Genome-wide QTL mapping of nine body composition and bone mineral density traits in pigs.

BACKGROUND: Since the pig is one of the most important livestock animals worldwide, mapping loci that are associated with economically important traits and/or traits that influence animal welfare is extremely relevant for efficient future pig breeding. Therefore, the purpose of this study was a genome-wide mapping of quantitative trait loci (QTL) associated with nine body composition and bone mineral traits: absolute (Fat, Lean) and percentage (FatPC, LeanPC) fat and lean mass, live weight (Weight), soft tissue X-ray attenuation coefficient (R), absolute (BMC) and percentage (BMCPC) bone mineral content and bone mineral density (BMD). METHODS: Data on the nine traits investigated were obtained by Dual-energy X-ray absorptiometry for 551 pigs that were between 160 and 200 days old. In addition, all pigs were genotyped using Illumina's PorcineSNP60 Genotyping BeadChip. Based on these data, a genome-wide combined linkage and linkage disequilibrium analysis was conducted. Thus, we used 44 611 sliding windows that each consisted of 20 adjacent single nucleotide polymorphisms (SNPs). For the middle of each sliding window a variance component analysis was carried out using ASReml. The underlying mixed linear model included random QTL and polygenic effects, with fixed effects of sex, housing, season and age. RESULTS: Using a Bonferroni-corrected genome-wide significance threshold of P < 0.001, significant peaks were identified for all traits except BMCPC. Overall, we identified 72 QTL on 16 chromosomes, of which 24 were significantly associated with one trait only and the remaining with more than one trait. For example, a QTL on chromosome 2 included the highest peak across the genome for four traits (Fat, FatPC, LeanPC and R). The nearby gene, ZNF608, is known to be associated with body mass index in humans and involved in starvation in Drosophila, which makes it an extremely good candidate gene for this QTL. CONCLUSIONS: Our QTL mapping approach identified 72 QTL, some of which confirmed results of previous studies in pigs. However, we also detected significant associations that have not been published before and were able to identify a number of new and promising candidate genes, such as ZNF608.

Evaluation of Foot and Claw Lesions and Claw Horn Growth in Piglets from Birth to End of Nursery.

The objective of this study is to evaluate foot lesions and claw horn growth of piglets from the day of birth until the end of the suckling period and describe the prevalence and extent in three different genetic lines. Therefore, bruising, dorsal horn lesions, claw horn growth, and weight gain were evaluated five times during growth, starting with the day of birth (day 0 of life) and ending with the end of nursery (day 68 ± 2 of life). Totally, 74 piglets of three genetic lines (German Landrace × Large White; Piétrain × Piétrain-Duroc; pure German Landrace) were examined. Bruising and dorsal horn lesions reached maximum levels at day 7 (±1) of life (with up to 91% of piglets having bruising marks and up to 94.1% of piglets having dorsal horn lesions). Differences among genetic lines were detected, with German Landrace × Large White crossbreds showing the highest percentage of bruising, but Piétrain × Piétrain-Duroc crossbreds showed the highest score for dorsal horn lesions at day 0. Until weaning (day 28 ± 1), front feet were more affected by bruising than hind feet (70.3% of the front feet and 64% of the hind feet showed bruising), but at the end of nursery (day 68 ± 2), hind feet showed a higher percentage of affected feet than front feet (65.5% vs. 41.3%). Several factors affect bruising scores in piglets, including body weight, age at examination, litter size, sex, parity, breed, and claw horn length. Additionally, significant differences for claw horn length were detected among the genetic lines from birth to end of nursery.

Magnetic Resonance Imaging Used to Define the Optimum Needle Length in Pigs of Different Ages.

Intramuscular injections result in tissue destruction and alteration. Therefore, it is necessary to evaluate the optimum injection point for intramuscular injections. As animals­especially pigs­vary in size and explicit information about injection depth is not available. To determine the predicted optimum injection depth, magnetic resonance imaging was used in pigs of different ages and weight groups. In total, 730 magnetic resonance images of 136 pigs were used to calculate the optimum injection depth for intramuscular injections. Four age groups were evaluated: <29 days of age, 29−70 days of age, 71−117 days of age and >170 days of age. For fattening pigs (71−117 days of age), the present study recommends a needle length of 20 mm (range: 40−58 mm). For younger pigs (<70 days of age), a needle length of 12 to 14 mm (range: 10−18 mm), and for older pigs (>170 days of age), a needle length of 30 mm (range: 25−37 mm) is recommended. However, more data are needed. Therefore, further studies are necessary, especially in the youngest (suckling pigs) and oldest (sows) age groups, as these are the groups mainly injected/vaccinated. Additionally, age and weight should be examined in more detail compared to fat distribution in the neck, genetics and the sex of the animal.

Comparison of American Quarter Horses Competing in Western Pleasure, Hunter under Saddle, and Reining Using Linear Traits.

To investigate differences in American Quarter Horses conformation suggesting specialization and subpopulations within the breed, a total of 45 horses were studied. These horses were classified according to their specific sport discipline: Western Pleasure (WPL, n = 15), Hunter under Saddle (HUS, n = 15), and Reining (RN, n = 15). Fifteen linear traits (comprising lengths, angles, and circumferences) were measured analogically and photometrically. Afterwards, 9 ratios and differences were calculated, so that a total of 24 traits were examined. The results showed significant differences between horses depending on their sport discipline. HUS horses were significant taller and were characterized by higher values in (nearly) all length traits; they were followed by WPL and RN horses. RN horses displayed the lowest values in length traits but the largest difference between height at withers and height at croup. Nine parameters were analyzed through correlations to the height at withers; all differences were significant, with high to moderate correlation coefficients. The detected differences between the groups support recent studies on the conformation and genotype of subpopulations within a breed and reveal new findings in relation to the selected disciplines. Image analysis worked well and provided reliable data; therefore, this method can be used to examine horses in a time-efficient manner, reducing the stress caused to the animal. Further studies are required to gain more information and to associate the features of AQH anatomic structures with successful performance.

Detection of Local Tissue Reactions after Anti-GnRF Injection in Male Pigs Assessed Using Magnetic Resonance Imaging.

This study aimed at evaluating the local tissue reaction of an anti-GnRF product, which is used for the so called "immunocastration" in male pigs. A total of 34 pigs were injected two times (including a booster injection) with an anti-GnRF product. Injection was performed using the prescribed safety vaccinator. Injection sites were evaluated three times post injection using magnetic resonance imaging. Two examinations were performed after the first injection and one after the booster. The local tissue reaction was evaluated three-dimensionally by semi-automatic analyses, by linear measurements of injection depth and length, and by scoring the kind of tissue affected. Results showed a long-lasting reaction in affected muscle tissue. Therefore, needle length should be discussed, and an evaluation of the injection site after slaughter should be performed include behavioural scorings post injection to evaluate the impact on animal well-being and carcass quality.

Lying Behaviour in Dairy Goats: Effects of a New Automated Feeding System Assessed by Accelerometer Technology.

The aim of this study was to evaluate lying behaviour in dairy goats before and after installation of an optimized automatic concentrated feeding system (AFS). A mixed-horned herd of Bunte Deutsche Edelziege was used. As many agonistic interactions between goats happen at the feeding place, a new automated feeding system was installed to better fulfil the needs of horned goats. Lying behaviour is an indicator to ascertain animal welfare of ruminants. In order to measure lying behaviour accelerometer technology was used and verified by video analyses. The results show an agreement of 99.62-99.93% per lying time by comparing accelerometers to video data. Over all goats, a mean ± SD lying time (LT) of 11.78 ± 1.47 h/d, a mean ± SD lying bout duration (LBD) of 0.51 ± 0.10 h/bout and a mean ± SD frequency of lying bouts (FLB) of 24.35 ± 5.57 were found. Lying behaviour follows a seasonal progression with significant lowest LBD and highest FLB in summer. With the old AFS significant differences in LBD and FLB were detected between horned and hornless goats, but with the new AFS results were adjusted. Findings suggest that changes in feeding management do not affect the general seasonal progression of lying behaviour but can affect the behavioural differences between horned and hornless dairy goats.

"Sex" and body region effects on bone mineralization in male pigs.

Lameness in pigs is one of the major reasons for culling and early losses in pigs. This can be linked to osteoporosis due to pathologic alterations in bone mineral density (BMD) or bone mineral content (BMC) and may also be linked to the sex. Dealing with the ban on piglet castration without anaesthesia in Germany 2021, we have three male "sex" types: entire boars (EB), immunocastrated boars (IB), and surgically castrated boars (SB). The hypothesis of the present study is that BMC or BMD varies between different male sex types. If sex has an effect on bone mineralization (BMC or BMD) and if this affects leg health, it could result in more lameness and problems during fattening in the negatively affected sex type. The present study evaluated bone mineralization (in terms of BMD and BMC) and body composition traits using dual-energy X-ray absorptiometry (DXA) three times during growth at 30, 50, and 90 kg live body weight. Nine body regions were analysed for bone mineral traits and compared for different male sex types and the fattening season. Significant differences were found regarding BMD (and BMC) among EB, IB, and SB for whole-body BMD (BMC). Additionally significant differences were found in the front and lower hind limbs, where SB showed a significantly higher BMD compared to EB, with IB in between. Additionally regional differences were detected among the groups. Further studies are needed to evaluate the effect of these differences in bone mineralization on leg health.

Vaccine safety testing using magnetic resonance imaging in suckling pigs.

Safety testing is one major part of the licensing procedure for veterinary vaccines and demands a large number of animals. Magnetic resonance imaging (MRI) was tested as an alternative, which may lead to a reduction in numbers of animals required for safety testing, and, correspondingly to a detailed description of the three-dimensional extent of the local tissue reaction repetitively in live pigs. In previous pig studies the following questions arose:To answer these questions the following study was performed by comparing two vaccine groups of suckling piglets (8 animals per group; A and B) with two control groups (4 animals per group; C and D). One control group was injected with a saline solution (C) and the other was only tattoo marked (D). The animals were examined using MRI at days 1, 8, 15, 22, 29, 36, and 43 post vaccination, ending with a final pathomorphologic examination. Pathomorphologic examination confirmed MRI findings. Saline solution does not result in a local tissue reaction as detected after injecting vaccines. Tattoo marking causes no local tissue reaction, neither in MRI nor in pathomorphologic examination. Therefore, MRI can be used as an alternative method for safety testing of vaccines in pigs of different age categories offering repetitive measurements of local tissue reactions. Involved cells might be examined only in a final pathomorphologic examination at the end of the trial on a reduced number of animals.

Metabolic syndrome and extensive adipose tissue inflammation in morbidly obese Göttingen minipigs.

OBJECTIVE: The worldwide prevalence of obesity has increased to 10% in men and 15% in women and is associated with severe comorbidities such as diabetes, cancer, and cardiovascular disease. Animal models of obesity are central to experimental studies of disease mechanisms and therapeutic strategies. Diet-induced obesity (DIO) models in rodents have provided important insights into the pathophysiology of obesity and, in most instances, are the first in line for exploratory pharmacology studies. To deepen the relevance towards translation to human patients, we established a corresponding DIO model in Göttingen minipigs (GM). METHODS: Young adult female ovariectomized GM were fed a high-fat/high-energy diet for a period of 70 weeks. The ration was calculated to meet the requirements and maintain body weight (BW) of lean adult minipigs (L-GM group) or increased stepwise to achieve an obese state (DIO-GM group). Body composition, blood parameters and intravenous glucose tolerance were determined at regular intervals. A pilot chronic treatment trial with a GLP1 receptor agonist was conducted in DIO-GM. At the end of the study, the animals were necropsied and a biobank of selected tissues was established. RESULTS: DIO-GM developed severe subcutaneous and visceral adiposity (body fat >50% of body mass vs. 22% in L-GM), increased plasma cholesterol, triglyceride, and free fatty acid levels, insulin resistance (HOMA-IR >5 vs. 2 in L-GM), impaired glucose tolerance and increased heart rate when resting and active. However, fasting glucose concentrations stayed within normal range throughout the study. Treatment with a long-acting GLP1 receptor agonist revealed substantial reduction of food intake and body weight within four weeks, with increased drug sensitivity relative to observations in other DIO animal models. Extensive adipose tissue inflammation and adipocyte necrosis was observed in visceral, but not subcutaneous, adipose tissue of DIO-GM. CONCLUSIONS: The Munich DIO-GM model resembles hallmarks of the human metabolic syndrome with extensive adipose tissue inflammation and adipocyte necrosis reported for the first time. DIO-GM may be used for evaluating novel treatments of obesity and associated comorbidities. They may help to identify triggers and mechanisms of fat tissue inflammation and mechanisms preventing complete metabolic decompensation despite morbid obesity.

Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver.

OBJECTIVE: Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. METHODS: CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue. RESULTS: GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2. CONCLUSION: GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.

Magnetic resonance imaging to detect local tissue reactions after vaccination in sheep in vivo.

OBJECTIVES: Vaccination is one of the most effective methods to keep up the health status in humans and in livestock. Therefore, farm animals are vaccinated several times during their lifetime. Although vaccines are being checked regarding their local reactogenicity, side effects occur frequently-especially in the case of the application of adjuvanted products. Many reports exist about local reactions in sheep. The present study aimed at testing MRI as a method to document injection site reactions three-dimensionally. DESIGN: Two groups of Merino lambs (n=16 each) were vaccinated subcutaneously into the left neck side. Two different, licensed inactivated vaccines were used. Both groups of lambs were anaesthetised and scanned using MRI at days 1, 3, 8, 15, 22 and 29 after vaccination. SETTING: The study was performed on a commercial-like farm. PARTICIPANTS: Thirty-two Merino lambs entered the experiment, 16 male and 16 female ones (one animal died at day 22 after vaccination). At first examination day they were approximately three months old. PRIMARY AND SECONDARY OUTCOME MEASURES: Volume differences were measured between vaccination and control neck side to evaluate the time pattern of local tissue reactions. RESULTS: Local tissue reactions were visible on the skin surface and also appeared in deeper tissue layers on MRI. These deeper reactions would not have been found without MRI or, alternatively, without sacrificing the animals. Some of these extensive local reactions lasted for more than 29 days. CONCLUSIONS: The in vivo MRI results proved suitable to record local tissue reactions in terms of three-dimensional extent over a longer period of time in large farm animals without the need to sacrifice test animals. A three-dimensional MRI examination of the injection site during regulatory licensing studies offers an objective evaluation that could be used in a benefit-risk assessment of veterinary vaccines. TRIAL REGISTRATION NUMBER: District Government of Upper Bavaria:55.2-1-54-2532-2-13.

Assessment of local reaction to vaccines in live piglets with magnetic resonance imaging compared to histopathology.

The safety of veterinary vaccines is assessed in clinical trials in Europe. The assessment of the local tissue reaction to vaccination by magnetic resonance imaging (MRI) could reduce the number of animals needed because repeated examinations can be performed in the same animal over time. The present study compared the evaluation of local tissue reactions to vaccination using MRI in live pigs with histopathology of porcine tissue, the current gold standard in regulatory safety testing. Eight piglets each were administered one of two commercial vaccines into marked injection sites. All animals were sedated and scanned repeatedly by MRI using a contrast agent up to day 29 after vaccination. On day 29, the animals were euthanized and underwent a pathological examination. The MRI results were compared with the pathomorphological findings at the injection site by regression analysis. The MR images and the pathological examinations yielded matching results concerning the sizes of the affected tissue volumes or areas. The use of MRI for regulatory safety testing can reduce the number of animals needed to 8 per examination group. The volume of a local reaction and its progression over time can be evaluated and documented. If persistent lesions develop a final pathomorphological examination is needed to identify the kind and local distribution of the reaction.

Safety testing of veterinary vaccines using magnetic resonance imaging in pigs.

Safety testing of veterinary vaccines requires the use of a large number of animals to investigate possible local and systemic reactions. This includes amongst others the pathological examination of the injection site in frequent intervals. For this a selected killing of animals in frequent intervals is inevitable. To reduce the number of animals needed for this kind of safety testing, magnetic resonance imaging (MRI) was used to detect and quantify possible inflammatory reactions after vaccination in vivo. Sixty four pigs were subdivided into 4 experimental groups (n=16); two groups consisting of 12 weeks old pigs and 2 of 6 month old pigs at vaccination day. The pigs were vaccinated with four licensed products (each group receiving one vaccine) and examined up to 6 times using MRI during a period of 5 weeks. The MRI images were evaluated semi-automatically comparing the volumes of altered signal intensities at the vaccination side (VS) with the volumes of the signal intensities at the control side (CS). A paired t-Test was used to identify significant differences (p<0.05) between VS and CS. The results show that MRI allows a 3D-quantification of the extent of local reactions in vivo, scanning the same animals at several points of time after vaccination. MRI is a suitable alternative method for non-invasive safety testing of injectable medicines and can therefore be used as an alternative method to reduce animal numbers for safety testing purposes.