The Estimation of Genetic Parameters for Chronic Progressive Lymphedema and Body Traits in the Rhenish German Draught Horse.

Chronic progressive lymphedema (CPL) is a prevalent and progressive disease in Rhenish German draught horses. The objective of our follow-up study was to evaluate the heritability of this disease in Rhenish German draught horses using pedigree-based and genomic relationship matrices. We employed linear and threshold animal models. Models included the random animal effect and effects of breeding association, coat colour, sex, and age within sex, and farm-related factors, on CPL scores. In addition, we estimated heritabilities in models assuming censoring for data when horses were below an age of 1-15 years. The heritabilities of CPL scores across all ages were 0.595 ± 0.131 and 0.482 ± 0.105 in the threshold and linear animal model with pedigree-based relationship matrices, respectively. The restriction of data to horses with a minimum age at examination or accounting for censored data in younger animals showed an increase in heritabilities of CPL scores up to 0.788 ± 0.168 (threshold model) and 0.752 ± 0.153 (linear model) at an age of 7-8 years. Analyses including genomic relationship matrices yielded very similar estimates, but with smaller standard errors than pedigree-based analyses. Heritabilities in threshold models for CPL prevalence (CPL-bin-score) and the number of affected limbs (CPL-bin-sum) were 0.176-0.189 ± 0.061-0.064 and 0.375-0.433 ± 0.164-0.170, respectively. We were able to show moderately to highly positive genetic correlations between the CPL score and cannon bone circumference (0.529-0.825), height at withers (0.338-0.555), and skinfold thickness (0.241-0.517). Using the dichotomous trait for the CPL score and the genomic relationship matrix resulted in corresponding estimates of 0.868, 0.793, and 0.784, respectively. This study showed the great importance of additive genetic variation influencing the expression of chronic progressive lymphedema in Rhenish German draught horses. Therefore, further research is warranted to implement breeding programmes in a small breeding population that exploit the potential of additive genetic differences among animals for reducing the prevalence and severity of lesions of this incurable disease.

Genotyping KIF1C (c.608G>A) Mutant Reveals a Wide Distribution of Progressive Ataxia in German Charolais Cattle.

Bovine progressive ataxia in Charolais cattle was first described in the 1970s; then, cases were reported in Charolais worldwide. A homozygous loss-of-function mutation within the KIF1C gene (c.608G>A) was found to be responsible for this neurodegenerative disease. The aim of this study was to determine whether the mutated KIF1C allele segregates in the German Charolais population and whether the estimated breeding values for growth and muscle conformation are associated with the mutated genotypes. Genetic test results of the KIF1C:c.608G>A variant were available for 1315 Charolais cattle from 35 herds located in Germany. In addition, 324 samples from eight other beef cattle breeds were tested for the mutated KIF1C allele. We were able to demonstrate that the KIF1C mutation is common, with a frequency of 11.75% in the German Charolais population. All but two of the eight (2/8 = 25%) homozygous mutated individuals showed clinical signs consistent with progressive ataxia. The estimated breeding values of muscle conformation in 200- and 365-day-old animals indicated a significant superiority for homozygous mutated animals when compared either with heterozygous or homozygous wild-type genotypes; this was also the case for heterozygous genotypes in comparison with homozygous wild-type genotypes. For the estimated breeding values of daily weight gain in 200- and 365-day-old animals, the significant differences between homozygous mutated and heterozygous or wild-type genotypes were in favour of the homozygous mutant animals. There were no differences in the estimated maternal breeding values among all three KIF1C genotypes. For the first time, two German Angus cattle carrying the KIF1C mutation heterozygous were detected. The breeders' survey highlighted that increased awareness would facilitate increased conviction among breeders of the need for genetic testing in order to eliminate the lethal KIF1C allele.

Radiological Methods for the Imaging of Congenital Malformations of C6-T1, the First and Second Sternal Ribs and Development of a Classification System, Demonstrated in Warmblood Horses.

There are conflicting data in studies on malformations of the cervicothoracic (C-T) junction (C6 to T2, including the first and second ribs), but evidence is mounting that they can be of clinical significance for horses. The objectives of this study were to establish a radiographic protocol for imaging the C-T junction in the field and to classify the radiographic variations found in 39 warmblood horses presented for clinical evaluation due to behavioral or performance issues. Malformations of the ventral lamina of C6 and transposition onto the ventral aspect of C7 were seen in 37/39 (94.9%) horses for both conditions. Rib anomalies were found among the horses with C6 and C7 malformations. A missing first rib, unilateral or bilateral, was found in 3/35 (11.4%) horses, a unilateral shortening of the rib in 17/35 (48.6%) horses, a bilateral shortening of the ribs in 12/35 (34.3%) horses, bifid ribs in 3/35 (8.6%) horses, and only 4/35 (11.4%) horses had a normal length of the first rib on both sides. There was a moderately to highly significant association between the grades of left and right malformations of C6 and C7 and first ribs as well as between C6 and C7. A large number of malformations were visualized radiographically at the C-T junction using the newly described methods of latero-lateral and oblique radiographic projections, which allows for these features to be identified in living horses.

Single Nucleotide Polymorphisms Associated with AA-Amyloidosis in Siamese and Oriental Shorthair Cats.

AA-amyloidosis in Siamese and Oriental shorthair cats is a lethal condition in which amyloid deposits accumulate systemically, especially in the liver and the thyroid gland. The age at death of affected cats varies between one and seven years. A previous study indicated a complex mode of inheritance involving a major locus. In the present study, we performed a multi-locus genome-wide association study (GWAS) using five methods (mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB and ISIS EM-BLASSO) to identify variants associated with AA-amyloidosis in Siamese/Oriental cats. We genotyped 20 affected mixed Siamese/Oriental cats from a cattery and 48 healthy controls from the same breeds using the Illumina Infinium Feline 63 K iSelect DNA array. The multi-locus GWAS revealed eight significantly associated single nucleotide polymorphisms (SNPs) on FCA A1, D1, D2 and D3. The genomic regions harboring these SNPs contain 55 genes, of which 3 are associated with amyloidosis in humans or mice. One of these genes is SAA1, which encodes for a member of the Serum Amyloid A family, the precursor protein of Amyloid A, and a mutation in the promotor of this gene causes hereditary AA-amyloidosis in humans. These results provide novel knowledge regarding the complex genetic background of hereditary AA-amyloidosis in Siamese/Oriental cats and, therefore, contribute to future genomic studies of this disease in cats.

Effect of Individual Rate of Inbreeding, Recent and Ancestral Inbreeding on Wool Quality, Muscling Conformation and Exterior in German Sheep Breeds.

This study provides comprehensive results on the current status of inbreeding depression for traits upon which sheep are selected for the herdbook in Germany. A total of 30 sheep breeds from the OviCap national database met the inclusion criteria for the present analysis regarding the depth and completeness of pedigrees and the number of animals with phenotypic data. We analyzed heritabilities and inbreeding depression for the three breeding objective traits of wool quality, muscling conformation and exterior. Heritabilities were across all breeds of moderate size, with estimates of 0.18 for wool quality and muscling conformation and of 0.14 for exterior. The models employed to estimate linear regression slopes for individual and ancestral inbreeding rates also account for non-genetic effects and the additive genetic effect of the animal. Inbreeding depression was obvious for all three traits when we averaged the estimates across all 30 sheep breeds. Inbreeding depression was significant for wool quality for only a few breeds, whereas for muscling conformation, 14/30 breeds achieved significant estimates. A 1% increase in inbreeding decreased the mean of all three traits across all sheep breeds by 0.33% of their standard deviation. Positive effects due to ancestral inbreeding were only significant in very few breeds in the different traits. Across all 30 sheep breeds, there were indications that purging effects (a reduction in negative effects of inbreeding depression due to selection for heterozygotes) may play a role for the exterior. The results of this study should help for reviewing breeding programs, particularly for sheep breeds with critical effective population sizes and increasing rates of inbreeding, with regard to the selection policy and selection intensity applied.

Historic Horse Family Displaying Malformations of the Cervicothoracic Junction and Their Connection to Modern German Warmblood Horses.

Malformations of the equine cervicothoracic junction affect the C6 and C7 cervical vertebrae, the T1 thoracic vertebra and in variable extent the first and second sternal ribs. To date, the clinical impact of this malformation, its prevalence and mode of inheritance in equine populations are not yet determined. We examined five skeletons for signs of malformation of the cervicothoracic junction, including three skeletons from widely used Thoroughbred stallions affected with the malformation and two skeletons serving as a comparison. The three affected historical horses were the Thoroughbred stallions Der Loewe XX, Birkhahn XX and their common great grandsire Dark Ronald XX. Malformations of C6 and C7 showed a large variation between the three stallions, as Dark Ronald XX, Der Loewe XX and Birkhahn XX were affected uni-laterally at C6 and C7, uni-laterally at C6 and bi-laterally at C6 and C7, respectively, with varying grades. In order to evaluate whether or not these malformations are incidental, we took a random sample of 20 living German Warmblood horses, which are distant descendants of these stallions. This sample consisted of ten controls and ten horses with malformations of C6/C7. Blood proportions of the historical sires in the modern Warmblood horses ranged from 0.10 to 6.25%. The contribution to inbreeding in each individual horse of our selected horse group by those sires was expressed as a percentage of the total inbreeding coefficient and ranged from 0.01 to 17.96%, demonstrating their influence on the modern Warmblood. In the present study, we were able to describe the variability of the malformation of C6/C7 within a horse family including historic and modern horses. Additionally, we detected variations appearing in connection with malformations of the cervicothoracic junction that have not been described in the literature yet. This is the first time that the malformations of C6 and C7 have been described within a familial context, providing hints on inheritance in Thoroughbreds and Warmbloods. It is worthwhile to carry out further studies in a larger setting to gain more comprehensive insights into the inheritance of the malformation and the role of important ancestors.

Inbreeding Depression and Purging for Meat Performance Traits in German Sheep Breeds.

This study provides estimates on genetic parameters, inbreeding depression and purging for meat performance measures from 25 German sheep breeds. All German meat, merino sheep breeds and breeds of other breeding directions with a sufficient number of pedigree and performance data were included in this study. Phenotypic traits retrieved from the national database OviCap were evaluated: daily weight gain, meatiness score and ultrasound measurements for muscle and fat thickness. We employed animal models to estimate heritability, variance and covariance components for these meat performance traits as well as inbreeding depression and purging. The heritabilities, on average, reached estimates of 0.55, 0.34, 0.53 and 0.61 for daily weight gain, meatiness score and ultrasound measurements for muscle and fat thickness, respectively. We estimated the linear regression slopes for the individual rate of inbreeding, new and ancestral inbreeding, as well as the inbreeding coefficient and its interaction with the inbreeding coefficient of Ballou, employing animal models with non-genetic effects and the additive genetic effect of the animal. Across all breeds, inbreeding was only significant for daily weight gain, whereas for all other traits, estimates were not significant. Within sheep breeds, we found significant inbreeding depression for daily weight gain in German Mutton Merino and German Blackheaded Mutton as well as for the meatiness score in German Whiteheaded Mutton. Significant effects for purging, based on ancestral inbreeding and the interaction effect of the classical inbreeding coefficient with the inbreeding coefficient of Ballou, were not obvious either across or within any sheep breed. A 1% increase in inbreeding significantly decreased the phenotypic trait median of daily weight gain across all sheep breeds by 0.50% and 0.70% of phenotypic and genetic standard deviation, respectively. Purging effects due to ancestral inbreeding were not significant in any breed or across breeds. The results of this study may indicate that inbreeding depression may be more harmful in traits under stronger selection than in traits that exert low selection pressure. The results of this study demonstrate the different effects that result in meat performance traits due to inbreeding. With increasing rates of inbreeding and critical effective population sizes, selection intensity for breeding objectives has to be critically reviewed for each sheep breed. Inbreeding depression and purging should be evaluated in order to prevent a decrease in trait means due to inbreeding and to determine whether detrimental alleles are eliminated.

Impact of Inbreeding and Ancestral Inbreeding on Longevity Traits in German Brown Cows.

A recent study on the population structure of the German Brown population found increasing levels of classical and ancestral inbreeding coefficients. Thus, the aim of this study was to determine the effects of inbreeding depression and purging on longevity traits using classical and ancestral inbreeding coefficients according to Kalinowski (2002) (Fa_Kal, FNew), Ballou (1997) (Fa_Bal), and Baumung (2015) (Ahc). For this purpose, uncensored data of 480,440 cows born between 1990 and 2001 were available. We analyzed 17 longevity traits, including herd life, length of productive life, number of calvings, lifetime and effective lifetime production for milk, fat, and protein yield, the survival to the 2nd, 4th, 6th, 8th, and 10th lactation number, and the culling frequencies due to infertility, or udder and foot and leg problems. Inbreeding depression was significant and negative for all traits but for culling due to udder and to foot and leg problems. When expressed in percentages of genetic standard deviations, inbreeding depression per 1% increase in inbreeding was -3.61 to -10.98%, -2.42 to -2.99%, -2.21 to -4.58%, and 5.13% for lifetime production traits, lifetime traits, survival rates, and culling due to infertility, respectively. Heterosis and recombination effects due to US Brown Swiss genes were positive and counteracted inbreeding depression. The effects of FNew were not significantly different from zero, while Fa_Kal had negative effects on lifetime and lifetime production traits. Similarly, the interaction of F with Fa_Bal was significantly negative. Thus, purging effects could not be shown for longevity traits in German Brown. A possible explanation may be seen in the breed history of the German Brown, that through the introgression of US Brown Swiss bulls ancestral inbreeding increased and longevity decreased. Our results show, that reducing a further increase in inbreeding in mating plans is advisable to prevent a further decline in longevity due to inbreeding depression, as purging effects were very unlikely in this population.

SNP-Based Heritability of Osteochondrosis Dissecans in Hanoverian Warmblood Horses.

Before the genomics era, heritability estimates were performed using pedigree data. Data collection for pedigree analysis is time consuming and holds the risk of incorrect or incomplete data. With the availability of SNP-based arrays, heritability can now be estimated based on genotyping data. We used SNP array and 1.6 million imputed genotype data with different minor allele frequency restrictions to estimate heritabilities for osteochondrosis dissecans in the fetlock, hock and stifle joints of 446 Hanoverian warmblood horses. SNP-based heritabilities were estimated using a genomic restricted maximum likelihood (GREML) method and accounting for patterns of regional linkage disequilibrium in the equine genome. In addition, we employed GREML for family data to account for different degrees of relatedness in the study population. Our results indicate that we were able to capture a larger proportion of additive genetic variance compared to pedigree-based estimates in the same population of Hanoverian horses. Heritability estimates on the linear scale for fetlock-, hock- and stifle-osteochondrosis dissecans were 0.41-0.43, 0.62-0.63, and 0.23-0.25, respectively, with standard errors of 0.11-0.14. Accounting for linkage disequilibrium patterns had an upward effect on the imputed data and a downward impact on the SNP array genotype data. GREML for family data resulted in higher heritability estimates for fetlock-osteochondrosis dissecans and slightly higher estimates for hock-osteochondrosis dissecans, but had no effect on stifle-osteochondrosis dissecans. The largest and most consistent heritability estimates were obtained when we employed GREML for family data with genomic relationship matrices weighted through patterns of regional linkage disequilibrium. Estimation of SNP-based heritability should be recommended for traits that can only be phenotyped in smaller samples or are cost-effective.

Prevalence of Chronic Progressive Lymphedema in the Rhenish German Draught Horse.

Chronic progressive lymphedema (CPL) is a common disease in Rhenisch German draught horses. The objective of our study was to evaluate the prevalence of this disease and its progression with age in 493 Rhenish German draught horses from different regions in Germany. We employed generalized linear models with binomial, multinomial, and normal distributions to analyse the effects of breeding association, coat colour, sex and age within sex, time of examination, limb on which CPL was recorded, and farm-related factors on disease scores. The occurrence and severity of CPL were significantly influenced by breeding area, sex, coat colour, and interaction of age by sex. Males had significantly higher CPL-scores than geldings (1.92-fold) and females (5.02-fold) as well as a faster (1.85-fold) disease progression per year of life than females (1.44-fold) and geldings (1.25-fold). Regression on age within sex was still significant when age was restricted to a minimum of 7 years in the analysis. Bay horses exhibited 1.77-fold and 2.19-fold higher CPL-scores than chestnut and black horses. Keeping horses on pasture and keeping bedding clean reduced CPL-scores, whereas feeding of hay silage and concentrates during winter increased CPL-scores. Our analysis revealed significant regression coefficients of cannon bone circumference on CPL-scores. Relationships with hoof measures and skinfold diameter at the neck were not found. In conclusion, differences among breeding districts and sexes by age had the largest impact on CPL prevalence and scores. Disease progression was evident up to a mean age of 16 years in males and 20 years in females. The identification of risk factors and their effects on CPL should help to reduce the occurrence and progression of CPL.

Genetic Diversity and Trends of Ancestral and New Inbreeding in German Sheep Breeds by Pedigree Data.

In Germany, many autochthonous sheep breeds have developed, adapted to mountain, heath, moorland, or other marginal sites, but breeds imported from other countries have also contributed to the domestic breeds, particularly improving wool and meat quality. Selective breeding and the intense use of rams may risk losing genetic diversity and increasing rates of inbreeding. On the other hand, breeds with a low number of founder animals and only regional popularity may not leave their endangered status, as the number of breeders interested in the breed is limited. The objective of the present study was to determine demographic measures of genetic diversity and recent as well as ancestral trends of inbreeding in all autochthonous German sheep breeds and sheep of all breeding directions, including wool, meat, and milk. We used pedigree data from 1,435,562 sheep of 35 different breeds and a reference population of 981,093 sheep, born from 2010 to 2020. The mean number of equivalent generations, founders, effective founders, effective ancestors, and effective founder genomes were 5.77, 1669, 123.2, 63.5, and 33.0, respectively. Genetic drift accounted for 69% of the loss of genetic diversity, while loss due to unequal founder contributions was 31%. The mean inbreeding coefficient, individual rate of inbreeding (∆Fi), and realized effective population size across breeds were 0.031, 0.0074, and 91.4, respectively, with a significantly decreasing trend in ∆Fi in 11/35 breeds. New inbreeding, according to Kalinowski, contributed to 71.8% of individual inbreeding, but ancestral inbreeding coefficients showed an increasing trend in all breeds. In conclusion, in our study, all but one of the mountain-stone sheep breeds and the country sheep breed Wald were the most vulnerable populations, with Ne < 50. The next most endangered breeds are exotic, country, and heath breeds, with average Ne of 66, 83, and 89, respectively. The wool, meat, and milk breeds showed the highest genetic diversity, with average Ne of 158, 120, and 111, respectively. The results of our study should help strengthen conservation program efforts for the most endangered sheep breeds and maintain a high genetic diversity in all sheep breeds.

A Missense Mutation in the Collagen Triple Helix of EDA Is Associated with X-Linked Recessive Hypohidrotic Ectodermal Dysplasia in Fleckvieh Cattle.

Mutations within the ectodysplasin A (EDA) gene have been associated with congenital hypotrichosis and anodontia (HAD/XHED) in humans, mice, dogs and cattle. We identified a three-generation family of Fleckvieh cattle with male calves exhibiting clinical and histopathological signs consistent with an X-linked recessive HAD (XHED). Whole genome and Sanger sequencing of cDNA showed a perfect association of the missense mutation g.85716041G>A (ss2019497443, rs1114816375) within the EDA gene with all three cases following an X-linked recessive inheritance, but normal EDAR and EDARADD. This mutation causes an exchange of glycine (G) with arginine (R) at amino acid position 227 (p.227G>R) in the second collagen triple helix repeat domain of EDA. The EDA variant was associated with a significant reduction and underdevelopment of hair follicles along with a reduced outgrowth of hairs, a complete loss of seromucous nasolabial and mucous tracheal and bronchial glands and a malformation of and reduction in number of teeth. Thermostability of EDA G227R was reduced, consistent with a relatively mild hair and tooth phenotype. However, incisors and canines were more severely affected in one of the calves, which correlated with the presence of a homozygous missense mutation of RNF111 (g.51306765T>G), a putative candidate gene possibly associated with tooth number in EDA-deficient Fleckvieh calves.

Tracking footprints of artificial and natural selection signatures in breeding and non-breeding cats.

Stray non-breeding cats (stray) represent the largest heterogeneous cat population subject to natural selection, while populations of the Siamese (SIAM) and Oriental Shorthair (OSH) breeds developed through intensive artificial selection for aesthetic traits. Runs of homozygosity (ROH) and demographic measures are useful tools to discover chromosomal regions of recent selection and to characterize genetic diversity in domestic cat populations. To achieve this, we genotyped 150 stray and 26 household non-breeding cats (household) on the Illumina feline 63 K SNP BeadChip and compared them to SIAM and OSH. The 50% decay value of squared correlation coefficients (r2) in stray (0.23), household (0.25), OSH (0.24) and SIAM (0.25) corresponded to a mean marker distance of 1.12 Kb, 4.55 Kb, 62.50 Kb and 175.07 Kb, respectively. The effective population size (Ne) decreased in the current generation to 55 in stray, 11 in household, 9 in OSH and 7 in SIAM. In the recent generation, the increase in inbreeding per generation (ΔF) reached its maximum values of 0.0090, 0.0443, 0.0561 and 0.0710 in stray, household, OSH and SIAM, respectively. The genomic inbreeding coefficient (FROH) based on ROH was calculated for three length categories. The FROH was between 0.014 (FROH60) and 0.020 (FROH5) for stray, between 0.018 (FROH60) and 0.024 (FROH5) for household, between 0.048 (FROH60) and 0.069 (FROH5) for OSH and between 0.053 (FROH60) and 0.073 (FROH5) for SIAM. We identified nine unique selective regions for stray through genome-wide analyses for regions with reduced heterozygosity based on FST statistics. Genes in these regions have previously been associated with reproduction (BUB1B), motor/neurological behavior (GPHN, GABRB3), cold-induced thermogenesis (DIO2, TSHR), immune system development (TSHR), viral carcinogenesis (GTF2A1), host immune response against bacteria, viruses, chemoattractant and cancer cells (PLCB2, BAHD1, TIGAR), and lifespan and aging (BUB1B, FGF23). In addition, we identified twelve unique selective regions for OSH containing candidate genes for a wide range of coat colors and patterns (ADAMTS20, KITLG, TYR, TYRO3-a MITF regulator, GPNMB, FGF7, RAB38) as well as congenital heart defects (PDE4D, PKP2) and gastrointestinal disorders (NLGN1, ALDH1B1). Genes in stray that represent unique selective events indicate, at least in part, natural selection for environmental adaptation and resistance to infectious disease, and should be the subject of future research. Stray cats represent an important genetic resource and have the potential to become a research model for disease resistance and longevity, which is why we recommend preserving semen before neutering.

Risk factors associated with the infection of sheep with Dichelobacter nodosus.

Ovine footrot is a highly contagious foot disease caused by the gram-negative bacterium Dichelobacter nodosus (D. nodosus). In a recent report, we showed a prevalence of 42.9% D. nodosus positive swabs across Germany. In this follow-up study, we used real-time PCR results for D. nodosus and footrot scores of 9297 sheep from 208 flocks and collated these data with survey data on herd and animal characteristics and herd management. The aims of the present study were to investigate herd and animal factors associated with D. nodosus infection and footrot scores in individual sheep. Multivariable analyses with generalized mixed models showed that month of recording, breed, herdbook membership, use of antibiotics, and footbaths in the past 3-10 years, signs of footrot in the past 12 months and flock environment of the sheep, modelled as a random farm effect within region, were significant risk factors. Among the 21 different breeds, Romney had the lowest risk of D. nodosus infection, while Swifter had the highest risk and German Merino and German White Heath were the next breeds at highest risk of D. nodosus infection. The variance between farms in the prevalence of D. nodosus was large and accounted for 84% of the total variance in the mixed model analysis. We conclude that specific and as yet unknown effects influencing D. nodosus infections in flocks, as well as breed and weather, are the most important effects on D. nodosus infection in sheep, pointing towards the need to establish adequate infection control at farm level.

A comparison of strategies for generating artificial replicates in RNA-seq experiments.

Due to the overall high costs, technical replicates are usually omitted in RNA-seq experiments, but several methods exist to generate them artificially. Bootstrapping reads from FASTQ-files has recently been used in the context of other NGS analyses and can be used to generate artificial technical replicates. Bootstrapping samples from the columns of the expression matrix has already been used for DNA microarray data and generates a new artificial replicate of the whole experiment. Mixing data of individual samples has been used for data augmentation in machine learning. The aim of this comparison is to evaluate which of these strategies are best suited to study the reproducibility of differential expression and gene-set enrichment analysis in an RNA-seq experiment. To study the approaches under controlled conditions, we performed a new RNA-seq experiment on gene expression changes upon virus infection compared to untreated control samples. In order to compare the approaches for artificial replicates, each of the samples was sequenced twice, i.e. as true technical replicates, and differential expression analysis and GO term enrichment analysis was conducted separately for the two resulting data sets. Although we observed a high correlation between the results from the two replicates, there are still many genes and GO terms that would be selected from one replicate but not from the other. Cluster analyses showed that artificial replicates generated by bootstrapping reads produce it p values and fold changes that are close to those obtained from the true data sets. Results generated from artificial replicates with the approaches of column bootstrap or mixing observations were less similar to the results from the true replicates. Furthermore, the overlap of results among replicates generated by column bootstrap or mixing observations was much stronger than among the true replicates. Artificial technical replicates generated by bootstrapping sequencing reads from FASTQ-files are better suited to study the reproducibility of results from differential expression and GO term enrichment analysis in RNA-seq experiments than column bootstrap or mixing observations. However, FASTQ-bootstrapping is computationally more expensive than the other two approaches. The FASTQ-bootstrapping may be applicable to other applications of high-throughput sequencing.

Genetic Diversity and Trends of Ancestral and New Inbreeding in Deutsch Drahthaar Assessed by Pedigree Data.

Loss of genetic diversity and high inbreeding rates confer an increased risk of congenital anomalies and diseases and thus impacting dog breeding. In this study, we analyzed recent and ancestral inbreeding as well as other measures of genetic variability in the Deutsch Drahthaar (DD) dog population. Analyses included pedigree data from 101,887 animals and a reference population with 65,927 dogs born between 2000 and 2020. The mean equivalent complete generations was 8.6 with 69% known ancestors in generation 8. The mean realized effective population size was 92 with an increasing trend from 83 to 108 over birth years. The numbers of founders, effective founders and effective ancestors, as well as founder genomes, were 814, 66, 38 and 16.15, respectively. Thirteen ancestors explained 50% of the genetic diversity. The mean coefficient of inbreeding and individual rate of inbreeding (ΔFi) were 0.042 and 0.00551, respectively, with a slightly decreasing trend in ΔFi. Exposure of ancestors to identical-by-descent alleles explored through ancestral coefficients of inbreeding showed a strong increasing trend. Comparisons between new and ancestral inbreeding coefficients according to Kalinowski et al. showed an average relative contribution of 62% of new inbreeding to individual inbreeding. Comparisons among average coancestry within the parental population and average inbreeding in the reference population were not indicative of genetic substructures. In conclusion, the creation of the DD dog breed about 120 years ago resulted in a popular breed with considerable genetic diversity without substructuring into lines or subpopulations. The trend of new inbreeding was declining, while ancestral inbreeding through ancestors who were autozygous at least once in previous generations was increasing.

Serological Diversity of Dichelobacter nodosus in German Sheep Flocks.

Footrot is one of the major causes of lameness in sheep and leads to decreased animal welfare and high economic losses. The causative agent is the Gram-negative anaerobic bacterium Dichelobacter nodosus. The prevalence of D. nodosus in 207 sheep flocks across Germany was 42.9%. Based on the sequence variation in the type IV fimbrial gene fimA, D. nodosus can be subdivided into ten serogroups (A-I and M). There are commercially available vaccines covering nine serogroups, but the efficacy is low compared to bivalent vaccines. The aim of this study was to investigate the diversity of serogroups in Germany at the flock and animal levels. In total, we detected at least one serogroup in 819 samples out of 969 D. nodosus-positive samples from 83 flocks using serogroup-specific singleplex PCR for the serogroups A-I. Serogroup A was most prevalent at the animal level, followed by serogroups B, H and C. At the flock level, serogroups A and B had the highest prevalence, each with 64%, but only 40% of flocks had both. The average number of serogroups per animal was 1.42 (range one to five) and, per flock, 3.10 (range one to six). The serogrouping showed within-flock specific clusters but were widely distributed, with 50 different combinations across the flocks. The factors associated with the number of serogroups per animal and single serogroups were the load of D. nodosus, footrot score, sheep breed and flock. Our results indicate that efficient vaccination programs would benefit from tailor-made flock-specific vaccines and regular monitoring of circulating serotypes in the flock to be able to adjust vaccine formulations for nationwide progressive control of footrot in Germany.

Prevalence and segregation analysis of dermoid sinus in Rhodesian Ridgebacks.

Rhodesian Ridgebacks are dogs with a characteristic dorsal hair ridge with backward-growing hair. Dermoid sinus was reported as a prevalent congenital condition in ridged dogs and as a sporadic finding in other dog breeds. This condition presents as a tubular skin indentation to variable degree into underlying tissues on the dorsal midline or cranial or caudal to the ridge. In the present study, data from 12,700 puppies born to 1622 litter from Rhodesian Ridgebacks in 2001-2019 in Germany were analysed for the prevalence of dermoid sinus and ridgless animals. Data from litters with dogs segregating for dermoid sinus were used to test compatibility with an autosomal recessive Mendelian trait. Overall prevalence of dermoid sinus in 12,700 puppies was 2.53%. In 1269 litter with only ridged animals, prevalence was 2.81%. In 46 litters, segregation of ridgeless dogs was observed, and two ridgeless animals had dermoid sinus. The overall trend for dermoid sinus prevalence significantly decreased by -0.099% per birth year, whereas a significant trend for ridgelessness was not found. A more complex genetic determination for dermoid sinus seems likely, due to the insufficient fit of a monogenic autosomal recessive model of inheritance and a heritability estimate of 0.78 ± 0.11. Genetic correlation with ridgelessness was -0.11. In conclusion, our data revealed a decreasing trend for the prevalence of dermoid sinus and a complex genetic basis for this condition. The ridge locus was unlikely to determine the expression of dermoid sinus, given the low segregation ratio for dermoid sinus in a population selected for the ridge allele.

AA-amyloidosis in captive northern tree shrews (Tupaia belangeri).

A high prevalence of AA-amyloidosis was identified in a breeding colony of northern tree shrews (Tupaia belangeri) in a retrospective analysis, with amyloid deposits in different organs being found in 26/36 individuals (72%). Amyloid deposits, confirmed by Congo red staining, were detected in kidneys, intestines, skin, and lymph nodes, characteristic of systemic amyloidosis. Immunohistochemically, the deposited amyloid was intensely positive with anti-AA-antibody (clone mc4), suggesting AA-amyloidosis. The kidneys were predominantly affected (80%), where amyloid deposits ranged from mild to severe and was predominantly located in the renal medulla. In addition, many kidneys contained numerous cysts with atrophy of the renal parenchyma. There was no significant association between concurrent neoplastic or inflammatory processes and amyloidosis. The lack of distinctive predisposing factors suggests a general susceptibility of captive T. belangeri to develop amyloidosis. Clinical and laboratory findings of a female individual with pronounced kidney alterations were indicative of renal failure. The observed tissue tropism with pronounced kidney alterations, corresponding renal dysfunction, and an overall high prevalence suggests amyloidosis as an important disease in captive tree shrews.