Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89^T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89^T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.     

Analysing the growth of turbot (<Emphasis Type="Italic">Psetta maxima</Emphasis>) in a commercial recirculation system with the use of three different growth models

The growth data of a commercial aquaculture recirculation system were analysed to investigate the growth performance of reared turbot (Psetta maxima). Three common growth models (von Bertalanffy, Gompertz and Schnute) were fitted to the growth data documented over a time period of 6 years. To determine the most suitable model, three different criteria were used: (1) the Akaike index criterion, (2) the sum of squared residuals and (3) the average daily deviation between the estimated final weight and the observed final weight. The evaluation of the growth models showed that the Schnute model had the lowest Akaike index, the lowest sum of squared residuals and the lowest daily deviation between estimated and real weight of all tested growth models. The Schnute model produced sigmoid growth curves. The estimated growth coefficients were the most realistic ones in regard to biological interpretation. In contrast, the von Bertalanffy growth model and the Gompertz model estimated inaccurate exponential growth curves and are therefore unable to simulate the growth data as well as the Schnute model. The results indicate that the von Bertalanffy growth model is not the optimal model to simulate the present growth data and that the growth potential of reared turbot has probably not yet been fully exploited in the aquaculture system(s) examined (so far).     

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Müllerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values). This revised version was published online in July 2006 with corrections to the Cover Date.     

Relation between soil organic matter and yield levels of nonlegume crops in organic and conventional farming systems

The aim of this study was to evaluate the interaction between yield levels of nonleguminous crops and soil organic matter (SOM) under the specific conditions of organic and conventional farming, respectively, and to identify implications for SOM management in arable farming considering the farming system (organic vs. conventional). For that purpose, correlations between yield levels of nonlegume crops and actual SOM level (C_org, N_t, C_hwe, N_hwe) as well as SOM‐level development were examined including primary data from selected treatments of seven long‐term field experiments in Germany and Switzerland. Yield levels of nonlegume crops were positively correlated with SOM levels, but the correlation was significant only under conditions of organic farming, and not with conventional farming treatments. While absolute SOM levels had a positive impact on yield levels of nonlegumes, the yield levels of nonlegumes and SOM‐level development over time correlated negatively. Due to an increased demand of N from SOM mineralization, higher yield levels of nonlegumes obviously indicate an increased demand for OM supply to maintain SOM levels. Since this observation is highly significant for farming without mineral‐N fertilization but not for farming with such fertilization, we conclude that the demand of SOM‐level maintenance or enhancement and thus adequate SOM management is highly relevant for crop production in organic farming both from an agronomical and ecological point of view. Under conventional management, the agronomic relevance of SOM with regard to nutrient supply is much lower than under organic management. However, it has to be considered that we excluded other possible benefits of SOM in our survey that may be highly relevant for conventional farming as well.     

Effects of biochar compared to organic and inorganic fertilizers on soil quality and plant growth in a greenhouse experiment

Our contemporary society is struggling with soil degradation due to overuse and climate change. Pre‐Columbian people left behind sustainably fertile soils rich in organic matter and nutrients well known as terra preta (de Indio) by adding charred residues (biochar) together with organic and inorganic wastes such as excrements and household garbage being a model for sustainable agriculture today. This is the reason why new studies on biochar effects on ecosystem services rapidly emerge. Beneficial effects of biochar amendment on plant growth, soil nutrient content, and C storage were repeatedly observed although a number of negative effects were reported, too. In addition, there is no consensus on benefits of biochar when combined with fertilizers. Therefore, the objective of this study was to test whether biochar effects on soil quality and plant growth could be improved by addition of mineral and organic fertilizers. For this purpose, two growth periods of oat (Avena sativa L.) were studied under tropical conditions (26°C and 2600 mm annual rainfall) on an infertile sandy soil in the greenhouse in fivefold replication. Treatments comprised control (only water), mineral fertilizer (111.5 kg N ha^–1, 111.5 kg P ha^–1, and 82.9 kg K ha^–1), compost (5% by weight), biochar (5% by weight), and combinations of biochar (5% by weight) plus mineral fertilizer (111.5 kg N ha^–1, 111.5 kg P ha^–1, and 82.9 kg K ha^–1), and biochar (2.5% by weight) plus compost (2.5% by weight). Pure compost application showed highest yield during the two growth periods, followed by the biochar + compost mixture. biochar addition to mineral fertilizer significantly increased plant growth compared to mineral fertilizer alone. During the second growth period, plant yields were significantly smaller compared to the first growth period. biochar and compost additions significantly increased total organic C content during the two growth periods. Cation‐exchange capacity (CEC) could not be increased upon biochar addition while base saturation (BS) was significantly increased due to ash addition with biochar. On the other hand, compost addition significantly increased CEC. Biochar addition significantly increased soil pH but pH value was generally lower during the second growth period probably due to leaching of base cations. Biochar addition did not reduce ammonium, nitrate, and phosphate leaching during the experiment but it reduced nitrification. The overall plant growth and soil fertility decreased in the order compost > biochar + compost > mineral fertilizer + biochar > mineral fertilizer > control. Further experiments should optimize biochar–organic fertilizer systems.