The use of gelatine in long‐term storage (up to 48 hr) at 5°C preserves the pre‐freezing and post‐thawing quality of brown bear sperm

Sedimentation of spermatozoa occurs during long‐term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long‐term pre‐freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF‐ULE‐Bear extender (TesT‐fructose‐egg yolk‐glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 10⁶ sperm ml^?1) (Standard); (ii) final dilution at RT and cooling in a tube (FD‐Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD‐Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD‐Tube, FD‐Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 10⁶ sperm ml^?1, glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR‐14/propidium iodide‐PI‐; VIAB), acrosomal status (PNA‐FITC/PI; iACR) and apoptotic status (YO‐PRO‐1/PI; YOPRO‐) by flow cytometry. At pre‐freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO‐) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO‐, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post‐thawing, Gelatine sample had similar scores for YOPRO‐, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long‐term pre‐freezing storage at 5°C.     

Balloon Valvuloplasty for Treatment of Pulmonic Stenosis in English Bulldogs with an Aberrant Coronary Artery

Background: English Bulldogs (EB) with pulmonic stenosis (PS) sometimes have an aberrant coronary artery (CA) type R2A encircling the pulmonary artery (PA). Balloon valvuloplasty (BV) is treatment of choice for severe PS, but is considered to be contraindicated in dogs with aberrant CA. Hypothesis: Conservative BV in EB with aberrant CA is safe and improves clinical signs and quality of life. Animals: Four client‐owned EB with severe PS were retrospectively reviewed/analysed. Methods: Retrospective study: Case records, echocardiography, BV, and follow‐up investigations of EB diagnosed with severe PS and treated with BV were reviewed. The ratios of PA to aortic (Ao) velocity time integral (VTI) were calculated to assess progression/improvement of PS. Results: An aberrant CA was confirmed on angiography in all EB. Conservative BV was performed, using a balloon of the size of the PA annulus or smaller (0.6–1 × PA annulus size). All dogs survived the procedure, but only a mild reduction in pressure gradient was achieved. There was an improvement in PA to Ao VTI in 3 of 4 dogs (P≤ .017), which were free of evidence of congestive heart failure (CHF) 5, 10, and 15 months after BV. One dog that had right‐sided CHF when BV was performed died due to progressive right‐sided CHF within 3 months. Conclusions: Conservative BV in EB might be safe and might improve quality and quantity of life.     

Variation in Spanish cultivated einkorn wheat (<Emphasis Type="Italic">Triticum monococcum</Emphasis> L. ssp. <Emphasis Type="Italic">monococcum</Emphasis>) as determined by morphological traits and waxy proteins

A collection of 29 lines of Spanish einkorn wheat (Triticum monococcum L. ssp. monococcum) were analysed for morphological traits and waxy proteins. Four different botanical varieties were detected in the collection on the basis of the morphological traits. For the waxy proteins, no polymorphism has been described in previous works in wheat diploid species. In the current materials, two different alleles were found, namely Wx-A ^m 1a and Wx-A ^m 1a′. The allele Wx-A ^m 1a′ that presents less mobility than the Wx-A ^m 1a, has not been previously described and was only found in one line.     

Screening species of <Emphasis Type="Italic">Pilocarpus</Emphasis> (Rutaceae) as sources of pilocarpine and other imidazole alkaloids

Plants of the Pilocarpus genus (Rutaceae) are popularly known as jaborandi and are the only source of pilocarpine, an imidazole alkaloid used in eye-drops for the treatment of glaucoma as well as for the stimulation of sweat and lachrymal glands. Alkaloid extracts from leaf samples of seven species of Pilocarpus, from the states of São Paulo and Maranhão in Brazil, were analyzed using HPLC–ESI–MS/MS. The samples contained between 0.88 ± 0.04 and 1.00 ± 0.14% of alkaloids in relation to the dry weight of their leaves, with significant differences in results (P ≤ 0.05) found only between Pilocarpus microphyllus planted in the state of Maranhão and Pilocarpus spicatus, Pilocarpus trachyllophus, Pilocarpus pennatifolius and Pilocarpus jaborandi; as well as between Pilocarpus spicatus and Pilocarpus racemosus. Pilocarpine was not found in P. spicatus, whereas in the other species it ranged from 2.6 ± 0.1 to 70.8 ± 1.2% of total alkaloids. P. microphyllus planted in the state of Maranhão for pilocarpine extraction had the highest total alkaloid content, but it had only 35% of pilocarpine in relation to total alkaloids. Three other species contained more pilocarpine in relation to total alkaloids: P. jaborandi (70.8%), P. racemosus (45.6%) and P. trachyllophus (38.7%); and could be candidates for pilocarpine extraction. Differences in alkaloid content were significant for all these samples (P ≤ 0.05). Imidazole alkaloids were observed and partially characterized based on their retention times and high resolution mass. The seven species analyzed had different imidazole alkaloid profiles, but only one did not present quantifiable pilocarpine contents in its leaves. The Pilocarpus genus shows potential for the prospection of novel alkaloids.     

Genetic diversity analysis in Phaseolus vulgaris L. using morphological traits

Common bean (Phaseolus vulgaris) is an important export crop in Kyrgyzstan since the end of the twentieth century. Genetic diversity analysis of common bean populations is useful for breeding programs, as it helps to select genetic material to be used for further crossing. Twenty-seven common bean accessions were analyzed using 13 qualitative morphological traits. In some cases, obtained morphological data were combined with previously published results based on microsatellite markers. The similarity matrices generated from the molecular and morphological data were significantly correlated (r = 0.49, P < 0.01). Cluster analyses based on Dice’s similarity coefficient were constructed based on morphological data and the combined data set of morphology and microsatellite, and both grouped the 27 accessions according to their origin: 15 belonged to the Andean and 12 to the Mesoamerican gene pool. On average, the Andean accessions were less diverse than the Mesoamerican accessions. The average diversity based on the Shannon diversity index for the 13 qualitative morphological traits was 0.05. Overall, this study revealed that qualitative morphological markers are efficient in assigning modern cultivars to their gene pools of origin.     

Effect of processing and storage on the stability of flaxseed lignan added to dairy products

This study investigated the effects of processing and storage on the stability of purified, flaxseed-derived secoisolariciresinol diglucoside (SDG) added to milk prior to the manufacture of different dairy products. We analyzed the effect of high-temperature pasteurization, fermentation, and milk renneting as well as storage on the stability of SDG added to milk, yogurt, and cheese. Also, the stability of SDG in whey-based drinks was studied. Added SDG was found to withstand the studied processes well. In edam cheese manufacture, most of the added SDG was retained in the whey fraction and 6% was found in the cheese curd. SDG was also relatively stable in edam cheese during ripening of 6 weeks at 9 degrees C and in yogurt during storage of 21 days at 4 degrees C. Up to 25% of added SDG was lost in whey-based drinks during storage of 6 months at 8 degrees C. We conclude that SDG can be successfully supplemented in dairy-based products.     

Behavior of zinc from six organic fertilizers applied to a navy bean crop grown in a calcareous soil

The objective of this study was to compare the mobility, leaching, availability, and relative effectiveness of Zn from Zn-polyhydroxyphenylcarboxilate (Zn-PHP), Zn-HEDTA (Zn-N-2-hydroxyethyl-ethylenediaminetriacetate), Zn-EDDHSA [Zn-ethylenediamine-di-(2-hydroxy-5-sulfophenylacetate)], Zn-EDTA (Zn-ethylenediaminetetraacetate), Zn-S,S-EDDS (Zn-ethylenediaminedisuccinate), and Zn-EDTA-HEDTA sources by applying different Zn rates (5 and 10 mg kg(-1)) to a calcareous soil under greenhouse conditions. A lysimeter experiment was carried out for 60 days and using navy bean (Phaseolus vulgaris L.) as an indicator plant. The Zn available to the plant and easily leachable Zn were determined in soil by different single extractions, while the distribution of Zn in the soil was assessed by sequential speciation. The utilization of applied Zn by the navy bean was greatest when the Zn treatments were Zn-EDTA, Zn-EDTA-HEDTA, Zn-HEDTA, and Zn-EDDHSA. Both total Zn in the plants and soluble Zn in the plant dry matter (extracted with 1 mM 2-morpholino-ethanesulfonic acid) were positive and significantly correlated with the following: the amounts of Zn extracted with the three single extractions used to estimate soil available Zn and the amounts of Zn in the water soluble plus exchangeable and organically complexed fractions. The Zn-HEDTA, Zn-EDDHSA, Zn-EDTA-HEDTA, Zn-S,S-EDDS, and Zn-EDTA sources significantly increased the mobility of micronutrients through the soil with respect to the control and Zn-PHP source. The maximum Zn concentration obtained in the leachate fractions was 65 mg L(-1) (13% of Zn applied) for the Zn-S,S-EDDS chelate applied at a rate of 10 mg Zn kg(-1) soil. In the course of the crop, the soil pH + pe parameter increased significantly with experimental time.     

Leaching and efficiency of six organic zinc fertilizers applied to navy bean crop grown in a weakly acidic soil of Spain

Zinc contamination of groundwater from fertilizers applied to pulse crops is a potential problem, but the use of different types of organic chelates can minimize the contamination potential while still adequately feeding the crops. The objective of this study was to compare the leaching, distribution in fractions and availability, and relative effectiveness of Zn from six organic Zn fertilizers (zinc-ethylenediaminetetraacetate- N-2-hydroxyethylethylenediaminetriacetate (Zn-EDTA-HEDTA), Zn-HEDTA, zinc- S, S'-ethylenediaminedisuccinate (Zn- S, S-EDDS), zinc-polyhydroxyphenylcarboxylate, Zn-EDTA, and zinc-ethylenediaminedi(2-hydroxy-5-sulfophenylacetate) (Zn-EDDHSA)) applied to a navy bean ( Phaseolus vulgaris, L.) crop cultivated by applying different Zn levels, in a weakly acidic soil under greenhouse conditions. Zinc soil behavior was evaluated by diethylenetriaminepentaacetic acid-triethanolamine (DTPA-TEA), DTPA-ammonium bicarbonate (DTPA-AB), Mehlich-3, and BaCl 2 extractions and sequential fractionation. In all the fertilizer treatments, the percentage of labile Zn that remained in the soil was high with respect to the quantity of Zn applied, with values respectively ranging from 42 to 80% for Zn-EDDHSA and Zn-EDTA sources. A positive correlation with a high level of significance existed between the micronutrient concentration in the navy bean crop (total and soluble) and labile Zn fractions, available Zn, and easily leachable Zn ( r ranged from 0.89 to 0.95, P < 0.0001). The relatively high quantity of total Zn leached by applying Zn-EDTA and Zn-S,S-EDDS sources (11.9 and 6.0%, respectively, for the rate 10 mg of Zn kg(-1) of soil) poses a potential pollution risk for neighboring waters. It would seem recommendable to apply Zn-HEDTA or Zn-EDDHSA sources, even applied at the low rate (5 mg of Zn kg(-1) of soil), because they produced available Zn concentrations in the soil that were above the critical concentration and also produced high Zn concentrations in plants (139 and 106 mg of Zn kg(-1) of dry matter, respectively).     

Assessment of the plant growth promotion abilities of six bacterial isolates using Zea mays as indicator plant

Zea mays, one of the most important cereals worldwide, is a plant not only with food and energy value, but also with phytoremediation potential. The use of plant growth promoting (PGP) rhizobacteria may constitute a biological alternative to increase crop yield and plant resistance to degraded environments. In search for PGP rhizobacteria strains, 6 bacterial isolates were isolated from a metal contaminated site, screened in vitro for their PGP characteristics and their effects on the growth of Z. mays were assessed. Isolates were identified as 3A10^T, ECP37^T, corresponding to Chryseobacterium palustre and Chryseobacterium humi, and 1ZP4, EC15, EC30 and 1C2, corresponding to strains within the genera Sphingobacterium, Bacillus, Achromobacter, and Ralstonia, respectively. All the bacterial isolates were shown to produce indole acetic acid, hydrogen cyanide and ammonia when tested in vitro for their plant growth promoting abilities, but only isolates 1C2, 1ZP4 and ECP37^T have shown siderophore production. Their further application in a greenhouse experiment using Z. mays indicated that plant traits such as root and shoot elongation and biomass production, and nutrient status, namely N and P levels, were influenced by the inoculation, with plants inoculated with 1C2 generally outperforming the other treatments. Two other bacterial isolates, 1ZP4 and ECP37^T also led to increased plant growth in the greenhouse. These 3 species, corresponding to strains within the genera Ralstonia (1C2), Sphingobacterium (1ZP4), and to a strain identified as C. humi (ECP37^T) can thus be potential agents to increase crop yield in maize plants.