RESUMO
The aim of this study was to evaluate the effect of supplementing bovine semen freezing extender with different concentrations of iodixanol on post-thaw sperm characteristics. Six ejaculates of three Nellore bulls were pooled and diluted in commercial extender (BotuBov®) and then divided into 4 groups: control group (without adding iodixanol); groups G1.5, G3, or G6 according to the concentration of iodixanol solution (RedCushion®). After dilution, the samples were cooled and frozen. Post-thaw semen evaluation included sperm motility by CASA immediately after thawing and after 60 min of incubation at 37°C, flow cytometry analysis for integrity of plasma and acrosomal membranes, membrane destabilization and translocation of phosphatidylserine, mitochondrial membrane potential, and formation of intracellular anion superoxide ( O 2 - ), hydrogen peroxide (H2 O2 ), and membrane lipid peroxidation. The group G6 presented significantly higher (p < .05) total and progressive motility, percentage of plasma and acrosomal membrane integrity, and H2 O2 than control and group G1.5. Furthermore, group G6 showed lower (p < .05) lipid peroxidation than control. In addition, regardless of the concentration used, the percentage of spermatozoa without phosphatidylserine translocation was higher (p < .05) in all iodixanol supplemented groups. In conclusion, iodixanol supplementation preserved the motility and integrity of sperm membranes during cryopreservation and protected against lipid peroxidation.
Assuntos
Preservação do Sêmen , Sêmen , Masculino , Animais , Bovinos , Congelamento , Antioxidantes/farmacologia , Fosfatidilserinas , Motilidade dos Espermatozoides , Preservação do Sêmen/veterinária , Crioprotetores/farmacologia , Espermatozoides , Análise do Sêmen/veterinária , Criopreservação/veterinária , Suplementos NutricionaisRESUMO
This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.
Assuntos
Acrosina , Proteômica , Acrosina/metabolismo , Animais , Cães , Masculino , Melhoramento Vegetal , Proteômica/métodos , Sêmen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Albumina Sérica/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Seminal plasma has several components that protect the sperm cells and assist in the fertilization process. In contrast, the exact role carried out by seminal plasma during the cooling of canine semen remains controversial. Moreover, concerning the long estrus period, the possibility to store chilled semen at 5°C for more than 72 hours and maintain good sperm quality for additional inseminations could increase fertilization rates. Thus, this study aimed to evaluate the seminal plasma influence on quality and oxidative stress of the extended canine semen stored at 5°C for 7 days. Three ejaculate pools from eight healthy dogs were collected by digital manipulation of the penis. The sperm kinetics, sperm vitality (eosin/nigrosin stain), integrity of plasma and acrosomal membranes, morphology, superoxide and hydrogen peroxide production, mitochondrial potential, lipid peroxidation, and oxygen reactive species production (induced and spontaneous thiobarbituric acid [thiobarbituric acid reactive substances, TBARS] assay) were evaluated every 48 hours (M0, M48, M96, and M168) until 7 days (168 hours) in cooled extended (TRIS egg yolk) semen of dogs at 5°C with (+SP) or without (-SP) autologous seminal plasma. No statistical difference was found for sperm kinetics in cooled samples with +SP and -SP during the experimental time period, except for the progressive motility of +SP samples that was higher at M48 than M96 (p = 0.023). The seminal plasma did not influence any other evaluated sperm characteristics. Finally, our results demonstrated that the presence or lack of seminal plasma during cooling the semen of dogs does not influence sperm quality at 5°C. Moreover, the components of the semen extender may contribute to maintaining good sperm quality and low reactive oxygen species production during the long period of the dog's semen cooling, even after semen centrifugation.
Assuntos
Preservação do Sêmen , Animais , Cães , Gema de Ovo , Masculino , Espécies Reativas de Oxigênio , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.
Assuntos
Búfalos , Bovinos , Proteoma , Proteínas de Plasma Seminal/análise , Animais , Masculino , Sêmen/química , Especificidade da EspécieRESUMO
Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process.