Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition.

The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 µM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos.

Prion-like Doppel gene polymorphisms and scrapie susceptibility in Portuguese sheep breeds.

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion-like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.

Doppel gene polymorphisms in Portuguese sheep breeds: insights on ram fertility.

Transgenic knockout of the gene encoding the prion-like protein Doppel leads to male infertility in mice. The precise role of Doppel in male fertility is still unclear, but sperm from Doppel-deficient mice appear to be unable to undergo the normal acrosome reaction necessary to penetrate the zona pellucida of the oocyte. The objective of this study was to characterize Doppel (Prnd) gene polymorphisms in eight Portuguese sheep breeds and to determine a possible relationship between these polymorphisms and ram fertility. Ovine genomic DNA of 364 animals of different breeds (Bordaleira entre Douro e Minho, Churra Badana, Churra Galega Mirandesa, Churra Mondegueira, Merino da Beira Baixa, Merino Branco, Saloia and Serra da Estrela) were analysed by multiple restriction fragment-single-strand conformation polymorphism (MRF-SSCP). This analysis revealed a synonymous substitution G-->A in codon 26 of Prnd gene. Churra Galega Mirandesa and Saloia breeds were more polymorphic (P=0.005 and P=0.04, respectively) than the overall population, while Serra da Estrela and Merino Branco animals were less polymorphic (P=0.007 and P=0.04). No polymorphism was found in Churra Mondegueira breed. Semen from 11 rams of Churra Galega Mirandesa breed (7 homozygous wildtype GG and 4 heterozygous GA) routinely used in the Portuguese Animal Germoplasm Bank was collected and frozen for fertility tests. A classification function was estimated, using data from post-swim-up semen motility and concentration and Day 6 embryo production rate, allowing the identification of the Doppel homozygous GG genotype with 86.7% of accuracy. This preliminary study detected the presence of only one polymorphism in codon 26 of Prnd gene in the Portuguese sheep breeds. In the polymorphic Churra Galega Mirandesa breed, GG genotype could be characterized through a model using three fertility traits, suggesting a relationship with male reproduction. Any future research should investigate not only AA genotype and its influence on ram fertility but also the possible consequences of the European Community selection program to eradicate Scrapie on the Prnd genotypes and indirectly on sheep breed's viability and preservation.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs

A direst agglutination test (DAT) and an immunofluorescence (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrabida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres ò1:320 and ò1:128). Of 34 samples collected form apparently healthly dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25 per cent. Considering these and previous studies in the adjancent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be high magnitude.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs.

A direct agglutination test (DAT) and an immunofluorescence technique (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrábida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres > or = 1:320 and > or = 1:128). Of 34 samples collected from apparently healthy dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25%. Considering these and previous studies in the adjacent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be of high magnitude.

Serodiagnosis of canine visceral leishmaniasis in Portugal: comparison of three methods.

Sera collected in Portugal from 43 dogs were screened for specific antibodies to Leishmania donovani antigens. Three different techniques were compared: an indirect immunofluorescence assay (IFA), a direct enzyme-linked immunosorbent assay (ELISA) and a competitive-ELISA (C-ELISA) using two species-specific monoclonal antibodies, D2 and D13. By IFA, 22 of the sera examined showed positive reactions, compared with 26 by ELISA or 27 by C-ELISA. There was no direct correlation observed between the serum titre by IFA and the strength of the reaction in ELISA or inhibition in C-ELISA. However, a good correlation was observed between sera identified as positive (95.5%) by all three techniques. Western blotting on leishmanial membranes showed that common antigens with Mr of 26,000 and 70-84,000 were recognized by all infected dog sera, regardless of the serum titre. In large scale studies, ELISAs are preferred to IFA for the rapid diagnosis of canine visceral leishmaniasis because of their greater simplicity.

Canine leishmaniasis: pathological and ecological factors influencing transmission of infection.

Canine leishmaniasis was studied in 1,823 dogs from the Lisbon metropolitan region. The breeds most affected were doberman and German shepherd, independent of sex and use. Young adult (12.2%) and older dogs (14.7%) had higher prevalences of infection. Parasitological confirmation of serological diagnosis was higher in dogs with indirect fluorescent antibody test titer greater than or equal to 1:512, indicating that parasitological patency is a late event. Exposure of Leishmania in lymph nodes is more efficient for parasitological confirmation (75.4% of cases). Frequent signs of disease were enlarged lymph nodes and onychogriphosis. However, 53.8% of the dogs with significant antibody titers (greater than or equal to 1:128) showed no symptom, suggesting that canine leishmaniasis has a prolonged asymptomatic period. This study confirmed the importance of the dog as the reservoir of visceral leishmaniasis.

Hepatozoonosis in foxes from Portugal.

Hepatozoon, sp. is described for the first time in foxes (Vulpes vulpes silacea) in Portugal. Of 301 foxes examined, 143 (48%) were infected. The gametocyte was the predominant stage of the life cycle and was found in every organ except the bone marrow, where schizonts were the most abundant stage. The morphological similarity of this parasite's gametocytes to Hepatozoon canis is emphasized.

Kala-azar in Portugal. V. The sylvatic cycle in the enzootic endemic focus of Arrabida.

Leishmania parasites were found in three of 43 foxes examined in the Arrabida region of the Setúbal peninsula in Portugal, this brings the total number of cases of vulpine leishmaniasis to four in 71 animals examined in this area, a prevalence rate of 5.63%. The three parasitized foxes found in this study were all symptomless and NNN cultures inoculated with tissue extracts were positive for all three animals. The immunofluorescence antibody test (IFAT) was positive with titres greater than or equal to 1/128 in the three infected foxes. The isoenzyme electrophoretic profile obtained from these positive cultures showed the strains to be identical with others isolated in Portugal from man, dog and fox, as well as with strains from elsewhere in the Mediterranean (zymodeme 1). The prevalence rate of 5.63% is probably sufficient to maintain endemicity. The presence of a semi-autonomic sylvatic cycle in the area seems likely. Hepatozoon was found in 28 foxes (65.11%).