Jorge André Matias Martins

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.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

Ovarian follicular response to different hormonal stimulation treatments in Canindé goats

The objective of the present study was to evaluate the effect of different hormonal stimulation treatments on the antral follicular population of naturalized Canindé goats. Adult goats (n=17) having estrous cycles at regular intervals were treated with intra-vaginal sponges containing 60 mg medroxyprogesterone acetate for 11 days, combined with an application of 50 μg d-cloprostenol on the Day 8 of treatment. For ovarian stimulation, goats were distributed into the following experimental groups: (a) multiple doses (MD), with a total of 120 mg NIH-FSH P1 in five intramuscular injections (30/30; 20/20 and 20 mg) at 12-h intervals; (b) three doses (TD), with a total of 120 mg NIH-FSH P1 in three intramuscular injections (60; 40 and 20 mg) at 24 h intervals; (c) one dose (OD), which consisted of the use of 70 mg NIH-FSH P1 combined with 200 IU eCG administered intramuscularly 36 h before sponge removal. In the MD and TD groups, FSH injections were begun on the Day 8 of progestagen treatment. The ovaries of all animals were observed by transrectal real time ultrasonography (TRU) during the follicular stimulation protocols. All follicles ≥2 mm were counted, measured and classified according to greatest diameter. The ultrasonographic assessment of the ovaries provided for well-defined ovarian structures. At the time of insertion of the sponges (Day 0), significant differences were observed (P<0.05) for the mean number of large follicles between the treated groups. Meanwhile, on Day 11, the three treatments did not differ (P<0.05), regardless of the follicular category. The diameter of small follicles was similar in MD, TD and OD during the whole period of the study. In the TD group, diameter of the large follicles was less (P<0.05) on Day 10 when compared to MD and OD. However, these differences were not observed on Day 11. In conclusion, the three treatments produced comparable distribution of the follicular populations. However, the single dose treatment can be preferred because of its simplicity and efficacious follicular response.

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

UNLABELLED 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.

History of registered Gyr breed in Brazilian Northeast: population structure and genetic improvement of growth traits

This paper provides an evaluation of the population structure, phenotype and genetic trends of registered Gyr herd cattle in northeast Brazil. The study provides important baseline information for the management, conservation and potential population expansion of this economically and culturally important cattle breed. Pedigree data were analyzed for individuals born between 1964 and 2006. Body weight values were adjusted to 205 and 365 days of age for animals born between 1978 and 2006. Phenotypic change of zebu Gyr in northeast Brazil is solely due to environmental improvement. However, there is potential for artificial selection for weight gain in young cattle. Effective population size decreased during the 1990s and the average inbreeding coefficient increased during the studied period. An increase of the effective population size of Gyr in northeast Brazil is strongly recommended, along with an increase in the management of the mating process to prevent inbreeding and to maintain the genetic variability of the breed.

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.

Further evidences for the mode of action of the larvicidal m-pentadecadienyl-phenol isolated from Myracrodruon urundeuva seeds against Aedes aegypti.

Nowadays, dengue fever is considered the most important arbovirosis worldwide and its control is still based upon combating the vector Aedes aegypti. Besides monitoring of mosquito populations resistant to conventional insecticides, the search for new environmentally safe insecticides and conduction of molecular studies focusing on the elucidation of mode of action and possible resistance mechanisms are considered the key for a sustainable management of the mosquito vector. Thus, the present work aimed to assess changes in protein expression of 3rd-instar larvae of Ae. aegypti after exposure to the natural insecticide m-pentadecadienyl-phenol. Bidimensional electrophoresis followed by mass spectrometry resulted in identification of 12 proteins differentially expressed between control and treated groups. Larvae exposed to the toxic compound for 24h showed elevated detoxification response (glutathione-S-transferase), increased levels of stress-related proteins (HSP70) as well as evidence of lysosome stabilization to enable survival. Furthermore, expression of proteins involved in protection of peritrophic membrane and metabolism of lipids indicated systemic effect of toxic effects in treated larvae.

Leptin and insulin in the seminal plasma of zebu bulls in peripuberty

In order to determine the profile of leptin and insulin values in seminal plasma and their correlations with sperm patterns during the peripuberty, a total of 16 crossbred Gyr dairy bulls were monitored from 60 days before to 60 days after puberty, separated into two groups, early and regular, according to the onset of puberty. Hormone values were determined by radioimmunoassay after gel filtration in fast performance liquid chromatography and semen patterns, according to the Brazilian College of Animal Reproduction. Insulin concentrations in seminal plasma did not differ between groups, nor between the ages, with the pooling of the data showing average of 35.15±16.93, 36.60±26.21, 26.82±09.81, and 43.56±31.71 32.24±16.71 U/mL for the pubertal period of -60, -30, 0, +30 and +60 days, respectively. These values were not correlated with sperm parameters, but differed in the group of animals with the highest percentage of major defects (31.08±18.58 U/mL) compared with those with lower percentage of these sperm defects (40.01±25.37 U/mL). Leptin was not correlated with sperm parameters and did not differ between groups or stage of sexual development, with concentrations of 23.10±10.43, 24.35±9.42, 22.41±9.90, 23.76±9.69 and 24.99±11.42 ng/mL for the periods of puberty aforementioned, respectively, after being grouped. These results demonstrated the binding of insulin to the structural quality of the sperm cell, and this hormone is indicated in andrologycal evaluation of future breeding soundness.