Effect of Caffeine Added to the Activating Solution on Sperm Motility of Fresh and Thawed Semen of Pacu,Piaractus mesopotamicus,and Curimba,Prochilodus lineatus

The aim was to evaluate the effect of different concentrations of caffeine added in activating solution over sperm motility in fresh and thawed semen of pacu, Piaractus mesopotamicus, and curimba, Prochilodus lineatus. The activating solutions were prepared with sodium bicarbonate solution of 0.76% (NaHCO₃) and caffeine was added at concentrations of 2.5, 5.0, 10.0, and 20.0 mM. As control, a solution of NaHCO₃ 0.76 without caffeine was used. Eight males of pacu and 20 males of curimba were used. Aliquots of 200 μL of semen were diluted in 800 μL extender solution (DMSO 10% and BTS 5%), placed in 0.5 mL straws and cryopreserved for 7 d in a liquid nitrogen tank. There was a linear increase in sperm motility for fresh semen of pacu, and for curimba fresh and thawed semen (P < 0.05), due to the increase in the concentration of caffeine. There was a quadratic response for duration of motility for thawed semen of pacu and for fresh semen of curimba (P < 0.05), respectively. These results indicate that addition of caffeine in the activator solution can improve sperm motility parameters, however, is dependent on the species and concentration used.     

A rare Cryptosporidium parvum genotype associated with infection of lambs and zoonotic transmission in Italy

An outbreak of cryptosporidiosis occurred in a mixed sheep/cattle farm of Central Italy in October 2011. A total of 450 ovines (250 sheep and 200 lambs) and 140 bovines (130 cows and 10 calves) were housed in two separated units, at the time of the outbreak. About half of the lambs had diarrhea due to Cryptosporidium sp. with a mortality rate of 80%; calves were not infected. Genomic DNA was extracted from an archived slide and from fecal specimens, and the parasite was identified as Cryptosporidium parvum by PCR and sequence analysis at the CpA135 gene. Genotyping at the GP60 gene showed the presence of a very rare genotype, IIaA20G2R1. Shortly after the outbreak was identified, the son of the farm's owner, aged 18 months, experienced an acute gastroenteritis and was hospitalized due to recurrent episodes of diarrhea, fever, vomiting and lack of appetite. The feces tested negative for bacteria and viruses, whereas cryptosporidiosis was diagnosed by microscopy and an immunochromatographic test. Molecular typing identified the C. parvum genotype IIaA20G2R1 in the feces of the child. This is the first case of transmission of cryptosporidiosis in Italy involving lambs as source of oocysts infectious to humans.     

Genetic diversity in Tunisian olive accessions and their relatedness with other Mediterranean olive genotypes

Eighty-four olive accessions obtained from the National Conservatory of Boughrara-Sfax (Tunisia), previously evaluated for morphological traits, were analysed with 47 random amplified polymorphic DNA (RAPD) markers. They were compared with other olive genotypes originated from Eastern or Western Mediterranean. The highest and lowest similarities between genotypes, estimated by simple matching algorithm, were 0.98 and 0.40, respectively. A dendrogram based on Ward's method and a factorial correspondence analysis (FCA) showed that most of Tunisian accessions are closely related to olive genotypes originating from the Eastern Mediterranean and some are clustering with genotypes originated from the Western Mediterranean. These findings suggested multiple and complex origin of Tunisian olive. A comparative study between a previous morphological analysis and current RAPD assay was carried out and discussed.     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

Natural Stable Isotopes for Determination of Gastrointestinal Transit Time in Fish

This study evaluated the application of stable isotopes of carbon as an alternative and more accurate method to determine gastrointestinal transit time (GTT) in fish by comparing it to the inert marker method. The stable isotope method detects alterations of the normal carbon flow in a biological system by analyzing naturally occurring isotopes of carbon, contrary to studies based on conventional techniques that apply external markers to the diet to determine GTT through visual observation of the color change in feces. Therefore, 320 pacu, Piaractus mesopotamicus juveniles were reared in 32 tanks under two different temperatures (25 and 29 C). The pacu juveniles received two different diets, one based on ingredients derived from C₃ photosynthetic cycle plants and the other based on C₄ plant ingredients, both containing titanium oxide (TiO₂) as a marker. After 40 d, the isotopic signature of the diets was changed, and the marker was replaced by chromic oxide (Cr₂O₃). In the isotopic technique, the feces were analyzed to determine the exchange in the isotopic ratio of carbon δ¹³C. Both methods found that GTT was faster (nearly 6 h) in fish at 29 C when using the C₄/C₃ feeding strategy and slower in fish at 25 C using the C₃/C₄ strategy (15 h by inert marker and 18 h by the isotopic method). In conclusion, GTT determination in pacu juveniles using the stable isotope technique exhibits the same accuracy obtained with the inert marker method at temperatures suitable (nearly 29 C) for the metabolism of these animals.     

Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains

The Mycoplasma hyopneumoniae genome contains at least 22 regions with a variable number of tandem nucleotide repeats (VNTRs) within coding DNA sequences (CDSs). In this work, the VNTR-containing CDSs were analysed in order to evaluate their degree of variation, possible correlations with antigenic properties, and their potential to be used as a basis for a strain typing PCR assay. We have analysed the VNTRs in five M. hyopneumoniae strains (J, 7448, 7422, PMS, and 232), based on published genomic sequences and on amplified and sequenced DNA segments. These VNTRs are distributed among 12 genes, most of which encode putative surface proteins, including known adhesins. The number of repeat units in any of the VNTRs is highly variable among the analysed strains, but they are, without exception, translationally in frame, and, therefore, code for a variable number of aminoacid repeats (VNTARs). These VNTARs determine putative structural, physicochemical and antigenic variations in the corresponding proteins, with potential implications for aspects associated to M. hyopneumoniae pathogenicity, such as cell adhesion and interactions with the host immune system. Considering that the characterized VNTARs are relatively stable, at least in vitro, and their sizes are strain-specific, we have developed a VNTR-based PCR assay for M. hyopneumoniae strain identification, useful for enzootic pneumonia (EP) diagnosis, strain typing, and distinction of circulating field isolates from vaccine strains in animals vaccinated against EP.     

Assessment of the ideal ratios of digestible essential amino acids for pacu,Piaractus mesopotamicus,juveniles by the amino acid deletion method

The present study aimed to determine the ideal ratios of digestible essential amino acids (EAAs) for pacu (Piaractus mesopotamicus) juveniles by the amino acid (AA) deletion method. A completely randomized design which consisted of 11 treatments and three replicates each was used. The treatments included a control diet (CD) containing 55% of nonpurified natural ingredients and 45% of purified synthetic amino acids and ingredients, and other ten isonitrogenous and isoenergetic EAA limiting diets (LDs), each being deficient in 44.4 ± 0.02% of the respective EAA. Pacu juveniles with initial average body weight of 6.22 ± 0.09 g were distributed among 33 fiber glass tanks. Fish were fed with semipurified and extruded diets for 113 days two times a day until apparent satiation. The ideal ratio of each dietary EAA was calculated on the basis of the relationship between body N retention and amount of EAA deleted from the respective EAA LD. Based on the AA deletion method, the ideal ratios of digestible EAAs for pacu juveniles, relative to lysine requirement of 100% were estimated as: methionine 14.6%, threonine 35.0%, tryptophan 6.6%, arginine 62.8%, histidine 13.6%, isoleucine 26.3%, leucine 43.7%, phenylalanine 27.2%, and valine 35.8%.     

Post-logging loss of genetic diversity in a mahogany (Swietenia macrophylla King,Meliaceae) population in Brazilian Amazonia

Big-leaf mahogany (Swietenia macrophylla King, Meliaceae) is the most valuable widely traded timber species from the Neotropics. Mahogany has been severely overexploited throughout its natural range, which stretches from southern Mexico through Central and South America as far south as the Bolivian Amazon. We investigated effects of selective logging on the genetic diversity of a mahogany population in eastern Amazonia. Using eight hypervariable microsatellite loci, we estimated the number of alleles per locus (A), mean expected and observed heterozygosity (H_e, H_o), the number of distinct multilocus genotypes (G_o), and the inbreeding coefficient (f) for pre- and post-logging cohorts (adults and seedlings) collected over identical spatial scales. We found a significant reduction in the number of alleles, observed heterozygosity, and distinct multilocus genotype number in the post- compared to the pre-logging cohort. The loss of genetic diversity is likely to have occurred due to a reduction in the effective population size as a consequence of logging, which leads to the loss of alleles and limits mating possibilities. The results raise concerns about the conservation genetics of logged mahogany populations where a high proportion of adults are removed from the system. We suggest initiatives to promote the maintenance of genetic diversity as an integral part of the production system, allowing genetically sustainable use of this valuable timber resource.