Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation.

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM - 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Freeze-dried Platelet-rich Plasma and Stem Cell-conditioned Medium for Therapeutic Use in Horses.

This study investigated platelet-rich plasma (PRP) and adipose stem cell-conditioned medium (ASC-CM) use as a strategy to accelerate tissue healing. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were quantified in fresh and freeze-dried PRP and ASC-CM, and a stability test was performed in the freeze-dried samples (90 and 180 days of storage). A cell proliferation test was performed using equine mesenchymal stem cell culture in reconstituted PRP gel mesh after freeze-drying. In vivo PRP, ASC-CM applications, or their association were performed in induced wounds at 15 and 9-day intervals, according to the treatments: saline solution (control), PRP, ASC-CM, or ASC-CM + PRP. Horses were monitored through photographs and wound area measurements on days 5, 7, 15, and 24 after lesion induction. Skin biopsies were obtained on days 15 and 24 of the experiment. PDGF and VEGF quantification did not differ between fresh or freeze-dried treatments, was similar after freeze-drying or 90 days of storage, but showed a significant reduction after 180 days of storage. Comparing all treatments, no differences were observed in the histopathological analyses. For inflammation, fibroplasia, and collagen formation, only the time effect between the first and second biopsies was significant. The cell proliferation test revealed intense multiplication in the PRP gel mesh. Healing time was similar among all treatments. In conclusion, our results showed the possibility to produce and maintain freeze-dried PRP and ASC-CM for 90 days. Further studies are needed to better explore the in vivo therapeutic PRP and ASC-CM effects.

Sperm membrane proteins associated with the boar semen cryopreservation.

This study aimed to define sperm membrane protein markers of semen freezability of boars with the aid of a proteomic approach. Semen from fourteen adult boars were subjected to slow freezing and rapid thawing. After thawing, sperm vigor and motility were analyzed, and based on these results, animals were separated into two groups: good (GFEs) and poor freezability (PFEs). Sperm membrane proteins were extracted and subjected to two-dimensional electrophoresis. Stained gels were analyzed by computerized resources to indicate differentially expressed protein spots, that were identified by mass spectrometry. Six animals showed good freezability with average sperm vigor and motility of 2.2±0.8 and 41.8±22.9, respectively, whereas eight boars showed poor freezability, with 1.9±0.6 and 26.8±17.5 of sperm vigor sperm motility, respectively. An average of 263±62.2 spots per gel and 234.2±54.6 of spots consistently present in all gels were detected. The intensities of five spots were significantly different between groups. Fc fragment of IgG binding protein and lactadherin were more intense in the PFE group, while Arylsulfatase A and F-actin capping protein subunit alpha 1 were more expressed in the GEF group. Based on their functions and interactions with other proteins, we conclude that these four sperm membrane proteins may act as potential markers of boar semen freezability.

Proteomic analysis of responsive stem proteins of resistant and susceptible cashew plants after Lasiodiplodia theobromae infection.

Gummosis is an aggressive disease caused by the necrotrophic fungus Lasiodiplodia theobromae (Pat.) Griffon & Maubl that threatens commercial cashew orchads in Brazil. To study the molecular mechanisms involved in the cashew response to L. theobromae, a proteomic analysis of stems from the commercial cashew clone BRS 226 (resistant) was conducted at early times post-artificial infection. In addition, changes in the stem proteome profiles of gummosis resistant and susceptible cashew plants grown under field condition and naturally exposed to pathogen were also compared. After two-dimensional gel electrophoresis (2D-PAGE), 73 proteins showed statistically significant differences in spot abundance. Of these, 31 spots were identified in BRS 226 stems compared with mock-inoculated controls and 32 in stems collected from field-grown resistant and susceptible cashew plants. L. theobromae-responsive proteins were mainly involved in energy metabolism pathways, stress and defense, cell signaling and protein metabolism indicating modulation of various cellular functions upon fungal infection. As stress-inducing factors seem to be important for susceptibility to disease, the change in the abundance relative these proteins may possibly indicate an attempt to maintain cellular homeostasis, as resistance determinant factor, related with a possible role in the regulation of oxidative burst. These findings provide the first information about the cellular mechanisms acting in the Anacardium occidentale genotypes associated with the pathophysiological state of infection with L. theobromae. BIOLOGICAL SIGNIFICANCE: Gummosis caused by Lasiodiplodia theobromae, a necrotrophic fungus, is the major disease of cashew plants in the semi-arid conditions of northeastern Brazil. Although various studies were carried out on this pathosystem, there is no information available on the molecular mechanisms of plant defense related to the incompatible interaction of cashew with L. theobromae. Therefore, this original study comprises a differential proteomic analysis of cashew stems from: (i) resistant dwarf clone BRS 226 mock-inoculated (control) and artificially inoculated with L. theobromae; and (ii) cashew plants bearing resistant and susceptible traits to gummosis, originated from open pollination of BRS 226 in a commercial orchard with high disease incidence. The contribution of the reprogrammed proteins to molecular events triggered in cashew plants challenged by L. theobromae has a great relevance in the identification of the host candidate proteins linked to biological pathways that respond to L. theobromae infection. Furthermore this study may contribute to improve breeding programs aimed at selecting resistant/tolerant cashew clones toward this pathogen.

Seminal plasma proteins of adult boars and correlations with sperm parameters.

The present study was conducted to identify the major seminal plasma protein profile of boars and its associations with semen criteria. Semen samples were collected from 12 adult boars and subjected to evaluation of sperm parameters (motility, morphology, vitality, and percent of cells with intact acrosome). Seminal plasma was obtained by centrifugation, analyzed by two-dimensional SDS-PAGE, and proteins identified by mass spectrometry (electrospray ionization quadrupole time-of-flight). We tested regression models using spot intensities related to the same proteins as independent variables and semen parameters as dependent variables (P ≤ 0.05). One hundred twelve spots were identified in the boar seminal plasma gels, equivalent to 39 different proteins. Spermadhesin porcine seminal protein (PSP)-I and PSP-II, as well as spermadhesins AQN-1, AQN-3 and AWN-1 represented 45.2 ± 8% of the total intensity of all spots. Other proteins expressed in the boar seminal plasma included albumin, complement proteins (complement factor H precursor, complement C3 precursor and adipsin/complement factor D), immunoglobulins (IgG heavy chain precursor, IgG delta heavy chain membrane bound form, IgG gamma-chain, Ig lambda chain V-C region PLC3, and CH4 and secreted domains of swine IgM), IgG-binding proteins, epididymal-specific lipocalin 5, epididymal secretory protein E1 precursor, epididymal secretory glutathione peroxidase precursor, transferrin, lactotransferrin and fibronectin type 1 (FN1). On the basis of the regression analysis, the percentage of sperm with midpiece defects was related to the amount of CH4 and secreted domains of swine IgM and FN1 (r² = 0.58, P = 0.006), IgG-binding protein (r² = 0.41, P = 0.024), complement factor H precursor (r² = 0.61, P = 0.014) and lactadherin (r² = 0.45, P = 0.033). The percentage of sperm with tail defects was also related to CH4 and secreted domains of swine IgM and FN1 (r² = 0.40, P = 0.034), IgG-binding protein (r² = 0.35, P = 0.043) and lactadherin (r² = 0.74, P = 0.001). Sperm motility, in turn, had association with the intensities of spots identified as lactadherin (r² = 0.48, P = 0.027). In conclusion, we presently describe the major proteome of boar seminal plasma and significant associations between specific seminal plasma proteins and semen parameters. Such relationships will serve as the basis for determination of molecular markers of sperm function in the swine species.