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Institute for animal production system in mediterranean environment

Proteomic Analysis of bovine tissues and biological fluids  (2002)

To provide a complete molecular description of the fundamental mechanisms necessary for cell life, a series of studies on different organisms are now undergoing to characterize the whole pattern of proteins expression. Different Proteome projects have been activated in the world and their interest grows together with the increased availability of new genetic data. The scientific community realized that, once the entire genome of an organism have been sequenced, to understand the molecular machinery regulating its cellular processes is essential to identify the genes that are really expressed in each cell, tissues and/or biological fluids and the final structure of the functionally active polypeptide chains. No data have been reported before on the pattern of expression of the proteins in bovine tissues and/or biological fluids, although a number of nucleotide sequences (almost 5000) was deposited in databases. Aim of our investigation was to compensate this gap. A series of studies was performed on Holstein Friesian bovines, the most diffused breed in the world. Different tissues and biological fluids have been homogenized and the protein content was extracted according to conventional procedures. Two dimensional electrophoresis (2D-PAGE) experiments allowed the separation of the thousands polypeptide components occurring in each sample. On this basis, high resolution 2D-PAGE reference maps have been defined for liver, kidney, skeletal muscle, plasma and red blood cells on healthy animals in basal state. Each protein component was identified by advanced mass spectrometry procedures. These analysis was performed using matrix assisted laser desorption ionization (MALDI) and/or electrospray ionization (ESI) techniques on protein digests eventually solved by micro-chromatography separation.

Image - liver.jpg
Image - skeletal muscle.jpg
Document - focus.doc


Proteomic Analysis of bovine tissues and biological fluids  (2002)

Nonostante la limitatezza dei dati disponibili sul genoma bovino, rispetto a quelli relativi a uomo e topo, sono state identificate le proteine più abbondanti in ciascun tessuto. In particolare, è stato ottenuto esito positivo per diverse centinaia di specie proteiche che sono state così associate ai rispettivi geni. I dati ottenuti potranno in futuro risultare basilari per la corretta comprensione dei complessi processi fisiologici e/o metabolici che regolano l’adattamento di quest’animale consentendo, al tempo stesso, l’identificazione di componenti proteiche e quindi di attività enzimatiche peculiari per la sua vita. Sono in corso analisi degli stessi tessuti per animali portatori di malattie ricorrenti nell’allevamento. Tali ricerche stanno evidenziando una variazione del profilo proteomico specifica per ciascun tipo di infezione e si sta procedendo all’identificazione delle proteine associabili a ciascuno stato patologico. Tali marcatori molecolari consentiranno di formulare, in futuro, nuove ipotesi a livello molecolare per queste malattie. Va, inoltre, ricordato che diversi prodotti di lavorazione di fluidi e/o tessuti bovini sono quotidianamente assunti dall’uomo durante la sua alimentazione. Una completa caratterizzazione della componente proteica di ciascuna materia prima risulta di fondamentale interesse per qualsiasi considerazione di natura nutrizionale e salutistica per l’uomo. I dati fin qui ottenuti sono stati resi disponibili alla comunità scientifica creando un sito WEB dedicato di consultazione (http://www.iabbam.na.cnr.it/biochem), collegato agli altri 2D-PAGE databases presenti sulla rete e consorziati al WORLD-2D-PAGE database. L’utente, avvalendosi dei risultati da noi conseguiti, potrà, dopo aver realizzato il suo esperimento 2D-PAGE nelle condizioni sperimentali ivi descritte, analizzare direttamente on line i suoi esperimenti e confrontare i dati ottenuti con quelli presenti sul sito. Nuove proteine identificate nel tempo da parte del nostro laboratorio saranno rese disponibili sul sito.


The cytogenetic map in the genetic improvement of domestic river buffalo (Bubalus bubalis, 2n=50)  (2003)

Since the construction and publication of river buffalo standard karyotype with six different chromosome banding techniques, the laboratory of Animal Cytogenetics and Gene Mapping of the ISPAAM has been focalised part of its scientific activity in the genetic improvement of river buffalo, a species of great economic importance in the world, with about 130,000 million of heads, and in Italy, with about 220,000, mostly in Campania region (Southern Italy).
Our studies have been addressed to both clinical cytogenetics on female river buffaloes with reproductive disturbances (25% of these females showed sex chromosome abnormalities) and molecular cytogenetics by physical mapping of various loci by means fluorescence in situ hybridization (FISH) techniques and probe DNA containing both type I markers (expressed genes) and type II markers (microsatellites). A cytogenetic map with 293 loci of all 31 cattle syntenic groups has been published: 171 loci were of type I and 122 of type II. This maps extends our knowledge on the physical organization of river buffalo genome and is a useful tool for:
(a) comparative genome studies with other species (human, cattle) with richer genetic and cytogenetic maps and with all genes sequenced;
(b) assisted selection of reproducers with molecular markers;
(c) detection of chromosome abnormalities;
(d) selection of chromosome regions containing genes of interest (animal production and disease resistances).
In particular, the order of loci found in some homologous chromosomes of bovids allowed to reveal the first autosomal mutation which differentiated the autosomes of bovids. Indeed, a proximal region of chromosome 9 of cattle and river buffalo (subfamily Bovinae) has been translocated in the proximale region of chromosome 14 of goata e sheep (subfamily Caprinae) (Iannuzzi et al. 2001). Furthermore, the comparative cytogenetic maps of X-chromosomes of river buffalo (acrocentric), cattle (submetacentric) and sheep (acrocentric with visible short arms) have demonstrated that this chromosome had a more complex evolution by the use of centromeric transpositions (between river buffalo and cattle) and transpositions of at least 4 chromosome regions between subfamilies Bovinae and Caprinae(Iannuzzi et al. 2000).

Image - Figura 1b.
Image - Figura 1.
Document - Table 1.


Formulation of the Dairy Replacement Problem by using a Markovian Decision Process Approach and Linear Programming.  (2003)

The "replacement problem" has been frequently studied in literature utilising different methodologies. The method traditionally used to solve this problem is to formulate it as a decisional Markovian Process and to optimise it using Dynamic Programming (DP). There are, however, problems associated with the use of DP. The decision to cull is based solely upon the expected performance of the animal in question and that of it's replacement. Therefore, information concerning the cows performance in relation to the remaining herd has been ignored. This is especially relevant were the producer is trying to increase yields by genetic selection.
A developed methodology (Yates and Rehman, 1998) suggests the use of Linear Programming (LP) to solve the "replacement problem", but the model presented is simplified.
The purpose of this project was to include more initial states to simulate a greater variation in genetic merit and other representative aspects.
Important parameters evaluated are:
- the age at first calving which was shown to be a significant factor in the occurrence of mastitis;
- the estimation of conception rates, calf survival rates, calf mortality rate, culling rate, first service pregnancy rates, failure to conceive rates; - the prediction of annual milk yields, milk yield for all the different genetic states and for each parity and year; - breeding costs, milk and meat prices. The model was developed over a 15 years decision horizon. Whilst the genetic merit, in terms of milk production, of an individual animal is different from that of any other animal, it was possible to group animals into similar bands based on their production. In this model were taken into account two different production levels (high and low milk yield) and cows with 7 different parities (age in terms of lactations). The total herd increase in milk yield also depends upon the conception and mortality rates, known collectively as the "replacement rate". This genetic turn-over is therefore a gradual process dependent upon the past and current state of the herd and the one desired in the future. Before specifying the Markovian decision process involved in this model, it was first necessary to define all the possible genetic states over the 15 year period which are 584, the model so specified was constructed using XPRESS, a software package to solve Linear Programming problems. Other factors were included like land, capital and labour utilisation, nutritional requirements.
The proposed solution can be seen as an innovation to introduce in dairy farms to help the farmers in taking decisions to optimise the management from an economic point of view and taking into account various problems that can raise in a long term planning.


The cytogenetic map in domestic sheep (Ovis aries, 2n=54)   (2007)

The sheep (Ovis aries, 2n=54) is one of the most economically important livestock species, especially in the Mediterranean Countries. Although linkage and radiation hybrid maps are available, cytogenetic maps found in Web sites are still very poor and built, among other things, on old standard karyotypes. The recent construction of standard karyotypes in domestic bovids, including that of the sheep, based on both high resolution banded models, use of molecular markers and homologous chromosome nomenclatures, has allowed us to present an advanced cytogenetic map of the sheep with 452 loci, most of them mapped in our laboratory by FISH technique on R-banded chromosomes. This cytogenetic map will be very useful in clinical cytogenetics (study of chromosomal abnormalities and their relationship with fertility), molecular cytogenetics (comparison of cytogenetic maps - order of the loci- between related and unrelated species), evolutionary cytogenetics (study of chromosomal rearrangements that have differentiated species during their evolution) and physical anchoring to specific chromosomal regions of both linkage and radiation hybrid maps.

Document - An advanced sheep (Ovis aries, 2n =
Document - Tabella Loci Mappati


Environmental biomonitoring by cytogenetic test on animals grazing in risk areas   (2007)

Animals, especially those fed with natural pasture, represent real environmental sentinels. Indeed, being them fed with local plants and soil (taken together with grass) in place of breeding, give an idea of the degree of contamination of food and of the food chain. Indeed, many mutagenic substances present in the environment pass through animal products, to humans, laying the foundations for the onset of abnormal clinical picture (reproductive, hormonal and immune disorders). Check these animals mean, then, check the food chain and, indirectly, the human health. As known, many mutagenic leave clear signs at chromosomal level (gap, chromosomal and chromatid breaks, fragments) which denote genomes potentially exposed to genetic mutations (incorrect cellular divisions both at the mitosis and meiosis). Cytogenetic studies conducted on sheep reared in the province of Naples and exposed at low and high levels of dioxin have revealed a pronounced chromosomal fragility found to be, respectively, 4, 8 and 14 times in sheep exposed to 5, 39 and 51 pg/g fat of dioxins, respectively, than that found in sheep control (not exposed to dioxins). Even levels of sister chromatid exchanges (SCEs) were significantly higher (P<0.001) in sheep exposed to dioxins, compared to those found in the controls.

Image - Metafase con SCEs
Image - Metafase con rotture cromatidiche
Document - Tabelle diossine


 
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