CSN1S1, CSN3 and LPL: Three Validated Gene Polymorphisms Useful for More Sustainable Dairy Production in the Mediterranean River Buffalo.

The search for DNA polymorphisms useful for the genetic improvement of dairy farm animals has spanned more than 40 years, yielding relevant findings in cattle for milk traits, where the best combination of alleles for dairy processing has been found in casein genes and in DGAT1. Nowadays, similar results have not yet been reached in river buffaloes, despite the availability of advanced genomic technologies and accurate phenotype records. The aim of the present study was to investigate and validate the effect of four single nucleotide polymorphisms (SNP) in the CSN1S1, CSN3, SCD and LPL genes on seven milk traits in a larger buffalo population. These SNPs have previously been reported to be associated with, or affect, dairy traits in smaller populations often belonging to one farm. A total of 800 buffaloes were genotyped. The following traits were individually recorded, monthly, throughout each whole lactation period from 2010 to 2021: daily milk yield (dMY, kg), protein yield (dPY, kg) and fat yield (dFY, kg), fat and protein contents (dFP, % and dPP, %), somatic cell count (SCC, 103 cell/mL) and urea (mg/dL). A total of 15,742 individual milk test day records (2496 lactations) were available for 680 buffalo cows, with 3.6 ± 1.7 parities (from 1 to 13) and an average of 6.1 ± 1.2 test day records per lactation. Three out four SNPs in the CSN1S1, CSN3 and LPL genes were associated with at least one of analyzed traits. In particular, the CSN1S1 (AJ005430:c.578C>T) gave favorable associations with all yield traits (dMY, p = 0.022; dPY, p = 0.014; dFY, p = 0.029) and somatic cell score (SCS, p = 0.032). The CSN3 (HQ677596: c.536C>T) was positively associated with SCS (p = 0.005) and milk urea (p = 0.04). Favorable effects on daily milk yield (dMY, p = 0.028), fat (dFP, p = 0.027) and protein (dPP, p = 0.050) percentages were observed for the LPL. Conversely, the SCD did not show any association with milk traits. This is the first example of a confirmation study carried out in the Mediterranean river buffalo for genes of economic interest in the dairy field, and it represents a very important indication for the preselection of young bulls destined for breeding programs aimed at more sustainable dairy production.

Sequencing and Characterization of αs2-Casein Gene (CSN1S2) in the Old-World Camels Have Proven Genetic Variations Useful for the Understanding of Species Diversification.

The CSN1S2 gene encodes αs2-casein, the third most abundant protein in camel milk. Despite its importance in foals, human nutrition, and dairy processing, the CSN1S2 gene in camels has received little attention. This study presents the first complete characterization of the CSN1S2 gene sequence in Old-World camels (Camelus bactrianus and Camelus dromedarius). Additionally, the gene promoter, consisting of 752 bp upstream of exon 1, was analyzed. The entire gene comprises 17 exons, ranging in length from 24 bp (exons 4, 8, 11, and 13) to 280 bp (exon 17). Interesting was the identification of the exon 12 in both species. The promoter analysis revealed 24 putative binding sites in the Bactrian camel and 22 in dromedary camel. Most of these sites were typical elements associated with milk protein, such as C/EBP-α, C/EBP-ß, Oct-1, and AP1. The SNP discovery showed relatively high genetic diversity compared to other camel casein genes (CSN1S1, CSN2, and CSN3), with a total of 34 polymorphic sites across the two species. Particularly noteworthy is the transition g.311G>A in the CSN1S2 promoter, creating a new putative consensus binding site for a C/EBP-ß in the Bactrian camel. At the exon level, two novel variants were found. One was detected in exon 6 of the Bactrian camel (g.3639C>G), resulting in an amino acid replacement, p.36Ile>Met. The second variant was found in noncoding exon 17 of dromedary CSN1S2 (g.1511G>T). Although this mutation occurs in the 3'-UnTranslated Region, it represents the first example of exonic polymorphism in the CSN1S2 for this species. This SNP also affects the binding sites of different microRNAs, including the seed sequence of the miRNA 4662a-3p, highlighting its role as a regulatory factor for CSN1S2 gene. A PCR-RFLP was set up for genotyping a dromedary Tunisian population (n = 157), and the minor allele frequency was found to be 0.27 for the G allele, indicating a potential yield improvement margin. The interspersed elements (INEs) analysis revealed 10 INEs covering 7.34% and 8.14% of the CSN1S2 sequence in the Bactrian and dromedary camels, respectively. Furthermore, six elements (A, B, F, H, I, and L) are shared among cattle and camels and are partially found in other ruminants, suggesting a common ancestral origin of these retrotransposons. Conversely, elements C, D, E, and G are specific to camels.

A New AS-PCR Method to Detect CSN201 Allele, Genotyping at Ca-Sensitive Caseins Loci and Milk Traits Association Studies in Autochthonous Lazio Goats.

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.

Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins.

An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Post-translational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. αs2-CN perfectly exemplifies such a complexity. With 2 functional genes, CSN1S2 I and CSN1S2 II, made of 20 and 16 exons respectively, nearly 30 different molecules of this CN were detected in the milk of one Amiata donkey. A cryptic splice site usage, leading to a singular shift of the open reading frame and generating two αs2-CN I isoforms with different C-terminal sequences, was brought to light. Twenty different αs1-CN molecules with different phosphorylation levels ranging between 4 and 9P were identified in a single milk sample, most of them resulting from exon skipping events and cryptic splice site usage. Novel genetic polymorphisms were detected for CNs (ß- and αs-CN) as well as for whey proteins (lysozyme C and ß-LG I). The probable new ß-LG I variant, with a significantly higher mass than known variants, appears to display an N-terminal extension possibly related to the signal peptide sequence. This represents the most comprehensive report to date detailing the complexity of donkey milk protein micro-heterogeneity, a prerequisite for discovering new elements to objectify the original properties of donkey's milk.

Influence of the Casein Composite Genotype on Milk Quality and Coagulation Properties in the Endangered Agerolese Cattle Breed.

The aim of this study was the characterization of CSN1S1, CSN2 and CSN3 genetic variability in Agerolese cattle, and the investigation of the effect of casein composite genotypes (CSN1S1, CSN2 and CSN3) on quality and coagulation traits of the corresponding milk. To these purposes, blood and milk from 84 cows were sampled and analysed. Allele frequencies at CSN2 and CSN3 revealed no Hardy-Weinberg equilibrium in the population with a prevalence of allele A2 for CSN2 and allele B for CSN3. BBA1A2AB and BBA2A2AB composite genotypes were the most common in the population. BBA1A2AB showed a higher total solids and fat content (12.70 ± 0.16 and 3.93 ± 0.10, respectively), while BBA2A2BB showed the best coagulation properties (RCT 12.62 ± 0.81; k20 5.84 ± 0.37; a30 23.72 ± 1.10). Interestingly, the A2 allele of CSN2 was very widespread in the population; thus, it will be intriguing to verify if A2A2 Agerolese cattle milk and the derived cheese may have better nutraceutical characteristics.

Complete CSN1S2 Characterization, Novel Allele Identification and Association With Milk Fatty Acid Composition in River Buffalo.

The αs2-casein is one of the phosphoproteins secreted in all ruminants' milk, and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterized in detail in buffaloes with only two alleles detected (reported as alleles A and B), and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realized at the donor splice site of exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, six in the promoter, and three SNPs in the coding region (g.11072C>T, g.12803A>T, and g.14067A>G) with two of them responsible for amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at the donor splice site of exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E, and F) responsible for seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form, and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, and B2 are deleted alleles because all are characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes, and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acid composition was carried out. The SNP g.14067A>G showed a significant association (P < 0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker-assisted selection programs aiming for the improvement of buffalo milk fatty acid composition.

Casein Gene Cluster in Camelids: Comparative Genome Analysis and New Findings on Haplotype Variability and Physical Mapping.

The structure of casein genes has been fully understood in llamas, whereas in other camelids, this information is still incomplete. In fact, structure and polymorphisms have been identified in three (CSN1S1, αs1-CN; CSN2, ß-CN; CSN3, κ-CN) out of four casein genes, whereas controversial information is available for the CSN1S2 (αs2-CN) in terms of structure and genetic diversity. Data from the genome analysis, whose assembly is available for feral camel, Bactrian, dromedary, and alpaca, can contribute to a better knowledge. However, a majority of the scaffolds available in GenBank are still unplaced, and the comparative annotation is often inaccurate or lacking.Therefore, the aims of this study are 1) to perform a comparative genome analysis and synthesize the literature data on camelids casein cluster; 2) to analyze the casein variability in two dromedary populations (Sudanese and Nigerian) using polymorphisms at CSN1S1 (c.150G > T), CSN2 (g.2126A > G), and CSN3 (g.1029T > C); and 3) to physically map the casein cluster in alpaca. Exon structures, gene and intergenic distances, large insertion/deletion events, SNPs, and microsatellites were annotated. In all camelids, the CSN1S2 consists of 17 exons, confirming the structure of llama CSN1S2 gene. The comparative analysis of the complete casein cluster (∼190kb) shows 12,818 polymorphisms. The most polymorphic gene is the CSN1S1 (99 SNPs in Bactrian vs. 248 in dromedary vs. 626 in alpaca). The less polymorphic is the CSN3 in the Bactrian (22 SNPs) and alpaca (301 SNPs), whereas it is the CSN1S2 in dromedary (79 SNPs). In the two investigated dromedary populations, the allele frequencies for the three markers are slightly different: the allele C at CSN1S1 is very rare in Nigerian (0.054) and Sudanese dromedaries (0.094), whereas the frequency of the allele G at CSN2 is almost inverted. Haplotype analysis evidenced GAC as the most frequent (0.288) and TGC as the rarest (0.005). The analysis of R-banding metaphases hybridized with specific probes mapped the casein genes on chromosome 2q21 in alpaca. These data deepen the information on the structure of the casein cluster in camelids and add knowledge on the cytogenetic map and haplotype variability.

Casein composition and differential translational efficiency of casein transcripts in donkey's milk.

The amount of the four caseins (αs1, αs2, ß and κ-CN) in donkey milk was evaluated by Urea-PAGE analysis at pH 8.6, followed by immuno-detection with polyclonal antibodies, coupled to densitometric analysis. The results showed the percentage of each casein in decreasing order: ß (54.28) > αs1 (35.59) > αs2 (7.19) > κ-CN (2.79). The mRNA quantification of donkey casein transcripts, carried out by RT-qPCR, showed that the average percentage of corresponding gene transcripts (CSN2, CSN1S1, CSN1S2 I and CSN3) was 70.85, 6.28, 14.23 and 8.65, respectively. The observed translation efficiency, assessed as percentage of single milk casein fraction out of single percentage of transcript, was 0.76, 5.66, 0.50 and 0.32, respectively. The analysis of the sequences flanking the start codon, the codon usage frequencies and the coding sequence length might explain, at least in part, the differential transcriptional and translational rate observed among the casein transcripts.

A fast and reliable polymerase chain reaction method based on short interspersed nuclear elements detection for the discrimination of buffalo, cattle, goat, and sheep species in dairy products.

OBJECTIVE: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the α-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). METHODS: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). RESULTS: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources (0.25 ng/µL). CONCLUSION: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

Milk microRNA-146a as a potential biomarker in bovine tuberculosis.

In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

Genetic characterization of the oxytocin-neurophysin I gene (OXT) and its regulatory regions analysis in domestic Old and New World camelids.

Oxytocin is a neurohypophysial peptide linked to a wide range of biological functions, including milk ejection, temperament and reproduction. Aims of the present study were a) the characterization of the OXT (Oxytocin-neurophysin I) gene and its regulatory regions in Old and New world camelids; b) the investigation of the genetic diversity and the discovery of markers potentially affecting the gene regulation. On average, the gene extends over 814 bp, ranging between 825 bp in dromedary, 811 bp in Bactrian and 810 bp in llama and alpaca. Such difference in size is due to a duplication event of 21 bp in dromedary. The main regulatory elements, including the composite hormone response elements (CHREs), were identified in the promoter, whereas the presence of mature microRNAs binding sequences in the 3'UTR improves the knowledge on the factors putatively involved in the OXT gene regulation, although their specific biological effect needs to be still elucidated. The sequencing of genomic DNA allowed the identification of 17 intraspecific polymorphisms and 69 nucleotide differences among the four species. One of these (MF464535:g.622C>G) is responsible, in alpaca, for the loss of a consensus sequence for the transcription factor SP1. Furthermore, the same SNP falls within a CpG island and it creates a new methylation site, thus opening future possibilities of investigation to verify the influence of the novel allelic variant in the OXT gene regulation. A PCR-RFLP method was setup for the genotyping and the frequency of the allele C was 0.93 in a population of 71 alpacas. The obtained data clarify the structure of OXT gene in domestic camelids and add knowledge to the genetic variability of a genomic region, which has received little investigation so far. These findings open the opportunity for new investigations, including association studies with productive and reproductive traits.

New Insights on Water Buffalo Genomic Diversity and Post-Domestication Migration Routes From Medium Density SNP Chip Data.

The domestic water buffalo is native to the Asian continent but through historical migrations and recent importations, nowadays has a worldwide distribution. The two types of water buffalo, i.e., river and swamp, display distinct morphological and behavioral traits, different karyotypes and also have different purposes and geographical distributions. River buffaloes from Pakistan, Iran, Turkey, Egypt, Romania, Bulgaria, Italy, Mozambique, Brazil and Colombia, and swamp buffaloes from China, Thailand, Philippines, Indonesia and Brazil were genotyped with a species-specific medium-density 90K SNP panel. We estimated the levels of molecular diversity and described population structure, which revealed historical relationships between populations and migration events. Three distinct gene pools were identified in pure river as well as in pure swamp buffalo populations. Genomic admixture was seen in the Philippines and in Brazil, resulting from importations of animals for breed improvement. Our results were largely consistent with previous archeological, historical and molecular-based evidence for two independent domestication events for river- and swamp-type buffaloes, which occurred in the Indo-Pakistani region and close to the China/Indochina border, respectively. Based on a geographical analysis of the distribution of diversity, our evidence also indicated that the water buffalo spread out of the domestication centers followed two major divergent migration directions: river buffaloes migrated west from the Indian sub-continent while swamp buffaloes migrated from northern Indochina via an east-south-eastern route. These data suggest that the current distribution of water buffalo diversity has been shaped by the combined effects of multiple migration events occurred at different stages of the post-domestication history of the species.

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

BACKGROUND: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. METHODS: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. RESULTS: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. CONCLUSION: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.

A novel polymorphism in the oxytocin receptor encoding gene (OXTR) affects milk fatty acid composition in Italian Mediterranean river buffalo.

The oxytocin receptor, also known as OXTR, is a protein which functions as receptor for the hormone and neurotransmitter oxytocin and the complex oxytocin-oxytocin receptor plays an important role in the uterus during calving. A characterisation of the river buffalo OXTR gene, amino acid sequences and phylogenetic analysis is presented. The DNA regions of the OXTR gene spanning exons 1, 2 and 3 of ten Mediterranean river buffalo DNA samples were analysed and 7 single nucleotide polymorphisms were found. We focused on the g.129C > T SNP detected in exon 3 and responsible for the amino acid replacement CGCArg > TGCCys in position 353. The relative frequency of T allele was of 0·257. An association study between this detected polymorphism and milk fatty acids composition in Italian Mediterranean river buffalo was carried out. The fatty acid composition traits, fatty acid classes and fat percentage of 306 individual milk samples were determined. Associations between OXTR g.129C > T genotype and milk fatty acids composition were tested using a mixed linear model. The OXTR CC genotype was found significantly associated with higher contents of odd branched-chain fatty acids (OBCFA) (P < 0·0006), polyunsaturated FA (PUFA n 3 and n 6) (P < 0·0032 and P < 0·0006, respectively), stearic acid (C18) (P < 0·02) and lower level of palmitic acid (C16) (P < 0·02). The results of this study suggest that the OXTR CC animals might be useful in selection toward the improvement of milk fatty acid composition.

Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5' flanking region and 478/477 nucleotides at the 3' flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Sequential cross-species chromosome painting among river buffalo, cattle, sheep and goat: a useful tool for chromosome abnormalities diagnosis within the family Bovidae.

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.