The production of milk by dairy cows far exceeds the nutritional needs of the calf and is vital for the economical use of dairy cattle. High milk yield is a unique production trait that can be ...effectively enhanced through traditional selection methods. The process of lactation in cows serves as an excellent model for studying the biological aspects of lactation with the aim of exploring the mechanistic base of this complex trait at the cellular level. In this study, we analyzed the milk transcriptome at the single-cell level by conducting scRNA-seq analysis on milk samples from two Holstein Friesian cows at mid-lactation (75 and 93 days) using the 10× Chromium platform. Cells were pelleted and fat was removed from milk by centrifugation. The cell suspension from each cow was loaded on separate channels, resulting in the recovery of 9313 and 14,544 cells. Library samples were loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument. After filtering at the cell and gene levels, a total of 7988 and 13,973 cells remained, respectively. We were able to reconstruct different cell types (milk-producing cells, progenitor cells, macrophages, monocytes, dendritic cells, T cells, B cells, mast cells, and neutrophils) in bovine milk. Our findings provide a valuable resource for identifying regulatory elements associated with various functions of the mammary gland such as lactation, tissue renewal, native immunity, protein and fat synthesis, and hormonal response.
Many coat color, behavioral and morphological traits are specific and fixed across cat breeds, with several variants influencing these traits being common among different breeds. In the domestic cat, ...rexoid mutations have been documented in several breeds. In the Cornish Rex, four bp deletion in the
gene has been found to cause a frame shift and a premature stop codon. In addition to the rexoid coat, Cornish Rex cats also have a characteristic head, ear shape and body type. Analysis of the selection signatures in the Cornish Rex genome revealed several regions that are under selective pressure. One of these is located in CFA B4, in the region where the
gene is located. The
gene in Burmese cats disrupts the cranial morphogenesis and causes brachycephaly in the heterozygous state. In our study, we confirmed the presence of a deletion in
in 20 Cornish Rex and in four F1 hybrids between Cornish Rex and domestic cat. However, we did not confirm the presence of the deletion in
in Cornish Rex cats. Genome-wide selection signature analysis was performed using ROH islands and integrated haplotype score (iHS) statistics based on publicly available SNP array data of 11 Cornish Rex cats. The selection signatures were detected on chromosomes A1, A3, C2, B1, B4 and D1.
Local breeds retained unique genetic variability important for adaptive potential especially in light of challenges related to climate change. Our first objective was to perform, for the first time, ...a genome-wide diversity characterization using Illumina GoatSNP50 BeadChip of autochthonous Dreznica goat breed from Slovenia, and five and one local breeds from neighboring Austria and Italy, respectively. For optimal conservation and breeding programs of endangered local breeds, it is important to detect past admixture events and strive for preservation of purebred representatives of each breed with low or without admixture. In the second objective, we hence investigated the effect of inclusion or exclusion of outliers from datasets on genetic diversity and population structure parameters. Distinct genetic origin of the Dreznica goat was demonstrated as having closest nodes to Austrian and Italian breeds. A phylogenetic study of these breeds with other goat breeds having SNP data available in the DRYAD repository positioned them in the alpine, European and global context. Swiss breeds clustered with cosmopolitan alpine breeds and were closer to French and Spanish breeds. On the other hand, the Dreznica goat, Austrian and Italian breeds were closer to Turkish breeds. Datasets where outliers were excluded affected estimates of genetic diversity parameters within the breed and increased the pairwise genetic distances between most of the breeds. Alpine breeds, including Dreznica, Austrian and Italian goats analyzed here, still exhibit relatively high levels of genetic variability, homogeneous genetic structure and strong geographical partitioning. Genetic diversity analyses revealed that the Slovenian Dreznica goat has a distinct genetic identity and is closely related to the neighboring Austrian and Italian alpine breeds. These results expand our knowledge on phylogeny of goat breeds from easternmost part of the European Alps. The here employed outlier test and datasets optimization approaches provided an objective and statistically powerful tool for removal of admixed outliers. Importance of this test in selecting the representatives of each breed is warranted to obtain more objective diversity parameters and phylogenetic analysis. Such parameters are often the basis of breeding and management programs and are therefore important for preserving genetic variability and uniqueness of local rare breeds.
ABSTRACT A selection of commercially available antibodies, targeted against markers employed in studies of mammary gland biology, was tested to determine their reactivity in goat mammary tissue and ...the derived tissue cultures. Expression of the markers smooth muscle actin ( SMA ), selected keratins ( KRT ) 5, 14, 18 , and 19 , CD24 molecule ( CD24 ), epithelial cell adhesion molecule ( EPCAM ), mucin 1 ( MUC1 ), integrin subunit alpha 6 ( ITGA6; CD49F ), integrin subunit beta 1 ( ITGB1; CD29 ), cyclin dependent kinase inhibitor 1A ( CDKN1A; p21 ), membrane metalloendopeptidase ( MME; CD10 ), progesterone receptor ( PGR ), estrogen receptor 1 ( ESR1 ), and vimentin ( VIM ) was first assessed on mRNA level, using reverse transcription PCR (RT-PCR). The reactivity of the antibodies in the tissue sections and the derived tissue cultures was determined using immunofluorescence. The result of this study is a list of commercially available antibodies, raised mostly against human antigens, which also recognize orthologous goat antigens and are useful for characterization of different mammary cell types. Additionally, primers that are functional in detecting expression of mammary lineage markers in goat mammary mRNA isolates were validated. The suggested antibodies, PCR primers, and the described methods are of practical value for researchers interested in characterization and isolation of cell types comprising mammary tissue of goats and probably other ruminants.
Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems ...with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-specific markers. Characterization of the primary culture was performed in parallel with localization studies of the markers in the skin histological sections, using commercially available antibodies. Relative expression of typical differentiation stage-specific markers was determined in the established cell lines, using RT-qPCR. Basal (proliferating) keratinocytes were the predominant cell type in the established cell lines, but low expression of post-mitotic keratinocyte markers was also detected. Differences in marker expression were observed neither between the peK originating from two different animals nor between the peK established with two different methods (enzymatically or by explanting). The described methods in combination with the suggested characterization and differentiation markers are suitable for establishment of proliferating peK and evaluation of their differentiation status.
The Lipizzan horse breed was established in the 16
century in Lipica and later spread throughout Central and Eastern Europe. In addition to six classical stallion lines, 17 classical mare family ...lines were established in the old Lipica stud. Additional 44 mare family lines, including local lines, were established in other Lipizzan studs. The founders of the classical Lipizzan mare family lines date back to the period between the 18
and 20
century and came from various breeds, including the Karst, Spanish, Italian, Kladruber and Arabian horses. The history of the Lipizzan mare family lines is well described and supported by the availability of 44 nucleotide sequences of the mtDNA control region. In this study, the mtDNA control region of 23 Serbian Lipizzans, representing the established Lippizan mare family lines, as well as the local mare family line Zora, have been sequenced. The objective of this study was to test whether the Zora mare family line could be distinguished from other Lipizzan mare family lines according to the mtDNA sequence. Sequences were aligned to the entire set of mtDNA control region sequences representing 44 Lipizzan mtDNA haplotypes. Based on the alignment, a median-joining network was constructed. The individuals in this study formed a cluster with the existing Lipizzan mare family lines. However, a total of ten individuals belonging to the local Zora mare family line formed a compact cluster close to the mtDNA haplotypes, representing the classical mare family lines Wera, Betalka, and Monteaura. Additional pedigree data are needed to clarify the origin of the mtDNA haplotype variant identified in this study.
Cryptorchidism is one of the most frequent congenital birth defects in male children and is present in 2-4% of full-term male births. It has several possible health effects including reduced ...fertility, increased risk for testicular neoplasia, testicular torsion, and psychological consequences. Cryptorchidism is often diagnosed as comorbid; copresent with other diseases. It is also present in clinical picture of several syndromes. However, this field has not been systematically studied. The aim of the present study was to catalog published cases of syndromes which include cryptorchidism in the clinical picture and associated genomic information.
The literature was extracted from Public/Publisher MEDLINE and Web of Science databases, using the keywords including: syndrome, cryptorchidism, undescended testes, loci, and gene. The obtained data was organized in a table according to the previously proposed standardized data format. The results of the study were visually represented using Gephi and karyotype view.
Fifty publications had sufficient data for analysis. Literature analysis resulted in 60 genomic loci, associated with 44 syndromes that have cryptorchidism in clinical picture. Genomic loci included 38 protein-coding genes and 22 structural variations containing microdeletions and microduplications. Loci, associated with syndromic cryptorchidism are located on 16 chromosomes. Visualization of retrieved data is presented in a gene-disease network.
The study is ongoing and further studies will be needed to develop a complete catalog with the data from upcoming publications. Additional studies will also be needed for revealing of molecular mechanisms associated with syndromic cryptorchidism and revealing complete diseasome network.
The evaluation of the genetic structure of autochthonous pig breeds is very important for conservation of local pig breeds and preservation of diversity. In this study, 18 microsatellite loci were ...used to detect genetic relationship between autochthonous pig breeds Black Slavonian (BS), Turopolje pig (TP), and Croatian wild boar and to determine phylogenetic relationship among Croatian autochthonous pig breeds and certain Asian and European pigs using the mitochondrial DNA (mtDNA) D-loop sequence polymorphism. Relatively high degree of genetic variation was found between the observed populations. The analysis of mtDNA showed that haplotypes of the studied pig populations are different from the other European and Chinese haplotypes. BS pigs showed some similarities with Mangalitsa and Duroc breeds. The genetic distances of TP can be explained by high degree of inbreeding during the past century. Despite the European origin of Croatian pig breeds with some impact of Chinese breeds in the past, the results of present study show that genetic diversity is still pronounced within investigated breeds. Furthermore, the genetic diversity is even more pronounced between Croatian breeds and other European and Chinese pig breeds. Thus, conservation of Croatian pig breeds will contribute to overall genetic diversity preservation of pig breeds.
The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone ...receptor (PGR) was quantified in mammary tissue‐derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up‐regulation of ESR1 (1.36‐ to 12.35‐fold) and in down‐regulation of PGR (‐2.53‐ to ‐3.62‐fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER‐α) positive cells do not exclusively belong to the luminal lineage and that ER‐α and PGR can be expressed individually or co‐expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone‐responsive and represent a useful surrogate for mammary tissue in research experiments.
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