Development of an In Vitro Goat Mammary Gland Model: Establishment, Characterization, and Applications of Primary Goat Mammary Cell Cultures

Alternatives to animal experiments, based on in vitro methodologies, have been suggested and adopted in the last decades in order to completely substitute or to reduce animal numbers in in vivo assays. In this chapter we describe methods for establishment, maintenance, and characterization of primary goat mammary epithelial cell cultures (pgMECs) and possible applications for which the derived primary cell model can be used instead of in vivo experiments. The established cell lines were grown in vitro for several passages and remained hormone and immune responsive and capable of milk protein synthesis. Knowledge on goat mammary cells and their manipulation is applicable to different fields of research; for example, it could be used in basic research to study mammary development and lactation biology, in agriculture to enhance lactation yield and persistency or to produce milk with special characteristics, in biopharma to express recombinant proteins in goat milk, or in biomedicine to study lactation, mammary development, and pathology, including neoplasia. The established cells represent an adequate surrogate for mammary gland; were successfully used to study mammary gland immunity, lactation, and mammary stem/progenitor cells; and have a potential to be used for other purposes

Mining for Structural Variations in Next-Generation Sequencing Data

Genomic structural variations (SVs) are genetic alterations that result in duplications, insertions, deletions, inversions, and translocations of segments of DNA covering 50 or more base pairs. By changing the organization of DNA, SVs can contribute to phenotypic variation or cause pathological consequences as neurobehavioral disorders, autoimmune diseases, obesity, and cancers. SVs were first examined using classic cytogenetic methods, revealing changes down to 3 Mb. Later techniques for SV detection were based on array comparative genome hybridization (aCGH) and single-nucleotide polymorphism (SNP) arrays. Next-generation sequencing (NGS) approaches enabled precise characterization of breakpoints of SVs of various types and sizes at a genome-wide scale. Dissecting SVs from NGS presents substantial challenge due to the relatively short sequence reads and the large volume of the data. Benign variants and reference errors in the genome present another dimension of problem complexity. Even though a wide range of tools is available, the usage of SV callers in routine molecular diagnostic is still limited. SV detection algorithms relay on different properties of the underlying data and vary in accuracy and sensitivity; therefore, SV detection process usually utilizes multiple variant callers. This chapter summarizes strengths and limitations of different tools in effective NGS SV calling

Aortic Stenosis in Dogs and Cats: Past, Present and Future

Aortic stenosis is one of the three most common congenital heart defects in dogs and less frequent in cats. Most dogs or cats have subvalvular type of stenosis; valvular or supravalvular types are less frequent. Heart failure is seldom a consequence of aortic stenosis; most dogs with heart failure have a concurrent disease. The most common accompanying diagnosis is pulmonic stenosis, especially in the Boxer breed. Screening programs seem to have efficiently lowered the incidence of aortic stenosis in dogs. Genetic evidence for aortic stenosis has been shown in Golden Retriever, Newfoundland and Dogue de Bordeaux; however, the genetic background of aortic stenosis at molecular level remains unclear

History of Lipizzan horse maternal lines as revealed by mtDNA analysis

Sequencing of the mtDNA control region (385 or 695 bp) of 212 Lipizzans from eight studs revealed 37 haplotypes. Distribution of haplotypes among studs was biased, including many private haplotypes but only one haplotype was present in all the studs. According to historical data, numerous Lipizzan maternal lines originating from founder mares of different breeds have been established during the breed's history, so the broad genetic base of the Lipizzan maternal lines was expected. A comparison of Lipizzan sequences with 136 sequences of domestic- and wild-horses from GenBank showed a clustering of Lipizzan haplotypes in the majority of haplotype subgroups present in other domestic horses. We assume that haplotypes identical to haplotypes of early domesticated horses can be found in several Lipizzan maternal lines as well as in other breeds. Therefore, domestic horses could arise either from a single large population or from several populations provided there were strong migrations during the early phase after domestication. A comparison of Lipizzan haplotypes with 56 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for at least 11% of the Lipizzans. A distribution of haplotype-frequencies was unequal (0.2%–26%), mainly due to pedigree errors and haplotype sharing among founder mares

Genetska karakterizacija istarske koze: polazište dugoročnog očuvanja

Istrian goat is an autochthonous Croatian breed which inhabited Istrian peninsula and was important in milk production and human nutrition, especially for poor people. For centuries Istrian goat was a recognizable heraldic symbol of Istria, but in her real form almost disappeared from the breeding area. The revitalization and reaffirmation of Istrian goat began with several dozen remaining breeding animals, after a decade-long breeding ban. Genetic characterization of the Istrian goat population is necessary for providing insight into the state of genetically preserved structure within population as well as positioning Istrian goats within phylogenetically related breeds. Microsatellite and mtDNA analysis of reproductive individuals of Istrian goat and related breeds, Croatian White goat and Saanen goat was carried out. In the population of Istrian goat, higher allelic variability (nA = 9.7; AR = 7.4) were found as well as significant genetic distance (FST = 0.068 - 0.086) in relation to other two breeds. Such results indicated that Istrian goat constitutes a separate genetic identity. The observed ten haplotype sequences of the D-loop mtDNA also confirm the significant genetic richness of the maternal hereditary component. The observed haplotypes in the population of Istrian goat belong to lineage A. A smaller number of haplotypes shows similarity to the group of “white” goats, indicating traces of earlier limited but targeted crossing of Istrian goats. The genetic profile analysis of Istrian goats indicates a high level of genetic variability and provides guidelines for a long-term conservation program. The preserved genetic and promising potential of milk production of Istrian goat makes a significant basis for her economic reaffirmation. Orientation of the breed towards milk production could be an efficient strategy for its effective preservation.Istarska koza jedna je od autohtonih pasmina koja je nastanjivala područje Istarskog poluotoka i bila važna u proizvodnji mlijeka te prehrani, posebice siromašnog stanovništva. Kroz stoljeća je činila prepoznatljivi heraldički simbol Istre, no u svojem stvarnom obličju gotovo je iščezla iz uzgojnog područja. Revitalizacija i reafirmacija istarske koze započela je nakon višedesetljetne zabrane uzgoja na uzgojnoj bazi od nekoliko desetaka preostalih rasplodnih jedinki. Genetska karakterizacija preostale populacije istarske koze nužna je za uvid u stanje unutar populacijske genetske očuvanosti kao i pozicioniranje naspram filogenetski srodnih pasmina koza. Provedena je analiza strukture mikrosatelita i mtDNA rasplodnih jedinki istarske koze te srodnih pasmina koza, hrvatske bijele i sanske koze. U populaciji istarske koze naspram druge dvije pasmine koza utvrđena je veća alelna varijabilnost (nA = 9,7; AR = 7,4) te značajna genetska udaljenost (FST=0,068-0,086) što ukazuje da istarska koza čini zaseban genetski identitet. Zapaženih deset haplotipova sekvenci D-loop regije mtDNA također potvrđuje značajno genetsko bogatstvo maternalne nasljedne komponente. Uočeni haplotipovi u populaciji istarske koze pripadaju haplogrupi A. Manji broj haplotipova pokazuje srodnost naspram pasmina iz skupine bijelih koza, što indicira na tragove ranijih ograničenih ciljanih oplemenjivanja istarske koze. Analiza genetskog profila ukazuje na visoku razinu očuvanosti genetske varijabilnosti te nudi smjernice dugoročno održivog konzervacijskog programa. Očuvani genetski i izgledni proizvodni (mliječni) potencijal istarske koze čini značajnu osnovu njene gospodarske reafirmacije

Genomic characterization of the three Balkan Livestock Guardian Dogs

Balkan Livestock Guardian Dogs (LGD) were bred to help protect sheep flocks in sparsely populated, remote mountainous areas in the Balkans. The aim of this study was genomic characterization (107,403 autosomal SNPs) of the three LGD breeds from the Balkans (Karst Shepherd, Sharplanina Dog, and Tornjak). Our analyses were performed on 44 dogs representing three Balkan LGD breeds, as well as on 79 publicly available genotypes representing eight other LGD breeds, 70 individuals representing seven popular breeds, and 18 gray wolves. The results of multivariate, phylogenetic, clustering (STRUCTURE), and FST differentiation analyses showed that the three Balkan LGD breeds are genetically distinct populations. While the Sharplanina Dog and Tornjak are closely related to other LGD breeds, the Karst Shepherd is a slightly genetically distinct population with estimated influence from German Shepard (Treemix analysis). Estimated genomic diversity was high with low inbreeding in Sharplanina Dog (Ho = 0.315, He = 0.315, and FROH>2Mb = 0.020) and Tornjak (Ho = 0.301, He = 0.301, and FROH>2Mb = 0.033) breeds. Low diversity and high inbreeding were estimated in Karst Shepherds (Ho = 0.241, He = 0.222, and FROH>2Mb = 0.087), indicating the need for proper diversity management. The obtained results will help in the conservation management of Balkan LGD dogs as an essential part of the specific grazing biocultural system and its sustainable maintenance

Mitochondrial variability of Small Međimurje dog

There are six native dog breeds in Croatia recognized by the Federation Cynologique International (FCI) and one national dog breed Small Međimurje dog (MEDI) still unrecognized by the FCI. To promote breed we have analysed mitochondrial DNA control region (CR-mtDNA) sequence (551-bp) in 35 Small Međimurje dogs sampled in Međimurje County. After comparison with 33 worldwide distributed dog breeds (N=115 samples), three main canine CR-mtDNA haplogroups (A, B and C) were observed in Small Međimurje dogs. Median-joining tree showed that MEDI forms six haplotypes presented in haplogroup C (H3 is the most frequent in MEDI population), haplogroup A (haplotypes H2, H5 and H7) and in haplogroup B (haplotypes H4 and H6). The results presented in this study correspond to other mtDNA studies of native dog breeds. For the better genetic description of MEDI and for the optimal future breeding management, further analyses of nuclear genome are recommended