Marco Zedda

It is often difficult, if not impossible, to separate postcranial elements of species, such as sheep and goats. Distinguishing between the skeletal remains of these species is important in a variety of scientific fields, such as comparative anatomy, taxonomy, biomechanical engineering , as well as zooarchaeology and palaeontology. The aim of this study was to assess morphological and mor-phometric differences of microscopic and macroscopic characteristics of the femur of sheep and goats, to be used to distinguish between these species. Approximately one hundred sheep and goat femora were examined. Microscopic results indicated that osteon and Haversian canal diameters are parameters useful to the distinction between sheep and goats. Twelve macroscopical features, which successfully separated goat and sheep femora, were identified and discussed, four of which were described for the first time with a mathematical approach. These differences could be related to the behavioural and locomotion patterns of the two species.

Conservation of farm animal genetic resources is of fundamental importance for the study of the relationships among breeds. The aim of this study was to evaluate the usefulness of the nuclear fluorescence inter simple sequence repeat (FISSR) markers in order to shed light on the genetic biodiversity of domestic animals. Two modifications of the original technique were made so as to make it more suitable for routine needs. The modified FISSR protocol was tested on different breeds of goat and donkey from Sardinia, a Mediteranean island known for its biodiversity. The two species are affected by different management problems in Sardinia: goats need a traceability of local products from different breeds, whereas donkeys are drastically reduced in number. The primers used were found to be very informative suggesting that the modified FISSR can be successfully applied in studies on different breeds of animal species without expensive experimentations. This method could be of interest in many geographic regions where there are more breeds of the same species with similar morphological features and different genetic pattern. The strongest point of this method is its low cost.

The retractor penis muscle originates from the vertebrae in pigs, horses, cattle and goats; it arises from the rectum in sheep. In all the species considered, sensitive innervation was found. This was represented by encapsulated receptors. Nervous vegetative supply, made up of isolated or assembled ganglion cells, was also present. Among the numerous sensory nerve endings found in pigs, goats and sheep were Pacinian, Pacinian-like, Golgi-Mazzoni corpuscles and Krause's end bulbs (genital corpuscles). Few Pacinian and Pacinian-like corpuscles were found in cattle and horses. A hypothesis on the probable functional role of the observed receptorial apparatus was formulated.

Proprioceptive innervation of the external anal sphincter muscle and the organization of the vegetative and sensitive nerve components of the internal and sphincter muscle have been studied in different mammals. The findings of typical muscle spindles in the external anal sphincter muscle were constant in the pig, frequent in the goat and cow, rare in the sheep and horse and absent in the roe and rabbit. In the pig, muscle spindles were observed in the entire extension of the muscle, while in the sheep, goat, cow and horse, the receptors were found only in the cranial portion of the muscle. In all the species studied, the internal anal sphincter muscle had numerous ganglion cells, isolated or grouped, and rare Pacinian, Pacinian-like, and Golgi-Mazzoni corpuscles. Their functional role has been hypothesized.

A reconstruction of the morphological features of domestic pigs from two Roman settlements is here suggested by means of the study of skeletal and dental remains, with the aim at evaluating their degree of selection in comparison with wild boars. Material was formed by 111 bone and tooth fragments and was uncovered during the excavations of Polybius' House in Pompeii and of Roman buildings in the neighbourhood of Caralis harbour (Sardinia). The remains underwent morphological examination. The eruption of permanent teeth and ossification of epiphyseal cartilages let us establish that most animals were over 18-20 months. When possible, the determination of sex was made by detecting tusk features. The presence of anthropic signs on the bone surface provides some information about slaughtering and cooking procedure in the Roman period and supports the hypothesis that the animal remnants were food remains. Osteometric analysis was carried out on long and short bones and teeth through...

Neurons confined to the central nervous system usually fail to regenerate their axons after injury, although evidence of axonal re-growth has been reported. In this study, rat transcallosal fibres were severed in the midline in order to investigate the reactive plasticity responses in the frontal and occipital cortices of both sides. The expression of growth-associated proteins, tyrosinated alpha-tubulin and GAP-43, was monitored at different time-points post-injury. Protein levels dropped during the first days post-axotomy, but subsequently returned to control levels. This initial decrease could be due to degeneration, and the subsequent increase connected to the reactive neosynaptogenesis and fibre sprouting from surrounding ipsilateral neurons, which is responsible for the reinnervation of the denervated area. Although transcallosal neurons are usually considered poorly regenerative, their axotomy may therefore induce reactive events.

Bone microstructure of domestic herbivores is still not completely
understood. Indeed, works focused on the bone histology
of numerous Mammalian species frequently led to misunderstandings
because of the high number of variations such as the
kind of bone, section orientation, species, breed and age.
Moreover, attempts to identify the species in archaeozoological
studies by a mere qualitative approach have not been encouraging
and in recent years quantitative methods, based on image
processing and statistical analysis, have appeared. The present
study was undertaken to determine whether morphometrical
and morphological differences exist in the compact bone structure
of the femur and humerus between horses and cows. Measurements
such as area, perimeter, minimum and maximum
diameter of osteons and Haversian canals as well as the osteonal
density were carried out on cross sections of eight humeri and
eight femurs of the two herbivores investigated. In agreement
with other authors, the qualitative investigation confirmed that
the compact bone of horses and cows can be classified as dense
Haversian tissue. Osteons of the horse were more numerous and
composed of a higher number of well-defined lamellae when
compared with the cow. Diameter, perimeter and area of osteons
and Haversian canals were always higher in horses than in cows
and this pattern could be related to the different locomotor
behaviour of these animals.

OBJECTIVES: An important step of sexual differentiation is the conversion of
testosterone to estrogen by aromatase leading to masculinization and defeminization
of the fetal brain areas crucial for normal sexual behavior and reproduction.
Brain sexual differentiation occurs throughout a critical period starting from
different prenatal stages depending on the species. Such period goes on from
gestation day (GD) 30 to 100GD in the sheep. The fetal sheep brain is reported
to aromatize androgens to estrogens at 64GD. The main goal of this work was to
evaluate aromatase expression in sheep hypothalami during the whole period of
sexual differentiation (35GD, 55GD, 80GD, 115GD) and whether differences may
be observed depending on gestational stage and sex.
METHODS: Sections at the hypothalamic level underwent immunoperoxidase
technique employing anti-aromatase and anti-androgen receptor antibodies.
Samples from 35GD and 55GD were also processed with in situ hybridization
using aromatase cDNA probe. Blot analyses were performed to quantify possible
aromatase immunoexpression differences between sexes. For sexing, samples at
35GD and 55GD underwent DNA extraction and SRY amplification.
RESULTS: Our results revealed aromatase and androgen receptor immunoreactivity
along the whole period of sexual differentiation. Both molecules were detected
in many brain regions and markedly in the periventricular area. The highest
aromatase and androgen receptor amounts were observed at 35GD and 55GD,
when aromatase was more abundant in females than in males.
CONCLUSIONS: In conclusion, the sheep can be included among the species
where aromatase is highly expressed in the hypothalamus during the whole period
of sexual differentiation.

Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. Recent studies have
clearly demonstrated that autophagy has a greater variety of physiological and pathophysiological roles than
expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging,
elimination of microorganisms, cell death, tumor suppression and antigen presentation. MAP-LC3 is one of the
most common markers to evaluate autophagic processes.
In our study, the autophagic activity in neurons and astrocytes from sheep brain under starving conditions was
evaluated. In order to detect LC3 immunoreactivity, confocal analysis by double immunofluorescence was performed
together with the cell type markers: GFAP to identify astrocytes, 􀁅-III tubulin to identify neurons. The
results show that astrocytes are characterized by LC3-positive areas, which increase in a time-dependent manner.
In contrast, LC3 immunoreactivity was very weak in neurons. Therefore, it can be assumed that astrocytes show a
higher capability than neurons to cope with stress and exhibit a stronger autophagic response.

A lot of evidence testifies that aromatase is expressed in the
central nervous system where it has been detected not only in
hypothalamic and limbic regions but also in the cerebral cortex
and spinal cord. In physiological conditions, aromatase is
expressed exclusively by neurons, where it has been mainly
found in cell bodies, processes and synaptic terminals.
Moreover, primary cultured cortical astrocytes from female rats
are more resistant to oxidant cell death than those from
males, suggesting a protective role of estradiol. The aim of this
study was to evaluate changes in aromatase expression in
response to 3-nitro-L-tyrosine, a marker of oxidative stress, in
primary neuronal cell cultures from brains of 60-day old sheep
fetuses. Cells were identified as neurons by using class III b-
tubulin, a marker of neuronal cells. Two morphological types
were consistently recognizable: i) bipolar cells with an oval cell
body; ii) multipolar cells whose processes formed a wide net
with those of adjacent cells. In situ hybridization technique
performed on 60-day old fetal neurons revealed that in baseline
conditions aromatase gene expression occurs.
Importantly, cells exposed to 360 μM 3-nitro-L-tyrosine were
fewer and showed more globular shape and shorter cytoplasmic
processes in comparison to control cells. The immunocytochemical
study with anti-aromatase antibody revealed that
cells exposed to 360 μM 3-nitro-L-tyrosine were significantly
more immunoreactive than control cells. Thus, it can be postulated
that the oxidant effects of the amino acid analogue 3-
nitro-L-tyrosine could be counterbalanced by an increase in
aromatase expression that in turn can lead to the formation of
neuroprotective estradiol via aromatization of testosterone.

A lot of evidence testifies that aromatase is expressed in the
central nervous system where it has been detected not only in
hypothalamic and limbic regions but also in the cerebral cortex
and spinal cord. In physiological conditions, aromatase is
expressed exclusively by neurons, where it has been mainly
found in cell bodies, processes and synaptic terminals.
Moreover, primary cultured cortical astrocytes from female rats
are more resistant to oxidant cell death than those from
males, suggesting a protective role of estradiol. The aim of this
study was to evaluate changes in aromatase expression in
response to 3-nitro-L-tyrosine, a marker of oxidative stress, in
primary neuronal cell cultures from brains of 60-day old sheep
fetuses. Cells were identified as neurons by using class III b-
tubulin, a marker of neuronal cells. Two morphological types
were consistently recognizable: i) bipolar cells with an oval cell
body; ii) multipolar cells whose processes formed a wide net
with those of adjacent cells. In situ hybridization technique
performed on 60-day old fetal neurons revealed that in baseline
conditions aromatase gene expression occurs.
Importantly, cells exposed to 360 μM 3-nitro-L-tyrosine were
fewer and showed more globular shape and shorter cytoplasmic
processes in comparison to control cells. The immunocytochemical
study with anti-aromatase antibody revealed that
cells exposed to 360 μM 3-nitro-L-tyrosine were significantly
more immunoreactive than control cells. Thus, it can be postulated
that the oxidant effects of the amino acid analogue 3-
nitro-L-tyrosine could be counterbalanced by an increase in
aromatase expression that in turn can lead to the formation of
neuroprotective estradiol via aromatization of testosterone.

ince the chronic exposure to hyperglycemia can result in the production of high
concentration of reactive oxygen species with subsequent damage of several cell
structures such as the cytoskeleton. In order to antagonize the oxidative status
many substances have been tested as antioxidants. In the present work attention
has been focused on the possible nitrosative effect of hyperglycemia on microtubular
network of neuroblastoma and glioma mortalized cell lines, testing the
possible neuroprotective effect of testosterone.
METHODS: Neuroblastoma (C1300) and glioma (C6) cell lines were cultured in the
presence of 300mM (C1300) or 310mM (C6) d-glucose, with or without 50nM
testosterone. After 72hrs, morphology, growth rate, cell viability and catalase
activity were evaluated. In addition, with the aim to detect any changes in the
amount of tubulin isoforms, Western blot analysis was performed.
RESULTS: In d-glucose-exposed cells, it was found a down-regulation of tubulin
isoforms and an increase in 3-nitro-l-tyrosine and subsequent modifications in
cell morphology, growth rate, viability and catalase activity. All these changes
were more severe in neuroblastoma than in glioma cell line. When testosterone
was added to the medium, all the parameters were very similar to controls. This
neuroprotective action was well-detectable in C1300 cells, whereas testosterone
was not able to recover significantly in C6 cells.
CONCLUSION: Our results displayed: i) a selective action of high glucose on microtubules;
ii) a different sensitivity to oxidative stress in neuronal and glial cells; iii) a
different neuroprotective action of testosterone on neuronal and glial cells.

OBJECTIVES: Using undifferentiated mouse neuroblastoma cells (C1300), we have
previously observed that testosterone (T) exerts a neuroprotective action against
oxidative stress. Nitrogen intermediates induce the production of 3-nitro-l-tyrosine
(3NT), an amino acid analogue involved in many neurodegenerative disorders. The
aim of our work is to investigate T capability on C1300 cell differentiation. It is also
evaluated whether differentiation could mitigate the nitrosative effects of 3NT.
METHODS: The effects of both T and 3NT were studied on an undifferentiated cell
line of neural origin (C1300). For this purpose, cell cultures underwent morphometric
investigation, blot analyses and catalase activity assay. All data obtained were
expressed as mean ±SD and tested by one-way ANOVA or Student’s t test.
RESULTS: The results were compared with those gathered by means of N6,2’-Odibutyryl-
adenosine-3’,5’-cyclic-mono-phosphate (db-cAMP), a well-known
differentiating agent. T-exposed cells showed an irregular shape and exhibited
long branching cytoplasmic extensions, which were longer than in db-cAMP cells.
Moreover, T-exposure induced an increase in the expression of tyrosinated and
acetylated α-tubulin while 3NT-incorporation into tubulin was markedly reduced.
The action of antioxidant defence systems, namely catalase activity, was enhanced
in cells exposed to T.
CONCLUSION: This work highlighted the effects of db-cAMP on differentiation
and neuroprotection, but even indicated that T exposure induced differentiation
in C1300 cells and this process matches a significant neuroprotective effect. This
action seemed to be more effective than in db-cAMP-treated cells. T is suggested,
like other substances having antioxidant properties, to be of potential interest in the
experimental therapy of neuropathological conditions.

Mouflon (Ovis aries musimon) and sheep (Ovis aries aries) are
considered as the wild and domestic subspecies of the same species. A
comparative study on the microstructure of mouflon and sheep
femoral bone diaphysis is here reported. Bone microstructure is
described for the first time in the mouflon. More than 200 secondary
osteons from both subspecies were analyzed and qualitative evaluation
was followed by quantitative determination of perimeter, area, minimum
and maximum diameters of secondary osteons and Haversian
canals. The basic structural patterns observed in both subspecies can
be classified as plexiform and irregular Haversian tissue, in accordance
with what reported in the literature for most ruminants. The
presence of many secondary osteons in the mouflon means that the
bone also consists of dense Haversian bone tissue. Statistical analysis
demonstrated that mouflon secondary osteons are larger than in the
sheep and made of a greater number of lamellae. Since mouflon and
sheep are taxonomically closely related and their body size is very similar,
the qualitative and quantitative differences here reported could be
primarily explained on account of their different lifestyle. Indeed, the
habits of wildlife typical of mouflons may lead to the presence of wide
areas of dense Haversian tissue in that subspecies, as mechanical
stresses are known to be related to number and size of secondary
osteons. Finally, this analysis could provide a useful tool to recognize
bones from different species, in forensic exam and archaeozoological
studies as well

A high number of differences exist in bone histological features depending on
the species, breed, age and bone. Moreover, osteon distribution may vary in
the different sides of a bone as a consequence of different biomechanical
strains. The aim of this work was to study the distribution and morphology of
osteons in different sides of the equine femoral diaphysis with the attempt to
correlate them to the main strains operating on them. The following parameters
of secondary osteons and Haversian canals were measured in the transverse
sections of diaphyses: perimeter, area, minimum and maximum diameter,
eccentricity and osteon population density. A typical Haversian tissue was
observed with elliptic secondary osteons consisting in about 10 well-defined
lamellae surrounding a circular Haversian canal. Quantitative analysis displays
a different population density of secondary osteons depending on the side. The
caudal and medial sides, where compression strains are higher, have more secondary
osteons in comparison with the cranial and lateral sides, where tension
strains are prevalent. These data suggest that secondary osteon population density
may depend on the predominant strains. Even the elliptical shape of secondary
osteons may be related to biomechanical strains, as their major axes are
oriented cranio-caudally parallel to prevalent strains.

The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphological changes detected on mouse neuroblastoma (C1300) and rat glioma (C6) cell lines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both cell lines showed morphological changes, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 μM for C1300 and C6, respectively). Such effects were dose-dependent, though glioma cells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases.

The effects of aluminum(III) on microtubular meshwork have been investigated using cultured murine neuroblastoma cells grown in a medium containing aluminum lactate at defined metal concentrations (10–20 μM). A role of aluminum(III) in promoting neuronal plasticity events is suggested. These events including sprouting and neurite outgrowth are associated with an increased tyrosine-tubulin (Tyr-Tub) expression, which can be due to the enhanced needs of recently formed, highly dynamic microtubules typical of neuronal plasticity. After 48 and 72 h aluminum exposure, an upregulation of Tyr-Tub expression is detected and this is concentration-dependent. A high amount of Tyr-Tub is observed also in non-treated cells, although later than in aluminum-exposed cells. Thus, it is possible that aluminum(III) accelerates neuronal plasticity events, for which Tyr-Tub is confirmed to be a useful marker.