Marginal arid zones in the south Mediterranean are faced with the dramatic departure of their labor forces through migration. Interest in the capacity of the dromedary species to enhance desert ecosystems and to be a potential lever of economic development of these marginal zones only started to grow in the last two decades. Based on an empirical survey of 179 stakeholders in four Mediterranean countries, we explored the links of the stakeholders in the dromedary sector in two dimensions: horizontal links with peers and vertical links along the value chain and in resource management. Both descriptive statistics and social network analysis highlight the original organization of the dromedary sector around herders and their social and cultural organization at the territorial level. Therefore, even if milk production and processing start to constitute an opportunity for the young generation who do not necessarily have the financial capacity to invest in a large dromedary herd, this change towards milk valorization can only happen if it is linked with the traditional system based on mobility. Using a systemic approach and working toward multiple valorizations of dromedary products instead of only targeting milk productivity should be explored.

Milk is a primary protein source that has always played a role in mammalian health. Despite the inten-sification of research projects on dromedary and the knowledge of the genetic diversity at the casein loci, the genetic structure of the Tunisian camel popula-tion still needs exploration. This study sought to de-termine the genetic diversity of 3 casein gene variants in 5 Tunisian camel ecotypes: c.150G > T at CSN1S1 (alpha S1-casein), g.2126A > G at CSN2 (beta-casein), and g.1029T > C at CSN3 (& UKappa;-casein). The obtained results were compared with data published on Sudanese and Nigerian camels to establish the level of differentiation within and between populations. A total of 159 blood samples were collected from 5 Tunisian camel ecotypes and the extracted DNA was genotyped by PCR-RFLP. A streamlined genotyping protocol was also developed for CSN3. Results indicated that allele T was quite rare (0.06) at CSN1S1 for all ecotypes. Minor allele frequency was found for G (0.462) in CSN2 except for Ardhaoui Medenine ecotype who deviated from the av-erage CSN2 allele frequency of the total population. Al-lele C showed minor allele frequency of 0.384 in CSN3. Among the Tunisian population, GAT (0.343) was the most represented haplotype in all ecotypes except for Ardhaoui Medenine, where GGC (0.322) was the most frequent one. Significant differences in heterozygosity and local inbreeding were observed across the Tuni-sian, Sudanese, and Nigerian populations, although the global fixation index indicated that only 2.2% of the ge-netic variance is related to ecotype differences. Instead, phylogenetic analysis revealed a closer link between the Tunisian and Sudanese populations through a clade subdivision with 3 main branches among the ecotypes. This study represents the first attempt to understand casein gene variability in Tunisian camels; with further study, milk traits and genetic differentiation among populations can be associated with the history of camel domestication.

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-β, 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-β 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.

Alpha-Lactalbumin (alpha-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for alpha-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

Background/Objectives: The Y chromosome plays a crucial role in male fertility, sex determination, and spermatogenesis, yet it remains poorly characterized in Mediterranean river buffalo (Bubalus bubalis, 2n = 50) because of its high repeat content, extensive heterochromatin, and complex palindromic structures. Although a chromosome-level Y assembly is available for swamp buffalo (2n = 48), no equivalent reference exists for the river type. Methods: To address this gap, Y chromosomes from 10 Mediterranean buffalo bulls were isolated by laser microdissection following peripheral blood culture and whole-chromosome amplification. Probe specificity was verified by FISH, and amplified Y chromosomes were sequenced using Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Sequencing data were assembled and analysed through de novo assembly, repeat identification, sequence alignment, and variant detection. Comparative analyses included alignment to the swamp buffalo Y chromosome and annotation of Y-linked genes using the Bos taurus reference genome. Results: FISH confirmed the specificity of the isolated material, showing strong signals on the Y chromosome and on X/Y PAR and heterochromatic regions. Sequencing generated over 240 million paired-end reads, and de novo assembly produced 566,815 contigs. Repeat analysis identified 3.91% repetitive elements, mainly SINEs, while variant calling detected more than 23,000 variants. Comparative analyses mapped several contigs to the swamp buffalo Y chromosome and Y-linked genes. Annotation against the B. taurus genome identified 26 unique genes, including homologs shared with the X chromosome, and revealed MSY gene duplications, including 10 copies of TSPY and 3 of HSFY. Conclusions: These findings show that laser microdissection with NGS enables effective access to the buffalo Y chromosome, representing a milestone in the characterization of the river type genome, and providing a basis for studies on buffalo male fertility and breeding programs.

Background/Objectives: The domestic yak (Bos grunniens) is a livestock species of major relevance in high-altitude environments and an important model for studying adaptation and reproductive isolation within Bovini. Despite its close phylogenetic relationship with cattle (Bos taurus), yak × cattle hybrids show a marked sex-biased fertility pattern, with fertile females and generally sterile F1 males, suggesting that subtle chromosomal or genomic differences may underlie post-zygotic reproductive barriers. In this study, we performed a cytogenetic characterization of eight adult yak bulls imported and reared in Central Italy using conventional and molecular cytogenetic approaches. Results: GTG-, RBG-, RBA- and CBA-banding confirmed the yak diploid number as 2n = 60 and the fundamental number as NF = 62, with banding patterns highly comparable to the standardized cattle karyotype. CBA-banding showed an X chromosome lacking evident constitutive heterochromatin and a Y chromosome with distal C-positive blocks. Chromosome instability was low, with 3.75% abnormal metaphases, mainly represented by chromatid and iso-chromatid breaks, while the mean sister chromatid exchange (SCE) rate was 5.19 ± 2.14 per cell. Sequential Ag-NOR/RBA staining localized nucleolar organizer regions (NORs) at the telomeres of autosomes 2, 3, 4, 11 and 25, as in cattle. Zoo-FISH using bovine chromosome paints for X, Y, 5 and 15 showed complete hybridization to the corresponding yak chromosomes, and BAC-FISH mapped the Y-linked ZFY and SRY genes to positions homologous to those reported in cattle. A comparative bioinformatics analysis of available yak genome assemblies confirmed the overall genome-wide correspondence with cattle, while revealing chromosome orientation issues and small local inconsistencies that may be relevant for comparative mapping and probe design. Conclusions: Overall, at the resolution tested, these findings support broad macrostructural conservation of yak and cattle karyotypes and provide cytogenetic reference data for yak populations reared outside of their traditional range. The persistence of F1 male sterility despite this large-scale chromosomal conservation suggests that fine-scale sex chromosome differences, particularly involving pseudoautosomal regions, recombination boundaries, or heterochromatin organization, may deserve targeted investigation.