Antibacterial activity of some medicinal plants from San Juan, Argentina

The antibacterial activities and preliminary phytochemical screening of 13 plants used as folk medicine in San Juan, Argentina, are reported.     

Spawning and Larval Development of the Four‐Sided Sea Cucumber,Isostichopus badionotus (Selenka 1867), under Controlled Conditions

The objective of this study was to generate information for the development of Isostichopus badionotus culture protocols by determining optimal spawning methods, egg production, length and duration of larval stages, and larval habitat settlement preferences. Studies performed during the spawning seasons of 2010 and 2011 showed that this organism spawns between July and November without mechanical or chemical induction. The larval development has five well‐defined stages: early, mid and late auricularia, doliolaria, and pentactula. Juveniles (654.3 µm length) were obtained on average 25 days after fertilization (DAF), when larvae were incubated at 25 ± 1 C. No preference was observed for any of the materials used for settlement of the larvae. The easy adaptation of I. badionotus to the culture conditions and the competence of the larvae to complete metamorphosis while feeding solely on microalgae concentrates make this species an excellent candidate for farming. However, it is necessary to continue designing techniques for larval culture and to establish appropriate captivity conditions that allow multiannual reproduction.     

Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen–Thawed Stallion Spermatozoa

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E™). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen–thawed semen samples.     

Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics

In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen–thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen–thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1–S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1–FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1–S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.     

Drought-tolerant naturalized populations of Lotus tenuis for constrained environments

Abstract

A perennial forage legume species tolerant to water stress would be useful to improve pasture and animal productivity in the central zone of Chile. The growth, dry-matter (DM) production, and drought tolerance of twelve accessions of Lotus tenuis Waldst & Kit, Syn. L. glaber, naturalized in Chile were evaluated with the objective to select contrasting genotypes, tolerant and sensitive to drought, for future breeding programmes. The accessions were sown in pots of 1.3 L containing a volcanic soil (Andisol). A completely randomized design with all combinations of Lotus accessions and four water treatments [100, 70, 40, and 10% of soil water availability (SWA)] was used. The relative rate of stem elongation (RRSE), the shoots and roots DM production, the relative water content (RWC), and the specific leaf area (SLA) were measured. A drought-sensitivity index (DSI) and the interaction genotype×environment were calculated. The RRSE, the DM production, RWC, and SLA all showed a significant reduction (P<0.05) in the treatment with the higher water restriction (10% SWA). There were significant differences (P<0.05) within RRSE and DM production genotype means, but the RWC and SLA did not differ among populations. The DSI varied broadly among genotypes, from 0.49 to 1.34, and was correlated negatively with DM production under water stress (10% SWA). It was concluded that the Lotus tenuis populations showed water-stress tolerance genetic variability, with accessions Lt14 and Lt4 the most contrasting. These findings will permit us to identify chromosomal regions associated with drought-tolerant genotypes which will allow us to accelerate the development of cultivars adapted to water-restricted environments.     

CHARACTERIZATION OF THE PHYSIOLOGICAL RESPONSE OF THE HIGHLY-TOLERANT TOMATO CV. ‘PONCHO NEGRO’ TO SALINITY AND EXCESS BORON

The variety of tomato (Solanum lycopersicum) called ‘Poncho Negro’ by farmers represents an important source of genetic resources of agricultural interest, because it has managed to thrive at levels of salinity and excess B that other varieties of the same species find impossible. This work was conducted under controlled growth conditions in a greenhouse, evaluating and obtaining plant material that served for physiological, chemical, and biochemical determinations. Stress conditions were supplied by irrigation, using different concentrations of sodium chloride (NaCl; 75 and 150 mM) and excess boron (B; 5 and 20 mg L^?1). The rate of net carbon dioxide (CO₂) assimilation, the quantum efficiency of photosynthesis and the relative water content (RWC) were measured. In addition, B, sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺), soluble sugars, and proline were quantified. The results suggest that ‘Poncho Negro’ accumulated Na⁺ mainly in the roots, partly limiting its entry into the aerial parts. In addition, it should be noted that the interaction of B and salinity reduced the movement of Na⁺ to the leaves. The ability of cv. ‘Poncho Negro’ to minimize leaf Na⁺ accumulation, accumulate more leaf B than control plants, and maintain its K⁺ level, when grown with an excess of B, possibly allowed the observed increase in the rate of photosynthesis. In addition, these tomato plants used proline and soluble sugars as osmo-regulators under high-B and saline conditions. Under all stress conditions studied, this variety of tomato was able to regulate its water content, with RWC values of approximately 86%.     

Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca²⁺-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca²⁺ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca²⁺ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.