Proteomic analysis of seminal plasma from locally-adapted "Curraleiro Pé-Duro bulls" (Bos taurus): identifying biomarkers involved in sperm physiology in endangered animals for conservation of biodiversity.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

Meat quality assessment from young goats fed for long periods with castor de-oiled cake.

Diet can influence both the qualitative and quantitative traits of ruminant meat. This study evaluated the effects of castor de-oiled cake on the meat of mixed-breed male goat kids. After 165days of diet treatment, no alterations (p>0.05) were observed in the in vivo performance, anatomic components, dissection and proximate composition of the Longissimus dorsi muscle, as well as in the color and pH of the carcasses. However, diet had an effect (p<0.05) on energy metabolites, fatty acid profile, and expression of certain proteins of the Longissimus dorsi muscle. To conclude, this study showed that the establishment of castor de-oiled cake diet for a long period to goats led to alterations in meat quality, without compromising its consumption qualities.

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.

Membrane-associated proteins of ejaculated sperm from Morada Nova rams.

The objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 µg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function.

Sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the Brazilian Northeast: the season's influence on the cooling of semen / Caracrterísticas espermáticas e bioquímicas do plasma seminal de caprinos nas estações chuvosa e seca do Nordeste brasileiro: influência da estação no resfriamento do sêmen

The present study aimed to verify the caprine semen characteristics during dry and rainy seasons in the Brazilian Northeast, and the influence of these seasons on cooled semen. Seminal volume, concentration, percentage of motile cells, vigor and spermatic morphology, as well as biochemical profile (fructose, citric acid, P, Ca2+, Mg, total proteins and phospholipase A2 activity) were analyzed. It was observed a reduction (P<0.05) in normal sperm morphology, fructose, citric acid, P, Mg and total protein concentration during the dry season, which did not affect the motility, vigor, volume and sperm concentration. Phospholipase A2 activity was increased during the dry season (P<0.05). The analysis of the semen cooled at 4ºC during 48 hours showed reduction in total motility and vigor sperm during the dry season (P<0.05). Based on these results, we conclude that the best period of year for caprine semen cooling is the rainy season.

Verificou-se as características seminais de caprinos durante a época seca e a chuvosa no Nordeste brasileiro e a influência da época no resfriamento do sêmen. Foram mensurados volume, concentração espermática, porcentagem de espermatozoides móveis, vigor, morfologia espermática e características bioquímicas (frutose, ácido cítrico, fósforo, magnésio, proteínas totais e atividade da fosfolipase A2). Observou-se redução (P<0,05) no número de espermatozóides morfologicamente normais, frutose, ácido cítrico, fósforo, magnésio e proteínas totais durante a época seca que não influenciaram na motilidade, vigor, volume e concentração do sêmen. Entretanto, a atividade da fosfolipase A2 foi maior na época seca. Quando o sêmen foi submetido ao resfriamento a 4ºC durante 48 horas, houve redução (P<0,05) na motilidade total e no vigor espermático durante a época seca. Com base nesses resultados, conclui-se que o período chuvoso é melhor para resfriar sêmen de caprinos no Nordeste brasileiro.

Sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the Brazilian Northeast: the season's influence on the cooling of semen / Caracrterísticas espermáticas e bioquímicas do plasma seminal de caprinos nas estações chuvosa e seca do Nordeste brasileiro: influência da estação no resfriamento do sêmen

The present study aimed to verify the caprine semen characteristics during dry and rainy seasons in the Brazilian Northeast, and the influence of these seasons on cooled semen. Seminal volume, concentration, percentage of motile cells, vigor and spermatic morphology, as well as biochemical profile (fructose, citric acid, P, Ca2+, Mg, total proteins and phospholipase A2 activity) were analyzed. It was observed a reduction (P<0.05) in normal sperm morphology, fructose, citric acid, P, Mg and total protein concentration during the dry season, which did not affect the motility, vigor, volume and sperm concentration. Phospholipase A2 activity was increased during the dry season (P<0.05). The analysis of the semen cooled at 4ºC during 48 hours showed reduction in total motility and vigor sperm during the dry season (P<0.05). Based on these results, we conclude that the best period of year for caprine semen cooling is the rainy season.(AU)

Verificou-se as características seminais de caprinos durante a época seca e a chuvosa no Nordeste brasileiro e a influência da época no resfriamento do sêmen. Foram mensurados volume, concentração espermática, porcentagem de espermatozoides móveis, vigor, morfologia espermática e características bioquímicas (frutose, ácido cítrico, fósforo, magnésio, proteínas totais e atividade da fosfolipase A2). Observou-se redução (P<0,05) no número de espermatozóides morfologicamente normais, frutose, ácido cítrico, fósforo, magnésio e proteínas totais durante a época seca que não influenciaram na motilidade, vigor, volume e concentração do sêmen. Entretanto, a atividade da fosfolipase A2 foi maior na época seca. Quando o sêmen foi submetido ao resfriamento a 4ºC durante 48 horas, houve redução (P<0,05) na motilidade total e no vigor espermático durante a época seca. Com base nesses resultados, conclui-se que o período chuvoso é melhor para resfriar sêmen de caprinos no Nordeste brasileiro.(AU)