Genomic inbreeding coefficients using imputation genotypes: assessing the effect of ancestral genotyping in Holstein-Friesian dairy cows.

The objective of this study was to assess the effect of using or not the genotypes of the parents of a cow for imputing single nucleotide polymorphisms (SNP), on the estimation of genomic inbreeding coefficients of cows. Imputation (i.e., genotyped plus imputed) genotypes from 68,127 Italian Holstein dairy cows registered in the Italian National Association of Holstein, Brown and Jersey Breeders (ANAFIBJ) were analyzed. Cows were genotyped with the HD Illumina Infinium BovineHD BeadChip and GeneSeek Genomic Profiler HD-150K, and the MD GeneSeek Genomic Profiler 3, GeneSeek Genomic Profiler 4, GeneSeek MD and the Labogena MD. To assess differences among estimators genomic inbreeding coefficients were estimated with 4 PLINK v1.9 estimators (F, Fhat1, 2, 3), 2 genomic relationship matrix (grm) based estimators (Fgrm and Fgrm2; with the latter including also pedigree information) and one estimator of runs of homozygosity (ROH; FROH). Assuming that the correct genomic inbreeding coefficients should be those estimated from genotyped SNP, a comparison of the genomic inbreeding coefficients estimated either with the genotyped SNP or the SNP after imputation was made. Information on the presence or absence of genotypic information from sire, dam and maternal grandsire during the imputation was investigated. Genomic inbreeding coefficients estimated with genotyped SNP or SNP after imputation were consistent for F, Fhat3, Fgrm2 and FROH, when at least one of the parents was genotyped. Biased (mainly higher) genomic inbreeding coefficients of imputation SNP were observed in cows that were genotyped with MD SNP panels whose SNP were poorly represented in the selected imputation SNP data set and also did not have their parents genotyped compared with what expected based on actual genotype data. For cows genotyped with MD the estimators Fhat1, Fhat2 and Fgrm provided higher genomic inbreeding coefficients of imputation SNP even with both parents and the maternal grandsire genotyped. Overall, FROH was the most robust estimator, followed by F and Fhat3. Our findings suggest that SNP selection, parental genotyping and estimator should be considered for designing imputation strategies in dairy cattle for estimating genomic inbreeding with imputation SNP. For computing genomic inbreeding coefficients, it is recommendable to have at least one parent genotyped and use an ROH based estimator.

Microsatellite imputation using SNP data for parentage verification in four Italian sheep breeds.

Microsatellite markers (MS) have been widely used for parentage verification in most of the livestock species over the past decades mainly due to their high polymorphic information content. In the genomic era, the spread of genotype information as single-nucleotide polymorphism (SNP) has raised the question to effectively use SNPs also for parentage testing. Despite the clear advantages of SNP panels in terms of cost, accuracy, and automation, the transition from MS to SNP markers for parentage verification is still very slow and, so far, only routinely applied in cattle. A major difficulty during this transition period is the need of SNP data for parents and offspring, which in most cases is not yet feasible due to the genotyping cost. To overcome the unavailability of same genotyping platform during the transition period, in this study we aimed to assess the feasibility of a MS imputation pipeline from SNPs in four native sheep dairy breeds: Comisana (N = 331), Massese (N = 210), Delle Langhe (N = 59) and Sarda (N = 1003). Those sheep were genotyped for 11 MS and with the Ovine SNP50 Bead Chip. Prior to imputation, a quality control (QC) was performed, and SNPs located within a window of 2 Mb from each MS were selected. The core of the developed pipeline was made up of three steps: (a) storing of both MS and SNP data in a Variant Call Format file, (b) masking MS information in a random sample of individuals (10%), (c) imputing masked MS based on non-missing individuals (90%) using an imputation program. The feasability of the proposed methodology was assessed also among different training - testing split ratio, population size, number of flanking SNPs as well as within and among breeds. The accuracy of the MS imputation was assessed based on the genotype concordance as well as at parentage verification level in a subset of animals in which assigned parents' MS were available. A total of 8 MS passed the QC, and 505 SNPs were located within the ±2 Mb window from each MS, with an average of 63 SNPs per MS. The results were encouraging since when excluding the worst imputed MS (OARAE129), and regardless on the analyses performed (within and across breeds) for all breeds, we achieved an overall concordance rate over 94%. In addition, on average, the imputed offspring MS resulted in equivalent parentage outcome in 94% of the cases when compared to verification using original MS, highlighting both the feasibility and the eventual practical advantage of using this imputation pipeline.

Heritability and genetic correlations of total and differential somatic cell count with milk yield and composition traits in Italian Simmental cows.

Costs of production have deeply increased each year in the last decades, breeders are continuously looking for more cost effective and more efficient ways to produce milk. Despite the major signs of progress in productivity, it is fundamental to optimize rather than maximize the performances of the dairy cows. Mastitis is still a highly prevalent disease in the dairy sector which causes several economic losses and environmental effect. Its accurate and early diagnosis is crucial to improve profitability of dairy cows and contribute to a more sustainable dairy industry. Among mastitis reduction strategies, there is the urgent need to implement breeding objectives to select cows displaying mastitis resistance by investigating the genetic mechanisms at the base of the inflammatory response. Therefore, in this study we aimed to further understand the genetic background of the differential somatic cell count (DSCC), which provides thorough insights on the actual inflammatory status of the mammary glands. The objectives of this study were to estimate on a cohort of 20,215 Italian Simmental cows over a 3-yr period: (1) the heritability and repeatability values of somatic cell score (SCS) and DSCC, (2) the genetic and phenotypic correlations between these 2 traits and milk production and milk composition traits, (3) the heritability and repeatability values of SCS and DSCC within class of udder health status. Heritability was low both for SCS (0.06) and DSCC (0.08), whereas the repeatability values for these traits were 0.43 and 0.36, suggesting that the magnitude of cow permanent environmental effect for these traits is remarkable. The genetic and phenotypic correlation of SCS with DSCC was 0.612 and 0.605, respectively. Because both significantly differed from the unit, we must consider those traits as different ones. This latter aspect corroborates the need to consider the DSCC as a further indicator of inflammatory status which might be implemented in the Simmental breed genetic evaluation. It is worthy to mention that heritability estimates for SCS and DSCC were the highest in healthy cows compared with the other udder health classes. This implies that when the udder health status changes, it is most likely due to environmental factors rather than aspects related to the animal's genetics. In contrast, the highest additive genetic variance and heritability found for SCS and DSCC in the healthy group might reveal the potential to further implement breeding strategies to select for healthier animals.

Performance of Swedish Warmblood fragile foal syndrome carriers and breeding prospects.

BACKGROUND: Warmblood fragile foal syndrome (WFFS) is a monogenetic defect caused by a recessive lethal missense point mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 gene (PLOD1, c.2032G>A). The majority of homozygous WFFS horses are aborted during gestation. Clinical signs of affected horses include fragile skin, skin and mucosa lacerations, hyperextension of the articulations, and hematomas. In spite of its harmful effect, a relatively high frequency of WFFS carriers has been found in Warmblood horses, suggesting a heterozygote advantage. Thus, in this study our aims were to: (1) estimate the frequency of WFFS carriers in the Swedish Warmblood breed (SWB), (2) estimate the effect of WFFS carrier genotype on performance traits in two SWB subpopulations bred for different disciplines, and (3) simulate the potential effects of balancing selection and different selection strategies on the frequency of carriers. METHODS: In total, 2288 SWB sport horses born between 1971 and 2020 were tested for the WFFS mutation and had estimated breeding values (EBV) for ten traditional evaluating and 50 linear descriptive traits. RESULTS: The frequency of WFFS carriers calculated from a pool of 511 randomly selected SWB horses born in 2017 was equal to 7.4% and ranged from 0.0 to 12.0% among the whole set of tested SWB horses, starting from 1971 till 2020. The effect of the WFFS carrier genotype was significant for several EBV mainly related to movements and dressage traits and especially for horses not bred for the show jumping discipline. Using simulation, we showed that balancing selection can maintain a recessive lethal allele in populations such as the SWB breed over generations and that the frequency is expected to slowly decrease in absence of balancing selection. Finally, we showed that selection against carrier sires can result in a more rapid decrease of the frequency of the mutant allele over time. CONCLUSION: Further research is needed to confirm the apparent association between equine performance and the WFFS carrier genotype. Identification of such associations or new causative mutations for horse performance traits can serve as new tools in horse breeding to select for healthy, sustainable, and better performing horses.

Genomic inbreeding coefficients using imputed genotypes: Assessing different estimators in Holstein-Friesian dairy cows.

The objective of this study was to estimate inbreeding coefficients in Holstein dairy cattle using imputed SNPs data. A data set of 95,540 Italian Holstein dairy cows from the routine genomic evaluations of the Italian National Association of Holstein, Brown, and Jersey Breeders were analyzed, with 84,445 imputed SNP. Ten widely used genomic inbreeding estimators were tested, including 4 PLINK v1.9 estimators (F, FHAT1, FHAT2, FHAT3), 3 genomic relationship matrix (GRM)-based methods [VanRaden's first method with observed allele frequencies (FGRM) or with fixed frequencies at 0.5 (FGRM05), VanRaden's third method, allelic frequency free and pedigree regressed (FGRM2)], runs of homozygosity (ROH)-based estimators in a complete (FROH) and simplified version (FROH2), and proportion of homozygous SNP (FPH). Pairwise comparisons among them were made, including the comparison with traditional pedigree-based inbreeding coefficients (FPED). Our results showed variability among the genomic inbreeding estimators. Coefficients of FGRM and FHAT3 were >1, meaning that more variability has been lost than the variability that existed in the base population. Regarding the remaining ones, FGRM05, FROH, FROH2, and FPH provided coefficients within the [0,1] space and are considered comparable to FPED. Not comparable to FPED, yet with an interpretable value, can be considered the coefficients of F, FHAT2, and FGRM2. Estimators based on ROH had the highest correlation with pedigree-based coefficients (0.59-0.66), among all estimators tested. In this study, Spearman correlations were shown to possibly provide a clearer estimation of the strength of the relationship between estimators. We hypothesize that imputation might cause extreme genomic inbreeding values that deserves further investigation.

Genetic parameters estimation in an Italian horse native breed to support the conversion from agricultural uses to riding purposes.

Horses are nowadays mainly used for sport and leisure purposes, and several local breeds, traditionally used in agriculture, are exposed to the risk of extinction. The long-term survival of local horse breeds depends on strategies to both monitor their genetic diversity and to find their sustainable role in the equine market. Thus, several local horse breeds need to adapt their breeding objective to allow a modernization process. The Bardigiano is an example of such horse breeds; we, therefore, studied the existing evaluation protocol from a genetic standpoint to assess the protocol's suitability to convert the Bardigiano from an agricultural to a riding horse. To this end, we estimated genetic parameters for four conformation measurements, ten grading traits and 23 linear traits. For conformation measurements, the heritabilities ranged from 0.31 for cannon bone circumference to 0.63 for height at withers. For conformation and attitude grading traits, the highest heritability (0.34) was estimated for development and the lowest (0.09) for gaits. The heritabilities for linear traits ranged from 0.05 for the leg straightness to 0.32 for the coat colour. Genetic correlations between linear traits and corresponding grading traits varied considerably, ranging from -0.42 to 0.98. This study showed that the current evaluation protocol in the Bardigiano horse is appropriate for genetic evaluation. Genetic parameters estimation can, in turn, be used to develop novel breeding values to help this conversion. Our study paves the way to optimize the Bardigiano horse breeding programme, and it may help several other local horse breeds experiencing similar issues.

Signatures of selection in the genome of Swedish warmblood horses selected for sport performance.

BACKGROUND: A growing demand for improved physical skills and mental attitude in modern sport horses has led to strong selection for performance in many warmblood studbooks. The aim of this study was to detect genomic regions with low diversity, and therefore potentially under selection, in Swedish Warmblood horses (SWB) by analysing high-density SNP data. To investigate if such signatures could be the result of selection for equestrian sport performance, we compared our SWB SNP data with those from Exmoor ponies, a horse breed not selected for sport performance traits. RESULTS: The genomic scan for homozygous regions identified long runs of homozygosity (ROH) shared by more than 85% of the genotyped SWB individuals. Such ROH were located on ECA4, ECA6, ECA7, ECA10 and ECA17. Long ROH were instead distributed evenly across the genome of Exmoor ponies in 77% of the chromosomes. Two population differentiation tests (FST and XP-EHH) revealed signatures of selection on ECA1, ECA4, and ECA6 in SWB horses. CONCLUSIONS: Genes related to behaviour, physical abilities and fertility, appear to be targets of selection in the SWB breed. This study provides a genome-wide map of selection signatures in SWB horses, and ground for further functional studies to unravel the biological mechanisms behind complex traits in horses.

Inter- and intra-breed genome-wide copy number diversity in a large cohort of European equine breeds.

BACKGROUND: Copy Number Variation (CNV) is a common form of genetic variation underlying animal evolution and phenotypic diversity across a wide range of species. In the mammalian genome, high frequency of CNV differentiation between breeds may be candidates for population-specific selection. However, CNV differentiation, selection and its population genetics have been poorly explored in horses. RESULTS: We investigated the patterns, population variation and gene annotation of CNV using the Axiom® Equine Genotyping Array (670,796 SNPs) from a large cohort of individuals (N = 1755) belonging to eight European horse breeds, varying from draught horses to several warmblood populations. After quality control, 152,640 SNP CNVs (individual markers), 18,800 segment CNVs (consecutive SNP CNVs of same gain/loss state or both) and 939 CNV regions (CNVRs; overlapping segment CNVs by at least 1 bp) compared to the average signal of the reference (Belgian draught horse) were identified. Our analyses showed that Equus caballus chromosome 12 (ECA12) was the most enriched in segment CNV gains and losses (~ 3% average proportion of the genome covered), but the highest number of segment CNVs were detected on ECA1 and ECA20 (regardless of size). The Friesian horses showed private SNP CNV gains (> 20% of the samples) on ECA1 and Exmoor ponies displayed private SNP CNV losses on ECA25 (> 20% of the samples). The Warmblood cluster showed private SNP CNV gains located in ECA9 and Draught cluster showed private SNP CNV losses located in ECA7. The length of the CNVRs ranged from 1 kb to 21.3 Mb. A total of 10,612 genes were annotated within the CNVRs. The PANTHER annotation of these genes showed significantly under- and overrepresented gene ontology biological terms related to cellular processes and immunity (Bonferroni P-value < 0.05). We identified 80 CNVRs overlapping with known QTL for fertility, coat colour, conformation and temperament. We also report 67 novel CNVRs. CONCLUSIONS: This work revealed that CNV patterns, in the genome of some European horse breeds, occurred in specific genomic regions. The results provide support to the hypothesis that high frequency private CNVs residing in genes may potentially be responsible for the diverse phenotypes seen between horse breeds.

Exploring genetic diversity in an Italian horse native breed to develop strategies for preservation and management.

Genetic diversity is a key factor for both adaptation and response to selection. The loss of genetic diversity causes a decrease in individual fitness, and it has a dramatically negative effect on population lifespan in the long term. This study aimed at exploring the genetic diversity at pedigree level of the Bardigiano horse breed, which is a native breed from Italy shaped for living in rural areas. In 1977, the Bardigiano studbook was founded to preserve the breed and for improving its use for riding and draft purposes. Pedigree data contained 9,469 horses, of which 3,416 were alive. Demographic and genetic parameters were estimated on subpopulations to evaluate potential genetic diversity differences among breeding and nonbreeding animals, and animals showing different breeding values (EBVs) for an index combining 18 conformation traits. Throughout the studied period, inbreeding steadily increased, reaching in the last birth year cohort a mean value equal to 0.10. The rate of inbreeding per generation, assuming a mean generation interval of 8.74 years, was equal to 1.64%. Moreover, significant differences on both average relatedness and inbreeding among horses with high and low EBVs were shown. Our study unravelled the state of genetic diversity in the Bardigiano breed, highlighting that breeding strategies for optimizing the contribution of breeding animals in the coming generations are needed to ensure long-term survival of the Bardigiano horse breed.

Fecal microbiota characterization of an Italian local horse breed.

The Bardigiano horse is a traditional native Italian breed with a rich history and peculiar characteristics. Local breeds are proven to have unique genetic traits developed over generations to adapt to defined geographical regions and/or conditions. The specific microbial communities that coexist within these animals are unraveled by studying their microbiota, which permits a further step in the characterization of local heritage. This work aimed to characterize Bardigiano horse fecal microbiota composition. The data obtained were then compared with published data of a mix of athlete breeds to evaluate potential differences among local and specialized breeds. The study involved 11 Bardigiano mares between 3 and 4 years of age, from which stool was sampled for the study. Samples were processed for 16S rRNA sequencing. Data obtained were analyzed and plotted using R, RStudio, and FastTree software. The samples analyzed were similar to what literature has reported on horses of other breeds and attitudes at higher taxonomic levels (from phylum to genera). While at lower taxonomic levels, the difference was more marked highlighting specific families found in the Bardigiano breed only. Weight, province of origin, and breeding sites significantly affected microbiota composition (p-value ≤0.02, p-value ≤0.04, and p-value ≤0.05, respectively). The comparison with athlete breed showed a significant difference confirming that animal and environmental factors are crucial in determining fecal microbiota composition (p-value <0.001). Understanding the microbiota composition in local breeds like the Bardigiano horse is crucial for preserving biodiversity, managing animal health, and promoting sustainable farming practices.

Analysis of ddRAD-seq data provides new insights into the genomic structure and patterns of diversity in Italian donkey populations.

With more than 150 recognized breeds, donkeys assume relevant economic importance, especially in developing countries. Even if the estimated number of heads worldwide is 53M, this species received less attention than other livestock species. Italy has traditionally been considered one of the cradles of European donkey breeding, and despite a considerable loss of biodiversity, today still counts nine autochthonous populations. A total of 220 animals belonging to nine different populations were genotyped using the double-digest restriction site associated DNA (ddRAD) sequencing to investigate the pattern of diversity using a multi-technique approach. A total of 418,602,730 reads were generated and successfully demultiplexed to obtain a medium-density SNP genotypes panel with about 27K markers. The diversity indices showed moderate levels of variability. The genetic distances and relationships, largely agree with the breeding history of the donkey populations under investigation. The results highlighted the separation of populations based on their genetic origin or geographical proximity between breeding areas, showed low to moderate levels of admixture, and indicated a clear genetic difference in some cases. For some breeds, the results also validate the success of proper management conservation plans. Identified runs of homozygosity islands, mapped within genomic regions related to immune response and local adaptation, are consistent with the characteristics of the species known for its rusticity and adaptability. This study is the first exhaustive genome-wide analysis of the diversity of Italian donkey populations. The results emphasized the high informativeness of genome-wide markers retrieved through the ddRAD approach. The findings take on great significance in designing and implementing conservation strategies. Standardized genotype arrays for donkey species would make it possible to combine worldwide datasets to provide further insights into the evolution of the genomic structure and origin of this important genetic resource.

Donkeys assume relevant economic importance in several countries worldwide. However, the genetic structure of these populations is less investigated compared to other species. The aim of this study was to investigate the genetic background of nine different Italian donkey populations. A total of 220 animals were genotyped with about 27K markers extracted by the double-digest restriction site associated DNA sequencing. The consistency of the results across different approaches agreed with the demographic history, the origin, and previous results on the nine donkey populations, suggesting that our conclusions are robust. Moreover, the results of the present study highlighted low to moderate levels of admixture and, for some breeds, confirmed the success of proper management conservation plans.

Evaluating the accuracy of a six-lead smartphone-based electrocardiographic device compared with standard electrocardiography in brachymorphic dogs.

BACKGROUND: No previous study has evaluated the accuracy of a six-lead smartphone-based electrocardiographic (s-ECG) device in dogs. METHODS: This was a prospective multicentre study. Patients referred for cardiologic consultation were selected. In each patient, a 30-second electrocardiogram was simultaneously acquired with a novel six-lead s-ECG and a standard six-lead ECG machine (st-ECG). A board-certified cardiologist evaluated each recording. Nineteen ECG variables, including heart rate and rhythm, as well as quantitative and qualitative features of waves, segments and intervals, were analysed. Agreement between s-ECG and st-ECG was evaluated using Cohen's kappa coefficient and the Bland-Altman test. RESULTS: Seventy-five dogs were enrolled, and 140 ECG tracings were analysed. There was perfect agreement between the two methodologies for heart rate and rhythm classification, both in dogs with sinus rhythm and those with pathological rhythms. Although some disagreement was found when comparing measurements of quantitative variables obtained with the s-ECG and the st-ECG, none of the differences was of clinical relevance. LIMITATIONS: The sample size was limited, and the interobserver variability was not analysed. CONCLUSION: The six-lead s-ECG studied herein is comparable to the st-ECG for heart rate and rhythm assessment, and seems clinically acceptable for the interpretation of waves, segments and intervals in dogs.

Echocardiographic values in healthy Pugs: Effect of body weight, age, and sex.

Background: Transthoracic echocardiography represents the main noninvasive technique for evaluating cardiac morphology and function in dogs. In dogs with particular somatotypes, such as brachymorphic dogs, breed-specific echocardiographic values are needed for a proper echocardiographic interpretation. Nowadays, the Pug represents one of the most popular brachymorphic canine breeds worldwide. However, data on echocardiographic measurements in this breed are currently limited. Aim: We aimed to determine echocardiographic values in a population of apparently healthy Pugs, and to assess the possible effects of body weight (BW), age, and sex on selected echocardiographic variables, with particular emphasis on those related to the left-sided cardiac chambers. Methods: Apparently healthy Pugs underwent a full physical examination, a 1-minute six-lead electrocardiogram, and a complete transthoracic echocardiography. Twenty-four echocardiographic variables were measured by combining M-mode, two-dimensional and Doppler modalities, and relative values were determined by applying the statistical procedures recommended by the Clinical and Laboratory Standards Institute. Moreover, the effect of selected demographic variables on echocardiographic measurements was tested using a linear mixed model. Results: The investigation included 86 Pugs. Echocardiographic values were provided for each variable and compared with previous veterinary literature. A statistically significant effect of BW, age, and sex was documented for several of the tested variables. Doppler examination demonstrated a trivial pulmonary regurgitation in 24/86 (27.9%) Pugs. Moreover, a persistent left cranial vena cava was suspected in 4/86 (4.7%) dogs. Conclusion: Echocardiographic features of the Pug were addressed and echocardiographic values were made available for clinical use. Because our findings were obtained using a standardized echocardiographic analysis in a population of 86 healthy Pugs, they may act as a reliable guide for an accurate echocardiographic interpretation in this breed.

Exploring the Italian equine gene pool via high-throughput genotyping.

Introduction: The Italian peninsula is in the center of the Mediterranean area, and historically it has been a hub for numerous human populations, cultures, and also animal species that enriched the hosted biodiversity. Horses are no exception to this phenomenon, with the peculiarity that the gene pool has been impacted by warfare and subsequent "colonization". In this study, using a comprehensive dataset for almost the entire Italian equine population, in addition to the most influential cosmopolitan breeds, we describe the current status of the modern Italian gene pool. Materials and Methods: The Italian dataset comprised 1,308 individuals and 22 breeds genotyped at a 70 k density that was merged with publicly available data to facilitate comparison with the global equine diversity. After quality control and supervised subsampling to ensure consistency among breeds, the merged dataset with the global equine diversity contained data for 1,333 individuals from 54 populations. Multidimensional scaling, admixture, gene flow, and effective population size were analyzed. Results and Discussion: The results show that some of the native Italian breeds preserve distinct gene pools, potentially because of adaptation to the different geographical contexts of the peninsula. Nevertheless, the comparison with international breeds highlights the presence of strong gene flow from renowned breeds into several Italian breeds, probably due to historical introgression. Coldblood breeds with stronger genetic identity were indeed well differentiated from warmblood breeds, which are highly admixed. Other breeds showed further peculiarities due to their breeding history. Finally, we observed some breeds that exist more on cultural, traditional, and geographical point of view than due to actual genetic distinctiveness.

Genomic inbreeding coefficients using imputed genotypes: assessing differences among SNP panels in Holstein-Friesian dairy cows.

The objective of this study was to evaluate the effect of imputation of single nucleotide polymorphisms (SNP) on the estimation of genomic inbreeding coefficients. Imputed genotypes of 68,127 Italian Holstein dairy cows were analyzed. Cows were initially genotyped with two high density (HD) SNP panels, namely the Illumina Infinium BovineHD BeadChip (678 cows; 777,962 SNP) and the Genomic Profiler HD-150K (641 cows; 139,914 SNP), and four medium density (MD): GeneSeek Genomic Profiler 3 (10,679 cows; 26,151 SNP), GeneSeek Genomic Profiler 4 (33,394 cows; 30,113 SNP), GeneSeek MD (12,030 cows; 47,850 SNP) and the Labogena MD (10,705 cows; 41,911 SNP). After imputation, all cows had genomic information on 84,445 SNP. Seven genomic inbreeding estimators were tested: (i) four PLINK v1.9 estimators (F, Fhat1,2,3), (ii) two genomic relationship matrix (grm) estimators [VanRaden's 1st method, but with observed allele frequencies (Fgrm) and VanRaden's 3rd method that is allelic free and pedigree dependent (Fgrm2)], and (iii) a runs of homozygosity (roh) - based estimator (Froh). Genomic inbreeding coefficients of each SNP panel were compared with genomic inbreeding coefficients derived from the 84,445 imputation SNP. Coefficients of the HD SNP panels were consistent between genotyped-imputed SNP (Pearson correlations ~99%), while variability across SNP panels and estimators was observed in the MD SNP panels, with Labogena MD providing, on average, more consistent estimates. The robustness of Labogena MD, can be partly explained by the fact that 97.85% of the SNP of this panel is included in the 84,445 SNP selected by ANAFIBJ for routine genomic imputations, while this percentage for the other MD SNP panels varied between 55 and 60%. Runs of homozygosity was the most robust estimator. Genomic inbreeding estimates using imputation SNP are influenced by the SNP number of the SNP panel that are included in the imputed SNP, and performance of genomic inbreeding estimators depends on the imputation.

Demonstration of Parthenogenetic Reproduction in a Pet Ball Python (Python regius) through Analysis of Early-Stage Embryos.

Parthenogenesis is an asexual form of reproduction, normally present in various animal and plant species, in which an embryo is generated from a single gamete. Currently, there are some species for which parthenogenesis is supposed but not confirmed, and the mechanisms that activate it are not well understood. A 10-year-old, wild-caught female ball python (Python regius) laid four eggs without any prior contact with a male. The eggs were not incubated and, after 3 days, were submitted to the University of Parma for analysis due to the suspicion of potential embryo presence. Examination of the egg content revealed residual blood vessels and a small red spot, indicative of an early-stage embryo. DNA was extracted from the three deceased embryos and from the mother's blood, five microsatellites were analyzed to ascertain the origin of the embryos. The captive history data, together with the genetic microsatellite analysis approach, demonstrated the parthenogenetic origin of all three embryos. The embryos were homozygous for each of the maternal microsatellites, suggesting a terminal fusion automixis mode of development.

The role of inbreeding depression on productive performance in the Italian Holstein breed.

Inbreeding depression has become an urgent issue in cosmopolitan breeds where the massive genetic progress achieved in the latest generations is counterbalanced by a dramatic loss of genetic diversity causing increased health issues. Thus, the aim of this study was to estimate inbreeding depression on productive traits in Holstein dairy cattle. More precisely, we aimed to i) determine the level of inbreeding in 27,735 Italian Holstein dairy cows using pedigree and genotype data, ii) quantify the effect of inbreeding on 305-d in milk yield (MY; kg), fat yield (FY; kg), and protein yield (PY; kg) based on different statistical approaches, iii) determine if recent inbreeding has a more harmful impact than ancestral ones, and iv) quantify chromosomal homozygosity effect on productive traits. Quality control was performed on the autosomal chromosomes resulting in a final dataset of 84,443 single nucleotide polymorphisms. Four statistical models were used to evaluate the presence of inbreeding depression, which included linear regression analysis and division of FPED and FROH into percentile classes. Moreover, FROH was partitioned into i) length classes to assess the role of recent and ancestral inbreeding and ii) chromosome-specific contributions (FROH-CHR). Results evidenced that inbreeding negatively impacted the productive performance of Italian Holstein Friesian cows. However, differences between the estimated FPED and FROH coefficients resulted in different estimates of inbreeding depression. For instance, a 1% increase in FPED and FROH was associated with a decrease in MY of about 44 and 61 kg (P < 0.01). Further, when considering the extreme inbreeding percentile classes moving from the 5th lowest to the 95th highest, there was a reduction of -263 kg and -561 kg per lactation for FPED and FROH. Increased inbreeding, estimated by FPED and FROH, had also a negative effect on PY and FY, either fit as a regressor or percentile classes. When evaluating the impact of inbreeding based on runs of homozygosity (ROH) length classes, longer ROH (over 8 Mb) had a negative effect in all traits, indicating that recent inbreeding might be more harmful than the ancestral one. Finally, results within chromosome homozygosity highlighted specific chromosomes with a more deleterious effect on productive traits.

Inbreeding depression is a reduction in performance or health due to the mating of closely related individuals. The overall aim of this study was to investigate the level of inbreeding in the Italian Holstein dairy cow breed and quantify its negative effect on productive performances. The level of inbreeding was estimated by pedigree (FPED) and genomic data by looking at stretches of homozygosity (FROH). Both methods revealed a reduction in milk yield, fat yield, and protein yield when inbreeding increased. Moreover, the study demonstrated that FROH was able to capture more inbreeding depression compared to FPED. In addition, the more recent inbreeding had a stronger negative impact on productive performances compared to ancestral ones. Then, since the amount of runs of homozygosity can vary across the chromosomes of an individual, the effect of each chromosomal homozygosity region on productive traits was also evaluated. The chromosome-level results might be included in breeding programs to limit the accumulation of homozygosity in particular regions that appear to have a more detrimental effect on productive traits. Overall, this study highlights the importance of avoiding inbreeding in animal breeding programs to keep productive animals in the long term.

Sex Determination in Immature Sierra Nevada Lizard (Timon nevadensis).

Sex determination has a fundamental role in a captive breeding context, both for commercial reasons and in relation to animal welfare itself. However, this can be particularly difficult, especially in reptiles with little or no sexual dimorphism. Twenty-three clinically healthy young Sierra Nevada lizards (Timon nevadensis) were included in this study for sex determination. The first attempt at sexing was carried out by cloacal probing. A small, buttoned probe was inserted very gently into the hemipenal pouches, and the length of the inserted part was evaluated and measured. Subsequently, for each animal, a contrast medium was administered into the cloaca, and radiography was performed within 5 min. Through probing, 11 males and 8 females were recognized. The test was, however, equivocal in four subjects. In contrast radiography, 14 males and 9 females were identified. All the animals were rechecked after 8 months through an ultrasound examination, confirming 15 of the 14 previously male sexed animals based on contrast radiography. All the animals identified as female (n = 9) by contrast radiography were confirmed. From these results, it seems that contrast radiography may have major sensitivity in sex determination compared to probing. This technique could represent a valid and less invasive aid for sexing young lizards.

Keep Garfagnina alive. An integrated study on patterns of homozygosity, genomic inbreeding, admixture and breed traceability of the Italian Garfagnina goat breed.

The objective of this study was to investigate the genetic diversity of the Garfagnina (GRF) goat, a breed that currently risks extinction. For this purpose, 48 goats were genotyped with the Illumina CaprineSNP50 BeadChip and analyzed together with 214 goats belonging to 9 other Italian breeds (~25 goats/breed), whose genotypes were available from the AdaptMap project [Argentata (ARG), Bionda dell'Adamello (BIO), Ciociara Grigia (CCG), Di Teramo (DIT), Garganica (GAR), Girgentana (GGT), Orobica (ORO), Valdostana (VAL) and Valpassiria (VSS)]. Comparative analyses were conducted on i) runs of homozygosity (ROH), ii) admixture ancestries and iii) the accuracy of breed traceability via discriminant analysis on principal components (DAPC) based on cross-validation. ROH analyses was used to assess the genetic diversity of GRF, while admixture and DAPC to evaluate its relationship to the other breeds. For GRF, common ROH (more than 45% in GRF samples) was detected on CHR 12 at, roughly 50.25-50.94Mbp (ARS1 assembly), which spans the CENPJ (centromere protein) and IL17D (interleukin 17D) genes. The same area of common ROH was also present in DIT, while a broader region (~49.25-51.94Mbp) was shared among the ARG, CCG, and GGT. Admixture analysis revealed a small region of common ancestry from GRF shared by BIO, VSS, ARG and CCG breeds. The DAPC model yielded 100% assignment success for GRF. Overall, our results support the identification of GRF as a distinct native Italian goat breed. This work can contribute to planning conservation programmes to save GRF from extinction and will improve the understanding of the socio-agro-economic factors related with the farming of GRF.

Genome-wide scan reveals genetic divergence in Italian Holstein cows bred within PDO cheese production chains.

Dairy cattle breeds have been exposed to intense artificial selection for milk production traits over the last fifty years. In Italy, where over 80% of milk is processed into cheese, selection has also focused on cheese-making traits. Due to a deep-rooted tradition in cheese-making, currently fifty Italian cheeses are marked with the Protected Designation of Origin (PDO) label as they proved traditional land of origin and procedures for milk transformation. This study aimed to explore from a genetic point of view if the presence of such diverse productive contexts in Italy have shaped in a different manner the genome of animals originally belonging to a same breed. We analyzed high density genotype data from 1000 Italian Holstein cows born between 2014 and 2018. Those animals were either farmed in one of four Italian PDO consortia or used for drinkable milk production only. Runs of Homozygosity, Bayesian Information Criterion and Discriminant Analysis of Principal Components were used to evaluate potential signs of genetic divergence within the breed. We showed that the analyzed Italian Holstein cows have genomic inbreeding level above 5% in all subgroups, reflecting the presence of ongoing artificial selection in the breed. Our study provided a comprehensive representation of the genetic structure of the Italian Holstein breed, highlighting the presence of potential genetic subgroups due to divergent dairy farming systems. This study can be used to further investigate genetic variants underlying adaptation traits in these subgroups, which in turn might be used to design more specialized breeding programs.