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One main aspect of safeguard plans for livestock Autochthonous Genetic Types (AGTs) is to ensure economic sustainability of their farming. This encourages farmers to contribute to the pres- ervation of AGTs. The aim of this study was to characterize the genetic structure of four AGTs of endangered goats at the αS1, β and αS2 calcium sensitive caseins encoding genes CSN1S1, CSN2 and CSN1S2, respectively. The peculiar polymorphisms detected at these loci are associated with milk quali-quantitative charac- teristics and have been largely investigated in widespread dairy goat breeds. On the contrary, there are very few studies carried out in AGTs goats. This study was performed on 50 Neapolitan goats, typical of the Campania region, and on 61 goats which included the three breeds Monticellana, Capestrina and Grigia Ciociara, reared in Lazio region (Lazio AGTs goat). Genotyping of quantitative alleles at CSN1S1, CSN2 and CSN1S2 loci was performed according to previous studies. At the CSN1S1 locus a high variability was observed in all four AGTs. In particular, a higher frequency of the null allele N was observed in Neapolitan goats than Lazio AGTs goats (0.420 vs. 0.008). On the contrary a higher frequency of F allele, associated with a low αS1 casein content in milk, was observed in Lazio AGTs goat (0.442) than Neapolitan goat (0.120). All four AGTs do not carry the null alleles CSN1S1 01, CSN2 01 and CSN1S2 0 and the alleles associated with an intermediate content of αS1 (CSN1S1 E) and αS2(CSN1S2 D) caseins in milk. The genotyping data obtained pro- vide useful information regarding the productive aptitudes of the four AGTs studied. In fact, the high frequency of CSN1S1 N allele in Neapolitan goat, predisposes this breed to produce low-protein milk for fresh consumption while the high frequency of the allele CSN1S1 F in the Lazio AGTs goat addresses their milk to process- ing for the production of typical cheese with unique organoleptic and flavor characteristics. Developing safeguard plans that take these results into account will have a higher chance of success, with a consequent increase in the number of animals raised.

Pig from Casertana and Siciliana breeds are two endangered breeds raised in the southern-Italy (Campania and Sicily regions, respectively) and characterized to have a black skin. Special projects are trying to save and characterize both of them by using several approaches. In this study we report the preliminary results we obtained after a cytogenetic investigation we performed by using both C- and R-banding techniques and the sister chromatid exchange (SCE) test to verify their chromosome stability under the environmental conditions. Fifty-two pigs from Casertana breed and 19 pigs from Siciliana breed were investigated. All animals from both breeds showed a normal karyotype, with the exception of two male pig from Siciliana breed which were found heterozygous carrier of rob(15;17) (2n=37, XY), probably being hybrids with the wild pig (2n=36) present in the Nebrodi mountains where this breed is raised in Sicily. SCE-test applied on 42 pigs from Casertana breed (22 males and 20 females) and 19 pigs from Siciliana breed (8 males and 11 females) revealed no statistical differences between the SCE-mean number in Casertana pig (7.13±3.20) than that (6.87±3.12) achieved in Siciliana pig. Statistical differences were found between males (7.26±3.38) and females (6.59±2.90) of Siciliana pig breed, as well as between females of Casertana (7.24±3.26) and Siciliana (6.59±2.90) breeds, while no statistical differences were found between males of the breeds, as well as between males and females of Casertana breed.

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157-166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus-oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1×10(6)spermmL(-1). After 18-20h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400µgmL(-1), final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3-8 cells, and 9-32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO(2),7% O(2), and 88% N(2). On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400µgmL(-1) condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.-2 cells: 59.7±11.2, 52.9±26.3, 56.1±14.6, 66.0±2.8, 38.0±11.3; 3-8 cells : 40.3±11.2, 47.1±26.3, 43.9±14.6, 34.0±2.8, 59.9±9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300µgmL(-1) slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts.

The present study reports on the frequency of X-Y aneuploidy in the sperm population of two minor cattle breeds reared in Italy, namely Modicana and Agerolese, which are listed in the "Anagraphic Register of autochthonous cattle populations with limited distribution". More than 50 000 sperm nuclei from 11 subjects (5 and 6, respectively for each breed) have been analyzed by the fluorescent in situ hybridization with the Xcen and Y-chromosome specific painting probes. The fraction of X- and Y-bearing sperm was close to the 1:1 ratio in the Modicana breed, whereas in the Agerolese the Y-fraction was significantly higher (P < 0.002) compared to the X-counterpart. The mean rates of X-Y aneuploidy were 0.510 and 0.466%, respectively, in the two breeds; no significant differences were found among individual bulls within each breed. Average frequencies of disomic and diploid sperm were 0.425 and 0.085% in the former and 0.380 and 0.086% in the latter. In both breeds, (a) disomy was significantly more frequent than diploidy (P < 0.01), (b) YY disomy was significantly (P < 0.001) more frequent than XY or XX; (c) MI errors (XY disomy) were significantly (P < 0.01) less represented than MII (XX + YY disomy). Compared to the dairy (Italian Friesian and Brown) and meat (Podolian and Maremmana) breeds previously analyzed, the "minor" breeds investigated in the present study showed a significantly (P < 0.002) higher rate of X-Y aneuploidy (0.486 vs. 0.159 and 0.190%, respectively). Considering all the breeds analyzed -so far- and assuming no significant interchromosomal effect, the baseline level of aneuploidy in the sperm population of the species Bos taurus was estimated as 5.19%. Establishing the baseline level of aneuploidy in the sperm population of the various livestock species/breeds engaged in animal production could reveal useful for monitoring future trends of their reproductive health, especially in relation to management errors and/or environmental hazards.

The present study reports on the variation of the mitotic index in growing day 2- to day 7- IVP bovine embryos of the Agerolese breed of cattle. After IVM (24 h), COCs were transferred into 25/well with 300 ?l IVF-TALP and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45-80 %). The pellet was diluted in IVF medium and added to the COCs at the concentration of 1×106sperm/mL. Gametes were co-incubated for 20-22 h at 39 °C, in 5 % CO2 in air. After co-incubation, presumptive zygotes were vortexed to remove cumulus cells and randomly allocated in six groups, each into 400 ?l of SOF medium, with 30 ?l/ml essential amino acids, 10 ?l/ ml non-essential amino acids, 0.34 mM tri-sodium citrate, 2.77 mM myo-inositol, and 5 % BS. Zygotes were incubated in a humidified mixture of 5 % CO2, 7 % O2 and 88 %N2 in air at 39 °C for 20-22 h. Starting on Day 0 ( IVF day), embryos were taken out Chromosome Res (2012) 20:779-814 795 of the incubator at day 2(48 h),3 (72 h),4 (96 h), 5 (120 h), 6(144 h) and 7(168 h), examined under a stereomicroscope, treated in a lysing buffer (0.01 N HCl, 0.1 % Tween 20) for 30 s, transferred in a small droplet to a precleaned slide and fixed with methanolacetic acid (3:1). Out of 178 embryos (3,100 cells) analyzed, the mitotic index was 19.6 % (28/143 cells) at day 2, 18.6 % (41/221 cells) at day 3, 10.3 % (27/ 263 cells) at day 4, 7.1 % (26/367 cells) at day 5, 0.7 % (5/681 cells) at day 6 and 0.6 (9/1425 cells) at day 7. FISH analysis is undergoing and will be reported elsewhere. Acknowledgements: This work was supported by the RARECA- PSR 214 project of the Campania Region (Italy).

The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

Donkey chromosomes were earlier characterized separately by C-, G- and R-banding techniques. However, direct comparisons between G- and R-banding patterns have still not been carried out in this species. The present study reports this comparison at the 450-band level by using replication G- and R-banding patterns. Two sets of synchronized lymphocyte cultures were set up to obtain early (GBA+CBA-banding) and late (RBA-banding) BrdU incorporation. Slides were stained with acridine orange and observed under a fluorescence microscope. Reverse GBA+CBA- and RBA-banded karyotypes at the 450-band level were constructed. To verify G- and R-banding patterns in some acrocentric chromosomes, sequential GBA+CBA/Ag-NORs and RBA/Ag-NORs were also performed. The results of CBA-banding patterns obtained in 12 animals from 2 breeds showed a pronounced polymorphism of heterochromatin, especially in EAS1q-prox. Ideogrammatic representations of G- and R-banded karyotypes were constructed using only one common G- and R-banding nomenclature. In the present study both G- and R-banding patterns and relative ideograms are presented as standard karyotype for this species at the 450-band level.

Polychlorodibenzodioxins (PCDDs) and related substances including polychlorodibenzofurans (PCDF) and the dioxin-like polychlorobiphenyls (DL-PCBs) are powerful mutagens that can enter the food chain as the result of a number of industrial processes, waste incineration or illegal waste burning. Cytogenetic tests could be useful to reveal the presence of such mutagens in the food chain by simply monitoring food producing species. In previous studies we studied sheep exposed to dioxins and found a pronounced chromosome fragility in exposed herds, compared to unexposed one (control) by using both SCE and chromosome abnormality (AC) test. In this study we applied the SCE-test in both cattle and river buffaloes reared in dioxin-contaminated areas, although the WHO-TEQ levels of bulk milk were much lower than those earlier detected in sheep milk. Peripheral blood cultures were performed in samples from 15 dairy cows showing average milk values over (18.56 pg/g of fat as WHO-TEQ) than those permitted (6.0 pg/g of dioxins+furans+PCBs as WHO-TEQ) and the results were compared with samples from 16 dairy cows (1.75 pg/g of fat as WHO-TEQ) used as control. Significantly (P < 0.01) higher mean number of SCE/cell (7.10 ± 2.8) were found in dioxin exposed cows compared to those measured in the controls (SCE/cell=5.24 ± 2.51). The same test was applied on 26 river buffaloes showing average values of WHO-TEQ over (21.79 pg/g of fat as WHO-TEQ) than those achieved in the milk mass of 26 river buffaloes (1.30 pg/g of fat as WHO-TEQ) used as control. Opposite to cattle, no significant differences were found between mean number of SCE/cell in exposed vs. non exposed river buffaloes (8.73 ± 3.32 vs. 8.79 ± 3.40).

The domestic yak (Bos grunniens) is an important Bovidae species which in Asia plays a remarkable role for the economy of the Tibetan highlands (over 4000 m above sea level.). Despite its strategic and economical importance, this species has been almost neglected, from a cytogenetic point of view, as demonstrated by the few papers present in the literature. To fill this gap, we undertook a cytogenetic investigation by using blood samples of eight yak bulls recently imported into central Italy. CGR-banding, silver staining and FISH-techniques were applied. The preliminary results were as follows: (a) the chromosomal make-up of the yak was 2n=60,XY, (b) the animals investigated showed normal karyotype, (c) the frequency of chromosome/chromatid breaks was similar to that of cattle (3.7 vs 3.0 %, respectively), (d) the mean rate of SCE/cell at 10 2g/ml (f.c.) was significantly (Pe0.001) lower compared to that of cattle (5.2 vs 8.3, respectively), (e) the GTG-RBA-RBG banding patterns Y at the 400 bands level of resolution Y did not reveal structural differences compared to cattle, (f) the CBA-banding pattern was also similar to cattle, i.e. no C-bands on the subcentromeric X, while the Y- chromosome showed heterochromatic tips in the short arms. Zoo-FISH with bovine painting probes derived from microdissected chromosomes 5-X-Xcen and Y- upon yak metaphase chromosomes showed complete hybridization, thus confirming the close homology between the two species. In addition, FISH-mapping with bovine BAC-clones containing ZFY and SRY genes to the yak Y-chromosome also revealed the same localization as reported in cattle (Yp12.2 and Yq23dist, respectively). However, the fact that Bos taurus x Bos grunniens F1 male hybrids are sterile, while the females are normally fertile, suggests that the main difference between yak and cattle might be in the X-Y pseudoautosomal (PAR) region of the two species. Further investigation, therefore, is necessary with more effective cytogenetic techniques, such as high resolution banding, fine gene mapping analysis and DNA sequencing.

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.

The present study reports on the expression and localization of “fragile sites” (FS) on prometaphase chromosomes of two groups of river buffaloes (Bubalus bubalis, 2n=50; Mediterranean Italian breed), reared in two different farms, with the aim to characterize chromosome fragility in this species. Totally, 400 aphidicolin induced breakages were identified and localized on the standardized ideogram of the river buffalo karyotype. Preliminary results can be synthesized as follows: (a) aphidicolin showed a remarkable decondensing effect on chromosome structure, enabling further studies at high resolution level; (b) the chromosomal expression of the breakages was not different in the two groups of animals; (c) the most fragile chromosomes were the inactive-X, chromosomes 9, 8 and active-X, showing 42, 32, 31 and 30 breakages, respectively; (d) the breaks were localized in the RBG-negative bands (corresponding to eterochromatic regions) or at the band-interband regions; (e) the chromosomal distri- bution of the break sites was not random and only partially related to chromosome length. The study is in progress to determine the relative incidence of the fragile sites at chromosomal band level, in order to construct a ‘fragile-site map’ of river buffalo, which could be utilized for genetic improvement programs of the species.