A quick methodology to identify sexual seedlings in citrus breeding programs using SSR markers

In citrus breeding and genetics, it is very important to distinguish between zygotic and nucellar seedlings in order to eliminate unwanted genotypes. Usually, isozyme marker shave been employed to determine the genetic origin of young plants. In this work we propose the use of SSR markers as an alternative methodology and compare them with isozymes in this kind of screenings. Two different populations were analysed: one derives from an interspecific cross and the other from selfing. We conclude that, in most cases, microsatellites are more efficient than isozymic markers to identify the sexual origin of citrus seedlings, given their higher level of polymorphism and the scarce number of polymorphic isozymes in some populations. We describe a quick and efficient methodology for SSR analysis, including a fast DNA extraction in microcentrifuge tubes, and visualization through silver staining, which eliminates the need for a labelling step. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fruit characterization of Cocos nucifera L. (ARECACEAE) cultivars from the Pacific coast of Costa Rica and the Philippines

Tall coconut cultivars from the Pacific coast of Costa Rica and the Philippines (San Ramón, Tagnanán, and Laguna), were evaluated for fruit characteristics. Most of the introduced cultivars showed extremely large heterogeneity. A cluster analysis, based on the Ward method, classified the palms into four groups with high internal homogeneity. Some of the evaluated coconut palms from the Costa Rican Pacific area had nut characteristics similar to the San Ramon and Tagnanan palm groups but not to the Laguna group. At the association level used (semipartial R ² = 0.10), another group which included the remaining palms sampled from the Costa Rican Pacific coast was constituted.     

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle.

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5-10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.     

Ganglioneuroblastoma in a cat: a rare neoplasm treated with electrochemotherapy

An 8-year-old male castrated cat was referred for sudden onset of lameness. Physical examination revealed a 1x2x1cm mass originating from a footpad of the right hind leg. A diagnosis of ganglioneuroblastoma was suggested by the tumour appearance following histopathological staining with haematoxylin and eosin and haematoxylin/van Gieson. Immunohistochemical staining for glial fibrillary acidic protein (GFAP), vimentin, neuron-specific enolase (NSE), neurofilament and S100 further confirmed the diagnosis. The staging process did not indicate metastatic spread. The cat was treated with three sessions of electrochemotherapy (ECT) 1 week apart, following local injection of bleomycin. The tumour had completely regressed within 1 week of the third ECT application and remained in remission for 402 days at which time a small recurrence was noted. The animal was given a further session of ECT using intra-lesional cisplatin and again went into remission. It remained tumour free at 450 days. Electrochemotherapy is considered a safe and effective treatment for localised neoplasms of cats and dogs and warrants further investigation.     

Ascorbic acid–cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos

Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl‐β‐cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC‐CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC‐CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap‐frozen for gene expression analysis by RT‐qPCR. Besides, in vitro‐produced zygotes were cultured with 100 µM of AC‐CD and blastocysts were as well snap‐frozen for gene expression analysis. A group without AC‐CD (control^?) and other with CD (control⁺) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC‐CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC‐CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC‐CD, while in blastocysts derived from zygote cultured with AC‐CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC‐CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC‐CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.     

Current challenges in sheep artificial insemination: A particular insight

Ovine artificial insemination (OAI) is not commonly performed because of specific problems related to semen application techniques, leading to highly variable results. The ideal methodology (frozen‐thawed semen/vaginal route) is unfeasible under field conditions due to the cervix morphology of the ewe, which prevents the process of intrauterine insemination necessary to obtain acceptable results. Currently, OAI commercial programmes use superficial cervical insemination, CAI (vaginal), with chilled semen (15°C) and intrauterine insemination, LAI (laparoscopic), with frozen‐thawed semen. The ability to improve upon these contrasting techniques may be derived from examining certain poorly studied factors such as insemination time, productive state of females and alternatives of seminal preservation, some of which we reviewed in this work. This interim solution will remain in use until AI by the vaginal route with frozen‐thawed semen is developed, but it poses new challenges in optimizing the freezing of the sperm and adapting the cervical (CAI) and/or transcervical intrauterine AI (TCAI). In this review, we address the current problems and evaluate their methodological (mechanical) and chemical (dilation) alternatives. Currently, TCAI is a methodologically complex technique with poor fertility results, so further studies are needed to improve the logistics of this procedure and the results of its application.     

Leaf pruning intensities at flowering of banana (Musa AAA,cv. Grande Naine) did not influence fruit green and yellow life and quality

This study examined the effects of leaf pruning intensities at flowering on the green and yellow life and fruit quality of bananas (Musa AAA, cv. Grande Naine). The fruit from banana plants that retained 7, 9, 11 and 13 leaves after pruning were packed in carton boxes of 13.7 kg and stored in a cold room at 14 °C for 21 days to simulate transportation conditions. During this period, eight visual evaluations of fruit peel colour were made. Next, fruits were induced to commercial ripening using ethylene at 100 μl/ml. Four evaluations (every 2 days) on fruit firmness, soluble solids, titratable acidity, fruit weight and peel colour were made to assess fruit yellow life. No interaction between evaluations and number of leaves retained was found for pulp firmness, soluble solid percentage, fruit acidity, fruit weight and maturation grade. The fruit green life and peel colour was similar for plants retaining different number of leaves. After the application of ethylene, there were no differences in fruit firmness (P > 0.62), percentage of soluble solids (P > 0.24) nor in the percentage of acidity (P > 0.32). No difference in fruit weight (P > 0.07) and ripening grade (P > 0.17) were observed among plants retaining different number of leaves. The results suggest that in tropical commercial banana plantations, producing for international markets, it is possible to defoliate the banana plants to seven leaves at flowering without causing a reduction on the green and yellow life and quality of fruit.