Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Altered Expression of Transforming Growth Factor‐Beta Isoforms in Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors may contribute to follicular persistence. Transforming growth factor‐beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)‐induced COD. In the oestrous‐synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous‐synchronized control group with that in ACTH‐induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH‐induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.     

On-farm multi-contamination of pigs by food-borne bacterial zoonotic hazards: An exploratory study

Application of risk analysis to control pork-borne zoonoses on farms is a major aim of the current European food safety legislation. The prevalence, levels of contamination and variations in pig contaminations according to herds and batches must therefore be studied to define relevant methods for control of zoonoses. The aims of this exploratory study were to provide information on the prevalence and levels of infection of finisher/fattening pigs by Campylobacter spp., Clostridium perfringens, Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus, and to quantify the respective effects of batches and herds. One hundred and twenty-seven pooled fresh feces samples and 120 pooled cutaneous swab samples were tested from 37 batches in 14 farrowing-to-finishing farms. Campylobacter spp. was identified in all fecal samples on farms whereas L. monocytogenes was never found. C. perfringens was identified in 48% of samples, Salmonella in 2%, and S. aureus in 48%. The decomposition of variance of on-farm prevalence and levels of contamination and a general linear model showed a systematically significant effect of herd, and an effect of batch only for levels of contamination with Campylobacter and C. perfringens and for prevalence of S. aureus. The contamination status of pigs seems to be mainly explained by herd status linked to poor biosecurity measures. Further studies are needed to explain such correlations in order to define farm indicators for food-borne zoonotic hazards.     

Lectin‐Binding Pattern in Ovarian Structures of Rats with Experimental Polycystic Ovaries

Numerous experimental models in different species have been developed for the study of polycystic ovarian syndrome. In this study, we used a model of induction of polycystic ovaries (PO) in rats by exposure to constant light to study the distribution and variations of glycosylated residues present in the different ovarian structures. Seven biotinylated lectins were used (Con‐A, WGA, DBA, SBA, PNA, RCA and UEA‐I) on tissue sections, and detection was performed using the streptavidin/peroxidase method. In tissue sections was observed an increase in affinity for Con‐A in the granulosa and theca interna of growing follicles and cysts in animals with PO in relation to the control group. Follicular cysts showed higher affinity for WGA and RCA‐I which growing follicles in the same group, and there was a decrease in affinity for PNA in the cysts in relation to the growth of follicles in both groups. Atretic follicles in both groups showed greater labelling with lectins PNA, SBA and RCA‐I in relation to healthy follicles. It could also be noted that the zona pellucida of cystic follicles lost the affinity for the lectin Con‐A. There was no staining on follicles in any category with the lectins DBA and UEA‐I, although it was staining in the corpus luteum (control group) and in the mesothelium and interstitial glands of both groups with DBA. These observations probably reflect changes in the glycosaminoglycans present in the different ovarian compartments or in the glycosylation of cellular components essential for proper follicular dynamics.     

Protoplasts in the analysis of early plant-pathogen interactions: current applications and perspectives

Transient gene expression in protoplasts has been applied to the analysis of gene expression, promoter activity, subcellular localization of proteins, and genome editing. Its advantages have been realized in recent years in various physiological, biochemical, genetic, and molecular biological studies. The omics and systems biology studies of plant development and their interactions with the environment have revitalized this approach. Early plant-pathogen interaction represents one of the most unique signalling research areas. Here, we try to discuss some developments and current applications of the protoplast system. It is for sure that the system will significantly bridge the gap between high throughput approaches and functional analysis in the study of plant-pathogen interactions in the omics era.     

Constant Light Exposure Causes Dissociation in Gonadotrophin Secretion and Inhibits Partially Neuroendocrine Differentiation of Leydig Cells in Adult Rats

The objective of this work was to study the changes that occur in the Leydig cells of rats exposed to continuous light. The laboratory rat is considered a non-photoperiodic species because exposure to short photoperiod has little or no effect on the reproductive status. However, exposure of adult female rats to constant light induces polycystic ovaries, indicating that extreme changes in the photoperiod affect the reproductive function seriously. Adult male rats were placed under continuous light conditions for a duration of 15 weeks. After this period, the animals were killed and testicles were dissected and processed by routine histologic protocols. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) serum levels were determined by radioimmunoassay (RIA). The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A, S-100 protein, P substance, synaptofisin, neurofilament protein-200, gliofibrillary acidic protein and neurone-specific enolase were used. The mean LH serum concentration was significantly lower, while the mean FSH level was significantly higher in treated animals. The expression of S-100, NSE, CrA, SP and SYN was significantly lower in treated animals. In conclusion, the constant light exposure acting directly at the pituitary level decreases LH secretion. The increased FSH secretion may be due to a partial reduction of the negative androgen feedback in the pituitary gland. Moreover, the constant light exposure affects the expression of some immunomarkers in Leydig cells, possibly because of the changes found in the gonadotrophin level and feedback mechanism.     

Long-term agroecosystem experiments: assessing agricultural sustainability and global change

Long-term agroecosystem experiments can be defined as large-scale field experiments more than 20 years old that study crop production, nutrient cycling, and environmental impacts of agriculture. They provide a resource for evaluating biological, biogeochemical, and environmental dimensions of agricultural sustainability; for predicting future global changes; and for validating model competence and performance. A systematic assessment is needed to determine the merits of all known experiments and to identify any that may exist in tropical and subtropical environments. The establishment of an international network to coordinate data collection and link sites would facilitate more precise prediction of agroecosystem sustainability and future global change.     

The scale of saproxylic beetles response to landscape structure depends on their habitat stability

Context

Forest management and disturbances cause habitat fragmentation for saproxylic species living on old-growth attributes. The degree of habitat spatiotemporal continuity required by these species is a key question for designing biodiversity-friendly forestry, and it strongly depends on species’ dispersal. The “stability–dispersal” model predicts that species using stable habitats should have lower dispersal abilities than species associated with ephemeral habitat and thus respond to habitat availability at smaller scales.

Objectives

We aimed at testing the stability–dispersal model by comparing the spatial scales at which saproxylic beetle guilds using substrates with contrasted stability (from stable to ephemeral: cavicolous, fungicolous, saproxylophagous and xylophagous guilds) are affected by landscape structure (i.e. habitat amount and aggregation).

Methods

We sampled saproxylic beetles using a spatially nested design (plots within landscape windows). We quantified habitat availability (tree cavities, polypores and deadwood) in 1-ha plots, 26-ha buffers around plots and 506-ha windows, and analyzed their effect on the abundance and diversity of associated guilds.

Results

The habitat amount within plots and buffers positively affected the abundance of the cavicolous and the fungicolous guilds whereas saproxylophagous and xylophagous did not respond at these scales. The habitat aggregation within windows only positively affected the saproxylophagous species richness within plots and also on the similarity in species composition among plots.

Conclusions

Beetle guilds specialized on more stable habitat were affected by landscape structure at smaller spatial scales, which corroborated the stability–dispersal model. In managed forests, the spatial grain of conservation efforts should therefore be adapted to the target habitat lifetime.