samples from wild rats, laboratory rats and animals from holdings of wild-trapped rats. To conclude, the qPCR described in this study is an important molecular tool that is able to quantify S. moniliformis in tissue samples of animal origin. It represents a suitable tool for future establishment and evaluation of other molecular assays that are highly needed for a better understanding of epidemiology and pathophysiology of RBF. In experimental studies, it will also be useful for titration purposes since the quantification of the organism using classical plate counting technique is problematic and inaccurate.

The Combined Effect of IgG and Fe Supply and Feeding Management on Growth Rates of Calves on Eight Commercial Dairy Farms in Germany.

During the first days of a calf's life, the foundations are laid for successful growth and thus also for the later performance of the cows. The aim of the present study was to analyze the impact on the weight gain of newborn calves due to important management factors related to colostrum supply, iron supply, feeding regime and microbial load at first feeding. In spring 2017, information of 123 Holstein calves were analyzed with regard to the colostrum supply and management factors on eight commercial dairy farms located in Germany. Additionally, blood samples of newborn calves were analyzed for total immunoglobulin G (IgG) and serum iron content. Furthermore, Brix analysis and analysis of contamination by E. coli were performed on first colostrum samples from teats or buckets. Average daily weight gain of calves at days 14 and 50 was calculated. The colostrum IgG was estimated by Brix refractometer. The volume of initial colostrum supply and the time between birth and colostrum intake significantly (p < 0.05) influenced the serum IgG concentration. The serum IgG concentration, the serum iron concentration and the feeding regimen (restrictive or not restrictive feeding) had a significant effect (p < 0.05) on daily weight gains. In conclusion, this study shows that, in addition to a sufficient supply of immunoglobulins, other aspects, such as an adequate colostrum, iron and milk supply, play an essential role in calf growth rates.

Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries.

Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.

Prevalence and zoonotic transmission of colistin-resistant and carbapenemase-producing Enterobacterales on German pig farms.

The treatment of infections due to colistin-resistant (Col-E) and carbapenemase-producing (CPE) Enterobacterales challenges clinicians both in human and veterinary medicine. Preventing zoonotic transmission of these multidrug-resistant bacteria is a Public Health priority. This study investigates the prevalence of Col-E and CPE on 81 pig farms in North-West Germany as well as among 138 directly exposed humans working on these farms. Between March 2018 and September 2020, 318 samples of porcine feces were taken using boot swabs. Farm workers provided a stool sample. Both a selective culture-based approach and a molecular detection of colistin (mcr-1 to mcr-5) and carbapenem resistance determinants (bla OXA-48/bla VIM/bla KPC/bla NDM) was used to screen all samples. Isolates from farm workers and farms were compared using core genome multilocus-sequence typing (cgMLST) and plasmid-typing. CPE were cultured neither from porcine feces nor from human stool samples. In one stool sample, bla OXA-48 was detected, but no respective CPE isolate was found. Col-E were found in 18/318 porcine (5.7%) samples from 10/81 (12.3%) farms and 2/138 (1.4%) farmers, respectively. All Col-E isolates were Escherichia coli harboring mcr-1. Both farm workers colonized with Col-E worked on farms where no Col-E were detected in porcine samples. In conclusion, CPE were absent on German pig farms. This supports findings of culture-based national monitoring systems and provides evidence that even when improving the diagnostic sensitivity by using molecular detection techniques in addition to culture, CPE are not prevalent. Col-E were prevalent in porcine feces despite a recent decrease in colistin usage among German livestock and absence of colistin treatments on the sampled farms. Farmers carried Col-E, but zoonotic transmission was not confirmed.

Pilot Study on Alteration of LA-MRSA Status of Pigs during Fattening Period on Straw Bedding by Two Types of Cleaning.

In countries with professional pig husbandry in stables, the prevalence of livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) on farms has remained high or has further increased in recent years. Simple measures to reduce LA-MRSA among pigs have not yet been successfully implemented. The aim of this pilot study is twofold: first, to examine how the LA-MRSA status of LA-MRSA positive fattening pigs at the date of housing changes over the fatting period on straw bedding and, second, whether this change could be influenced by the quality of cleaning and disinfection (C&D). For this purpose, 122 animals are individually tested for LA-MRSA carriage at five sequential time points comparing pigs housed on a farm using straw bedding plus C&D (n = 59) vs. straw bedding plus simple cleaning (n = 63). At the time of housing, all animals in both groups are LA-MRSA positive. This status changes to 0% in the group with simple cleaning until the end of fattening and 28% in the C&D group. LA-MRSA in environmental and air samples is also reduced over the fattening period. The results indicate that keeping pigs on straw might be one way to reduce LA-MRSA during the fattening period with simple cleaning appearing to be more beneficial than C&D. Further investigations are necessary to determine the influencing factors more precisely.

Single Blinded Study on the Feasibility of Decontaminating LA-MRSA in Pig Compartments under Routine Conditions.

： In countries with intensive pig husbandry in stables, the prevalence of livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) on such farms has remained high in the last few years or has also further increased. Simple measures to reduce the LA-MRSA among pigs have not yet been successfully implemented. Earlier publications showed a decontamination of LA-MRSA was only possible with great effort. The aim of this study is to determine the suitability of routine cleaning and disinfection (C&D) for adequate LA-MRSA decontamination. For this purpose, at least 115 locations in a piglet-rearing compartment were examined before and after cleaning and disinfection. The sample locations were stratified according to accessibility for pigs and the difficulty of cleaning. The cleaning work was carried out routinely by farm employees, who were not informed about the sampling (single blinded). While before cleaning and disinfection, 85% of the samples from the surfaces were LA-MRSA positive, while only 2% were positive thereafter. All LA-MRSA-positive samples after cleaning and disinfection were outside the animal area. Air samples also showed no LA-MRSA after cleaning and disinfection. Conclusion: In well-managed livestock farms, decontamination of the LA-MRSA barn is quite possible; after C&D no LA-MRSA was detectable at animal height.

Animal Hygiene Indexes in Relation to Big-Five Personality Traits of German Pig Farmers Evaluated by Self- and Other-Rating.

Improving biosecurity in intensive livestock production has become an increasingly challenging task. Often, animal hygiene measures are implemented at lower levels than recommended. Therefore, veterinarians and farm advisors look for new approaches to improve their advisory process with farmers. In the current study it has been hypothesized that German pig farmers' big-five measured personality traits might correlate with farms' biosecurity level expressed by a "continuous animal hygiene index" and a "technical animal hygiene index." Hence, comprehensive data on the implementation of more than 100 hygiene measures were collected at farm level from a specific pilot sample of 42 pig farmers from a livestock intensive region in north-western Germany. In addition, big-five personality traits (BFI-S) were measured by self- and other-rating. Inter-rater reliabilities for personality traits indicated expected positive correlations apart from agreeableness (r S = -0.101). Regarding the self-rating, neuroticism was valued lowest ( x ¯ = 3.88 ± 1.18) and conscientiousness highest ( x ¯ = 5.68 ± 0.70). The animal hygiene indexes revealed medium biosecurity levels on the participating farms. Piglet breeders had a significantly higher value for the "continuous animal hygiene index" ( x ¯ = 63.00 ± 9.91%). Personality traits conscientiousness and openness showed correlations with the continuous and the technical animal hygiene index. Depending on the production systems as well as the rating perspectives, correlations varied. For one of the personality traits playing a direct role in social interaction-extraversion-the advisory process might function as a mediating factor. The current results show that clustering of single hygiene measures into indexes in the evaluation of pig farms' biosecurity level might have advantages. The preliminary results from this study should be validated in larger, more representative samples. Furthermore, structured and systematic consideration of personality traits of farmers adds an additional aspect to include individuality of farmers more systematically in complex advisory processes. Interaction of personality traits with characteristics of the advisory process should be further researched and should be included in a much broader socio-political understanding of what is involved in changing practices.

Start-up financing of professional pest control in pig farming in North Rhine-Westphalia in Germany.

BACKGROUND: Due to the risk of spreading epizootic diseases through rodents, pest control is mandatory in pig farming in European countries. However, there is limited research focused on rodent control practices, usage of anticoagulant rodenticides, and the acceptance of Pest Control Operators (PCOs) in pig farming in Germany. Therefore, the present study aims to investigate current control practices in pig holdings and to analyze the potential of a financial support on the implementation of professional pest control. RESULTS: Data were collected from monitoring records of PCOs and personal interviews with farmers and PCOs. 33 of 47 farmers, who were offered the possibility to outsource rodent control to PCOs supported by financial contribution of the North Rhine-Westphalian Animal Disease Fund (TSK) for a period of 2 years, joined the project.Despite the widespread opinion that the professional would not be beneficial - the authors figured out that farmers could financially benefit in time saved and by improved rodent control measures from the work of the PCOs. Costs of pest control measures per operation on average did not differ significantly between costs incurred by employment of PCOs (1.310 € per year) and calculated costs that arise by farmers themselves (1.217 € per year).All PCOs used Difenacoum and Brodifacoum against pest infestations. In doing so, the infestation with rodents was reduced and most of the participating farmers assessed the project as successful and employ the PCOs permanently. However, mapping the farm locations to resistance areas of the Rodenticide Resistance Action Committee (RRAC) shows that Brodifacoum was frequently used in areas which are marked as areas that are at low risk or rather have no risk for resistance. The environmental risks, however, are increased in these areas. CONCLUSION: The instrument of temporal start-up financing professional pest control allows ensuring the continuous engagement of PCOs after the project period. This could possibly lead to long-term effects on the individual farm hygiene and on disease prevention. Nevertheless, important research questions with regard to the application of anticoagulant rodenticides of farmers and PCOs in livestock farming and with regard to risk mitigation measures were generated, meriting further investigation.

Cell arrest and cell death in mammalian preimplantation development: lessons from the bovine model.

BACKGROUND: The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. METHODS AND FINDINGS: To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. CONCLUSIONS: In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

Highly sensitive saturation labeling reveals changes in abundance of cell cycle-associated proteins and redox enzyme variants during oocyte maturation in vitro.

Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2-D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4-7 images and 1291 spots in pH 6-9 images. Thirty-eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano-LC-MS/MS analysis. The ten spots which could be unambiguously identified include the Ca2+-binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14-3-3 epsilon, elongation factor-1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin-3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.

Leptin promotes meiotic progression and developmental capacity of bovine oocytes via cumulus cell-independent and -dependent mechanisms.

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy.

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.

Maturation of bovine oocytes in the presence of leptin improves development and reduces apoptosis of in vitro-produced blastocysts.

The series of events associated with oocyte growth and maturation determines the oocyte's ability to undergo successful fertilization, cleavage and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. Experiments were conducted to determine whether leptin treatment of oocytes during maturation affects their developmental capacity after fertilization and whether it has long-lasting effects on apoptosis and gene expression in the resulting blastocysts. Cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0 (control), 1, 10, or 100 ng/ml leptin or in medium supplemented with 10% (v/v) estrous cow serum (ECS). Addition of leptin during oocyte maturation had no effect on cleavage rate after fertilization. However, an increased proportion of oocytes that matured in the presence of 1 or 10 ng/ml leptin developed to blastocysts, which exhibited increased cell numbers. The proportion of apoptotic cells was reduced in blastocysts originating from leptin- or ECS-treated oocytes. Transcript levels of the genes encoding leptin receptor (LEPR), signal transducer and activator of transcription (STAT3), BCL2 associated X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP), were determined by reverse transcriptase-quantitative polymerase chain reaction analysis of expanded and hatched blastocysts. Depending on the dose used, leptin treatment of oocytes resulted in increased LEPR, STAT3, and BIRC4 mRNA levels and reduced BAX mRNA levels in blastocysts. In conclusion, leptin improved the ability of the oocyte to sustain embryonic development and had long-term effects on blastocyst apoptosis and transcript abundance of LEPR, STAT3, and apoptosis-associated genes.

Efficient transgenesis in farm animals by lentiviral vectors.

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.