Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800  g for 10 min and 2400  g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400  g for 3 min and freezing 2; M10 – centrifugation at 800  g for 10 min and freezing 2; R3 – centrifugation at 2400  g for 3 min and freezing 1; and R10 – centrifugation at 800  g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.     

Characterization of Some Italian Common Bean (<Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> L.) Landraces by RAPD,Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)₃GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.     

Formation of an Antral Follicle–like Structure of Bovine Cumulus–Oocyte Complexes Embedded Individually or in Groups in Collagen Gels

Culture techniques of antral follicle–like structure (AFLS) derived from cumulus–oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4–5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4–14 using the individual culture method were significantly higher (p < 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p < 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.     

Double Labelling Immunohistochemistry on the Nerve Fibres of Retractor Clitoridis Muscle of the Sow

Veterinary Research Communications -     

Ultrastructural and Enzymatic Activity of Membranous Vesicles Isolated from Canine Seminal Plasma

The objectives of this study were to verify the presence of membranous vesicles (MV) in canine seminal plasma by mean of transmission electron microscopy (TEM), to describe the ultrastructural characteristics and to identify some enzymatic activity associated with them. Semen samples, collected by digital manipulation from dogs with proven fertility, were pooled and used for membrane vesicles preparation according to conventional procedures. TEM observations showed the existence of vesicular membranous structures of more or less spherical shape with different sizes. These vesicles were surrounded by a single‐, double‐ or multiple‐layered laminar membranes. The mean vesicle diameter was 117.6 ± 86.9 nm ranging from 24.4 to 716.6 nm. Enzyme activity determinations showed the presence of adenosine deaminase, 5′‐nucleotidase, ADPase, ATPase, dipeptilpeptidase IV, alkaline phosphatase, total acid phosphatase and prostatic acid phosphatase, while the aminopeptidase activity was absent. In conclusion, results of this study, compatible with results from other mammals, showed for the first time the presence of MV, their ultrastructural and enzymatic characteristics in dog seminal plasma.     

Effect of a pilot washing system on dicofol levels in orange matrix

An efficient analytical method is described for the analysis of dicofol residues in pulp and orange peel. Samples are mixed with Celite and transferred to chromatographic columns prepacked with silica gel. Dicofol is eluted with ethyl acetate, and the extracts are analyzed by gas chromatography with electron capture detection. Mean recoveries for dicofol at levels of 0.5, 2.0, 5.0, and 10 mg/kg ranged from 87 to 95% with relative standard deviation values between 2.6 and 9.0%. To investigate the effect of a pilot washing system on dicofol residues in oranges, the analytical procedure was applied to samples submitted to different treatments with commercial formulations under field and laboratory conditions. The orange samples with and without washing were analyzed in duplicate, and the results indicated that washing under the described conditions did not allow a complete removal of dicofol residues from orange peel.