The β-casein gene (CSN2) encodes one of the major milk proteins involved in calcium and phosphorus transport and combines a highly conserved genomic organization with extensive genetic diversity, making it an informative model for investigating mammalian genome evolution. Although numerous studies have described CSN2 polymorphisms, particularly in domestic species, the broader evolutionary history of the locus remains poorly understood. Here, we investigated the evolutionary architecture of CSN2 across Bovidae and related Cetartiodactyla by integrating coding variation, non-coding polymorphisms, retroposon insertions and recombination analyses. Comparative genomic analyses identified previously undescribed caprine haplotypes, expanded the known spectrum of β-casein variation in domestic and wild Caprinae, and revealed multiple structural configurations of the locus, including distinct retroposon architectures and intronic insertion/deletion polymorphisms. Retroposon mapping and comparative analyses supported a hierarchical accumulation of structural variants, while also indicating ancestral polymorphism and lineage-specific retention in rapidly radiating caprine lineages. A locus-level phylogenetic analysis revealed two deeply divergent allelic lineages and provided evidence of ancient interallelic recombination predating the divergence of Capra and Ovis. Comparative analyses further showed that a two-amino-acid deletion previously associated mainly with Caprinae is more widely distributed across Bovidae than previously recognized. Overall, the CSN2 locus emerges as a multilayered evolutionary system shaped by structural variation, ancestral polymorphism, incomplete lineage sorting and ancient recombination, illustrating how the integration of coding, structural and retroposon variation can improve locus-level evolutionary reconstruction in Bovidae.

Congenital malformations, including cephalic anomalies such as diprosopia and dicephalia, represent rare but significant developmental abnormalities in humans and animals. While these conditions are primarily attributed to incomplete embryonic splitting, their genetic origins in domestic animals remain poorly understood. This study presents a detailed molecular and anatomical investigation of three cases of cephalic malformations in ruminants: two Holstein-Friesian calves and one Mediterranean Italian River Buffalo. Anatomical analyses classified the malformations as diprosopus in two cases and dicephalic in one. Notably, this is the first reported case of dicephalia in a female Mediterranean Italian River Buffalo. Molecular analysis using microsatellite markers and sex-linked genes (AMELX/Y and SRY) confirmed the monozygotic origin and the female sex of all cases, with identical genotypes detected in both heads of each calf.

The aim of this study was to characterize exon 1 of the mannose-binding lectin 2 (MBL2) gene in Comisana sheep and to evaluate its association with susceptibility to mastitis and udder-health–related traits. A total of 152 lactating Comisana ewes were analysed. MBL2 exon 1 was sequenced to identify genetic variants. Associations between MBL2 genotypes or alleles and somatic cell count (SCC), differential somatic cell count (DSCC), and mastitis occurrence were assessed. Additional analyses compared carrier versus non-carrier groups, and multivariable logistic regression models were used to adjust for age. Sequencing revealed three alleles (A1, C, and D), including a newly identified A1 variant that alters the encoded protein and was the most frequent allele in the population. No significant associations were observed between full MBL2 genotypes and SCC, DSCC, or mastitis. However, allele-based analyses showed that ewes carrying the A1 allele had a lower prevalence of mastitis compared with non-carriers. This association remained significant after adjustment for age in multivariable logistic regression models. These results provide new insights into MBL2 genetic variability in sheep and suggest that the A1 allele may contribute to increased resilience to mastitis. Further studies in larger and genetically diverse populations are warranted to confirm the role of MBL2 in udder health.

Over the past thirty years, the identification of species-specific molecular markers has significantly advanced our understanding of genetic diversity in both plants and animals. Among these, short InDels have emerged as vital genomic features, contributing more to sequence divergence than single nucleotide polymorphisms do in closely related species. This study aimed to identify specific InDels for Bos taurus, Bubalus bubalis, Capra hircus, and Ovis aries via an in silico approach and validated them in 400 individuals (100 for each species). We identified and characterized short, specific InDels in the sequences of the CSN1S1, CSN1S2, MSTN, and PRLR genes, which can be used for species identification of Capra hircus, Ovis aries, Bos taurus, and Bubalus bubalis, respectively. We developed a Tetraplex Specific PCR assay to enable efficient discrimination among these species. This study highlights the utility of InDels as biallelic, codominant markers that are cost-effective and easy to analyse, providing valuable tools for genetic diversity analysis and species identification.

This study investigates autosome evolution between river buffalo (Bubablus bubalis, BBU) and cattle (Bos taurus, BTA), two closely related species within the Bovidae family. Despite differences in chromosome numbers (2n = 60 in cattle and 2n = 50 in river buffalo), previous cytogenetic studies have shown high autosome similarity. However, standard banding techniques have limitations in detecting small-scale genomic rearrangements. Using molecular comparisons, this study identifies two previously undetected chromosomal inversions: a 30-Mb inversion on BBU7 (compared to BTA6) and a 4-Mb inversion on BBU14 (compared to BTA13). These findings were validated through bioinformatics analyses (genomic alignments and BLAST searches) and confirmed via fluorescence in situ hybridization technique. In addition, it has been shown that several river buffalo chromosomes are shown inverted in the genome assembly considered in this study (NDDB_SH_1). The study highlights that autosome evolution in Bovidae involves not only centric fusions but also cryptic intra-chromosomal rearrangements. These results contribute to a deeper understanding of genome evolution in closely related species and demonstrate the importance of high-resolution molecular techniques in uncovering hidden genomic changes.

In Italy, karyotype analysis is routinely performed on all young males intended for reproduction to screen for chromosome abnormalities. This practice gained prominence following the identification of a complex chromosome abnormality in a well-known bull named Magnifico and its progeny. This procedure significantly enhances the value of the Mediterranean Italian River Buffalo breed, particularly in the context of artificial insemination both within Italy and in other countries importing semen. In the current study, 159 young Mediterranean Italian River Buffalo (MIRB) males intended for reproduction, 30 females with fertility issues, 3 young females and a male co-twin were investigated by analyzing their karyotype using both CBA- and RBA-banding. All males displayed a normal karyotype (2n = 50, XY), although one animal displayed an abnormal X-chromosome with C-band polymorphism, as it is C-band negative when this chromosome normally shows the largest C-band among all chromosomes. This result was also confirmed by the FISH-mapping technique using three bovine BAC-clones containing SAT-I, SAT-III and SAT-IV. Cytogenetic investigation showed that seven females, along with one male co-twin, were affected by freemartinism (XX/XY leukocyte chimerism) and were subsequently removed from the farm. In two of these females, clinical and necroscopic examinations showed severe damage of the internal reproductive organs.

Mastitis represents one of the main challenges in dairy buffalo farming, with significant implications for animal health, milk quality, and farm profitability. Among its different forms, subclinical mastitis is difficult to detect due to the absence of standardised diagnostic tools. Epigenetic mechanisms, such as DNA methylation, have been associated with immune response and disease susceptibility in cattle, but remain unexplored in buffaloes. This study explores genome-wide DNA methylation profiles in Mediterranean Italian River Buffaloes, using nanopore sequencing technology, to identify potential epigenetic signatures associated with mastitis resistance. CpG sites showed a unimodal distribution, with most sites exhibiting high methylation levels. A total of 22 differentially methylated cytosines (DMCs) were identified, with 68% showing hypomethylation in the control group and 32% showing hypermethylation. Genomic annotation revealed that hypermethylated DMCs were predominantly located in intronic regions, while hypomethylated DMCs were largely enriched in distal intergenic regions. This study is the first to investigate DNA methylation changes associated with mastitis in Mediterranean Italian River Buffalo using nanopore sequencing. Distinct epigenetic patterns were identified between healthy and mastitic animals. This study provides a first epigenetic overview of mastitis in buffaloes and lays the groundwork for future investigations with larger cohorts to validate and extend these observations. Given the small sample size, these findings should be considered exploratory, but offer insights into the molecular basis of mastitis and may support the development of new diagnostic tools based on validated epigenetic signatures.

INTRODUCTION:The SOX9 gene encodes a transcription factor that acts downstream of the Y-linked SRY gene and plays a pivotal role in fetal testis development. Duplication of SOX9 or its regulatory sequences is a known cause of testicular or ovotesticular disorder of sex development (DSD) in chromosomal females (XX DSD). Numerous reports have described canine XX DSD, characterized by virilization (e.g., enlarged clitoris) and the presence of testes or ovotestes. This study aimed to identify SOX9 variants in a cohort of French Bulldogs with XX (SRY-negative) DSD. METHODS:In total, 27 DSD dogs were studied, including 19 with abdominal, spermatogenetically inactive testes; four with inactive testis and ovotestis; one with inactive testis and ovary; one with ovotestes; and in two dogs, histological analysis could not be performed. Moreover, 24 control females of the same breed, all with normal external female genitalia, were included. RESULTS:Three known DNA variants were identified in SOX9: a 3 bp insertion/deletion (CCT/---, rs852828782), a T>C SNP (rs22704771) in the 5' UTR, and an intronic T>G SNP (rs9183825). These variants were rare, and their distribution was similar in both cohorts. Additionally, the number of SOX9 gene copies was assessed using ddPCR. A single XX DSD case with additional skeletal malformations carried three copies of SOX9, while all other cases and control females had two copies. CONCLUSION:We conclude that SOX9 duplication is a rare cause of XX DSD in French Bulldogs, and that the identified sequence variants in this gene are not associated with the disorder.

This study investigates chromosomal damage in donkeys to assess the impact of long-term exposure to different concentrations of environmental genotoxic agents, which pose health risks to animals and humans by promoting DNA breaks. The genotoxic damage was assessed through aneuploidy, chromosomal aberrations (CAs), and sister chromatid exchanges (SCEs) tests in donkeys, crossbred Ragusano and Grigio Siciliano breeds, from three areas in the Sicily region (Italy) with different levels of air pollution. Donkeys from areas with higher concentrations of fine particulate matter and nitrogen dioxide showed significantly elevated levels of aneuploidy and chromosomal abnormalities compared to those from less polluted areas. These findings provide the first evidence in donkeys of the combined effects of long-term exposure to airborne pollutants on genomic stability. This study reinforces the potential use of donkeys as effective biomonitoring organisms for evaluating environmental health risks and genotoxic damage under different pollution conditions.

Splicing regulatory sequences are cornerstones for exon recognition. Mutations that modify them can severely compromise mRNA maturation and protein production. A wide range of mutations, including SNPs and InDels, can influence splicing regulatory signals either directly (e.g., altering canonical donor and acceptor dinucleotides) or indirectly (e.g., creating cryptic splice sites). CSN1S1 and CSN1S2 genes encode for the two main milk proteins, αs1 and αs2 caseins, respectively. They represent a remarkable and unique example of the possibilities for alternative splicing of individual genes, both due to the high number of alternative splices identified to date and for recognized allele-specific splicing events. To date, at least 13 alleles of CSN1S1 originating from mutations that affect canonical splice sites have been described in Bos taurus (CSN1S1 A, A1, and H), Ovis aries (E, H, and I), Capra hircus (D and G), Bubalus bubalis (E, F) and Camelidae (A, C, and D). Similarly, allele-specific splicing events have been described at the CSN1S2 locus in B. taurus. (CSN1S2 D), C. hircus (CSN1S2 D), B. bubalis (CSN1S2 B, B1, and B2), Equus asinus (CSN1S2 I B), and Camelidae. This review highlights that mutations affecting canonical splice sites, particularly donor sites, are significant sources of genetic variation impacting the casein production of the main dairy livestock species. Currently, a key limitation on this topic is the lack of detailed functional and proteomic studies. Future research should leverage advanced omics technologies like long-read transcriptomics and allele-resolved RNA sequencing to characterize these splicing mechanisms, guiding precision breeding strategies.

Gibber Italicus, song thrush (Turdus philomelus), and redwing (Turdus iliacus) are 3 bird species in which sexual dimorphism is not readily apparent. Therefore, molecular sexing is a valuable tool for breeding, selection, and conservation purposes. We compared DNA extraction by commercial kit and an alkaline method from feathers, then developed a molecular method for sexing these species using the P2/P8 and CHD1F/CHD1R primer pairs. Both protocols were successful in extracting DNA; the alkaline method is more cost-effective, whereas the commercial kit protocol is easier to standardize. All DNA samples were successfully amplified using both the P2/P8 and CHD1F/1R primer pairs. The use of the CHD1F/1R primer pair is notably advantageous as it produces fragments of different lengths of the sex chromosomes, making them easily distinguishable via electrophoresis. The sequence analysis of the amplicons obtained with the CHD1F/1R primer pair revealed specific single-nucleotide polymorphisms for song thrush and redwing, which could be used as markers to differentiate between the 2 species if required.

Microplastics are widespread pollutants in aquatic environments and pose significant risks to aquatic organisms, including species vital to aquaculture. The gilthead sea bream, extensively farmed in the Mediterranean, is frequently exposed to these contaminants, leading to potential long-term health consequences. Telomere length, a reliable marker of genomic integrity and cellular aging, offers a promising approach for assessing the biological effects of environmental stressors like microplastics. This study investigates the impact of microplastic exposure on telomere length in gilthead sea bream, evaluating its potential as a genomic biomarker for detecting microplastic-induced damage. Juvenile sea breams were divided into three groups: a control group and two experimental groups exposed to relatively low (25 mg/kg b.w./day) and high (250 mg/kg b.w./day) doses of polystyrene microplastics for 21 days. Telomere length was measured using qPCR, and statistical analyses were conducted to compare the T/S ratio between the groups. The results showed significantly shorter telomeres in fish exposed to both low and high doses of polystyrene microplastics compared to controls, with a clear dose-dependent effect (p < 0.05). These findings indicate that microplastic exposure compromises genomic stability in gilthead sea bream, supporting the use of telomere length as a rapid and sensitive biomarker for environmental monitoring in aquaculture. The study highlights the potential of telomere length as a valuable tool for evaluating fish health in polluted environments, contributing to the development of sustainable practices in aquaculture.

OBJECTIVE:The Bagnolese sheep is an authochtonous dual-purpose breed (milk and meat) reared in the Campania region, whose milk is used to produce Pecorino Bagnolese cheese. Genetic information on this sheep is extremely limited, especially regarding genes affecting productions. The aim of this study was to investigate milk production traits in Bagnolese sheep and the variability of diacylglycerol acyltransferase 1 (DGAT1) gene and its effects on milk production. METHODS:Milk quantity was recorded during the morning milking, while Kjeldahl and Gerber methods were used to assess protein and fat percentage (w/v) of collected milk samples. Two polymerase chain reaction-restriction fragment length polymorphism protocols using BamHI and MspI endonucleases for genotyping of g.5553C>T and g.8539C>T at DGAT1 locus, respectively, were set up. RESULTS:Bagnolese sheep milk shows high fat and protein concentrations. Genotyping revealed a high frequency of the g.5553C and g.8539C alleles (0.56 and 0.95, respectively). The association study between the single nucleotide polymorphism (SNP) g.5553C>T and milk traits showed that animals with the CT genotype had a higher percentage of fat produced per milking than those with the CC and TT genotypes (p<0.01). Similar results were found for protein yield percentage, with CT individuals being more productive than CC individuals (p<0.01). CONCLUSION:Bagnolese sheep milk parameters found are associated with high yields in the resulting dairy products. CT genotype at the SNP g.5553 of DGAT1 has shown a positive association with fat and protein milk yield percentage suggesting it could be considered a marker to improve productions of this breed. Finally, the new genotyping techniques used for this study enable a cheap and reliable characterization of two DGAT1 SNPs in sheep.

According to the Domestic Animal Diversity Information System (DAD-IS) of the FAO, Italy has one of the largest numbers of local small ruminant breeds among European countries. In Southern Italy, namely the Campania Region, Bagnolese and Laticauda sheep breeds and Cilentana goat breeds are considered endangered according to the DAD-IS. Conservation of endangered animal breeds is a goal of the European Union (EU). However, the role of infectious diseases as risk factors for endangered breeds has rarely been considered. Small ruminant lentiviruses (SRLV) infect sheep and goats, causing slow-progressive, persistent, and debilitating diseases that can lead to animal death and productivity loss. In this study, we investigated the presence of SRLV in Bagnolese, Laticauda, and Cilentana breeds using a commercial ELISA in parallel with an in-house ELISA. The results of the two tests were in good agreement (Cohen Kappa 0.84, 95 % CI = 0.76–0.93). Discrepancies between the two tests were resolved using western blotting. In total, 430 samples were tested (248 Bagnolese, 125 Laticauda, and 57 Cilentana). The apparent prevalence rates were 12.5 %, 6.4 %, and 1.7 % in Bagnolese, Laticauda, and Cilentana, respectively. In the molecular analysis of 11 proviral partial sequences, subtypes B2 and A24 were identified in two Bagnolese herds. Owing to the beneficial role of sheep and goat breeding in marginal areas, it is important to screen the entire population and implement control/eradication of SRLV infections in conjunction with each conservation program.

The 25th International Colloquium on Animal Cytogenetics and Genomics is dedicated to the memory of Dr James (Jim) Womack, a pioneer in gene mapping, especially in cattle [...]

Calcium-sensitive caseins are the main protein component of milk. In the goat, they are encoded by three genes (CSN1S1, CSN2, and CSN1S2) located on chromosome 6. A high number of alleles has been discovered for these genes in the goat species, responsible for changes in the milk’s qualitative and quantitative characteristics. This study aimed to develop an Allele-Specific PCR (AS-PCR), which allowed us to unequivocally detect goat carriers of the CSN201 allele. Subsequently, the calcium-sensitive casein loci genotype was investigated in three native goat breeds of the Lazio Region (Bianca Monticellana, Capestrina, and Ciociara Grigia). No individuals were carriers of the CSN1S101, CSN1S1E, CSN201, CSN1S2D, and CSN1S20 alleles, while a high frequency of the alleles CSN1S1F and CSN1S1A*,B* was observed. Association analyses between the different genotypes at the CSN1S1 locus and some milk traits, namely the fat and protein yielded and the fat, protein, solids-not-fat, and casein percentages without an effect on the milk yield, were observed.

Collision tumors (CT) consist of two independent neoplasms with distinct neoplastic populations. Disorders of sexual development (DSDs) are characterized by atypical sexual development leading to various abnormalities of the genital tract. Sex reversal (SR) syndromes are a type of DSD characterized by a discrepancy between chromosomal sex and gonadal development (testes/ovaries) and the presence or the absence of the SRY gene. A phenotypically female 8-year-old Jack Russell terrier dog was referred due to anomalous vaginal discharge and non-pruritic cutaneous bilateral symmetrical alopecia on the flanks. During abdominal palpation, a voluminous mass was detected in the left quadrant area, later confirmed by ultrasound. The owner decided to proceed with euthanasia and necropsy. In the abdominal cavity, the left gonad was increased in size, the right one and the uterus were decreased, and the vagina and vulva appeared to be thickened. Histologically, both gonads were revealed to be testes: the left one was affected by a double neoplastic component (sustentacular tumor and interstitial cell tumor), whereas the right gonad showed coarctated seminiferous tubules. PCR amplification of the genes SRY and AMELX revealed the absence of the MSY region of the Y chromosome. To the authors' knowledge, this is the first report describing a case of a testicular collision tumor in a DSD SRY-negative dog.

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.

Studies into telomere length in cattle are relatively recent and have focused mainly on the Holstein Friesian cattle breed, making it arduous to evaluate the correlation with ageing due to the early age of culling in this breed. Telomere length provides information about the productive lifespan and the quality of farm management, complying with the 'One Health' approach. This study evaluated telomere length in Agerolese cattle, an autochthonous dairy breed characterized by a long productive lifespan (13 years). Multiplex quantitative PCR estimated telomere length in DNA extracted from blood and milk matrices. Interestingly, the results showed longer telomeres in Agerolese (compared to the Holstein Friesian cattle control group), with a negative correlation between telomere length and increasing age and a synchronous trend between blood and milk samples, with a positive correlation between them.

The Pony of Esperia is an Italian autochthonous horse breed reared in the wild on the Aurunci and Ausoni Mountains. Currently, it is considered an endangered breed, as its population consists of 1623 animals. It is therefore essential to identify all aspects that can improve the management and economy of its breeding, favoring its diffusion. In this paper, the effects of intestinal strongyle infection on the chromosome stability of peripheral blood lymphocytes (PBLs) was evaluated through aneuploidy and chromosome aberration (gap, chromatid and chromosome breaks, and the number of abnormal cells) test. Statistical difference in the mean values of aneuploidy, cells with chromosome abnormalities, and chromosome and chromatid breaks were observed between ponies with high fecal egg counts (eggs per gram > 930) and those with undetectable intestinal strongylosis. The causes of this phenomenon and possible repercussions on the management of Pony of Esperia are discussed in the paper.