Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

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.

Genetic structure of goat breeds from Brazil and the United States: Implications for conservation and breeding programs.

The objective of this study was to assess genetic diversity among 5 Brazilian (155 animals) and 5 U.S. goat (120 animals) breeds using 23 microsatellite markers. Samples from the United States represented a broad geographic distribution whereas Brazilian samples were from the northeast region. Samples from Boer were common to each country's breed count. Expected and observed heterozygosity among breeds ranged from 0.55 to 0.72, suggesting ample genetic diversity in the breeds evaluated. United States Angora, U.S. Spanish, and Brazilian Nambi ranked highest for allelic richness, averaging 6.1, 7.1, and 6.5 alleles per locus, respectively. Angora and Spanish also ranked highest in private alleles (7 and 9, respectively). Using STRUCTURE, the U.S. Spanish were also found to share a common cluster assignment with Brazilian Nambi, suggesting that progenitor breeds may have been the same and passed through the Canary Islands or Cape Verde in route to the New World. When non-Boer breeds were pooled by country, the effect of the subpopulation compared with total population () = 0.05, suggesting minor genetic differences exist between countries. The lack of genetic structure among goat breeds when compared with other species (e.g., vs. ) suggests goat breeds may exhibit a plasticity that facilitates productivity across a wide range of countries and environments. Taken a step further, the concept of breed for meat goats may not be as relevant for goat production.

Time-dependence of lung injury in mice acutely exposed to cylindrospermopsin.

Cylindrospermopsin is a cyanobacterial toxin of increasing environmental importance, as it can lead to disease if orally or intravenously absorbed. However, its in vivo lung impairment has not been documented. Thus, we aimed at verifying whether cylindrospermopsin can induce lung injury and establish its putative dependence on the time elapsed since exposure. BALB/c mice were intratracheally injected with either saline (NaCl 0.9%, 50 µL, SAL group, n = 12) or a sublethal dose (70 µg/kg) of semi-purified extract of cylindrospermopsin (CYN groups, n = 52). Lung mechanics, histological and biochemical analyses, and cylindrospermopsin presence in lungs and liver were determined in independent groups at 2, 8, 24, 48, and 96 h after cylindrospermopsin instillation. There was a significant increase in static elastance at 24 and 48 h after exposure to cylindrospermopsin, while viscoelastic component of elastance and viscoelastic pressure rose at 48 h. Alveolar collapse augmented in CYN groups at 8 h. A significant increase in polymorphonuclear influx into lung parenchyma, as well as a higher myeloperoxidase activity started off at 24 h. Exposure to cylindrospermopsin increased lipid peroxidation and superoxide dismutase activity and reduced catalase activity in CYN groups. The toxin was detected in lungs and liver of all CYN mice. In conclusion, cylindrospermopsin exposure impaired lung mechanics, which was preceded by lung parenchyma inflammation and oxidative stress.

Relaçäo entre fatores sangüíneos de bovinos em bubalinos / Relationship between cattle blood factors in water buffaloes

Com o objetivo de verificar as relaçöes entre fatores sangüíneos de bovinos e bubalinos, foram tipificados 121 búfalos de rio (Bubalus bubalis bubalis), das raças Mediterrâneo, Murrah, Jafarabadi e também alguns animais sem raça definida (SRD). A tipagem foi realizada em placas de microtitulaçäo segundo técnicas usadas rotineiramente para tipagem de bovinos. Foram empregados 45 reagentes bovino-isoimunes, cobrindo dez sistemas de grupos sangüíneos. Ao final das leituras, os animais da raça Murrah apresentaram reaçöes positivas para os fatores A1, D, H, B, K, Q, T2, Y2, A', B', F', O', Q', C2, R2, X2, F, V, N', J, L e U1; os da raça Mediterrâneo para os fatores A1, D, H, I2, K, P, Q, A', B', F', O', P'2, C2, R2, X2, V, N', J, L e Z; os da raça Jafarabadi para os fatores A1, D, H, Z', K, P, Q, T1, Y2, A', D', E'2, K', O', Q', C2, R2, N', J, M1, S, U1 e R'; os SRD positivos para os fatores A1, D, H, Z', B, K, P, T2, Y2, A', B', D', E'2, F', I', O', Q', C2, R1, R2, N', J, M1, S, U1 e R'. Ressalte-se que os fatores sublinhados foram observados apenas naqueles respectivos grupamentos raciais. Os animais SRD mostraram 13 fatores comuns com os da raça Mediterrâneo, 21 com os da raça Jafarabadi e 15 com os da raça Murrah. Os resultados mostram relaçöes estreitas entre 35 fatores pertencentes a 10 sistemas. Tal fato é indicativo da possível utilizaçäo desses reagentes bovino-isoimunes com reaçöes positivas, em testes de parentesco de bubalinos. Ao mesmo tempo, mostram existir diferenças genéticas quanto aos fatores observados e suas freqüências nas subpopulaçöes analisadas, permitindo, ainda, a detecçäo de mestiçagem