Vitrification of testicular tissue from prepubertal cats in cryotubes using different cryoprotectant associations.

Protocols for the cryopreservation of testicular tissue are not yet established. In cats, few studies have been conducted on testicular vitrification using different cryoprotectant associations (CPAs). Thus, the objective of this study was to compare the effect of different CPAs on the vitrification of testicular tissue from prepubertal cats in cryotubes. We used 10 pairs of testicles, with each pair divided into 8 fragments that were distributed into different experimental groups. Two of these fragments were allocated into the control group (CG) and the other six were distributed according to the CPAs to be tested (dimethyl sulfoxide (DMSO)/glycerol (GLY), ethylene glycol (EG)/GLY, or DMSO/EG). The cryoprotectants were used at a final concentration of 5.6 M. The fragments were subjected to vitrification in cryotubes and after 1 week, they were warmed and processed for histomorphologic assessment, quantification of nucleolar organizer regions (NORs), and determination of cell viability. The DMSO/EG and EG/GLY groups presented the greatest cell separation from the cell basement membrane and the highest degrees of retraction of the basal membrane. In these aspects, DMSO/GLY did not differ from the CG and both were significantly superior to the other groups. In terms of cell distinction, visibility of the nucleus, and nuclear condensation, all the vitrified groups had significantly lower values than the CG, while the DMSO/GLY and EG/GLY groups did not differ between themselves. Through the quantification of NORs, the potential for cell proliferation of the CG was found to have a mean of 3.80, while DMSO/GLY presented a mean of 3.60, and thus there was no significant difference between these two groups. The proliferation potentials of both groups were significantly superior to that of the DMSO/EG (mean: 2.07) and EG/GLY (mean: 1.98) groups. In the CG and DMSO/GLY group, 91.8% and 64.2% of cells, respectively, were found to be viable. The cell viabilities of both groups were significantly superior to those of DMSO/EG (52.5%) and EG/GLY (57.10%). Vitrification in cryotubes combined with the use of the DMSO/GLY association was effective in maintaining the histomorphology, cell proliferation potential, and cell viability of testicular tissue from prepubertal cats after cryopreservation.

Proteomic characterization of canine seminal plasma.

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at -80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.

Proteins of the canine seminal plasma / Proteínas do plasma seminal canino

ABSTRACT: Studies have been performed to identify the proteins present in canine seminal plasma (SP) and relate them to sperm quality as well as to discover molecular markers of reproductive tract diseases. There is evidence that heparin-binding proteins, zinc-binding proteins, and lactoferrin as well as the matrix metalloproteinase, superoxide dismutase, catalase, and glutathione peroxidase enzymes are associated with canine sperm quality. Other studies indicate that prolactin and enzymes like arginine esterase, acid phosphatase, and alkaline phosphatase could be successfully used as biomarkers of reproductive disorders. Thus, the present literature review aims to address aspects related to proteins of the canine SP, their influence on fertility, and their importance as biomarkers of reproductive disorders.

RESUMO: Pesquisas têm sido realizadas para identificar as proteínas presentes no plasma seminal canino, com o intuito de relacioná-las com a qualidade espermática, bem como buscar por marcadores moleculares de patologias do trato reprodutivo. Há evidências de que as proteínas ligadoras de heparina, ligadoras de zinco, a lactoferrina, bem como as enzimas matrix metalloproteinase, superoxide dismutase, catalase e a glutationa peroxidase estão relacionadas com a qualidade seminal canina. Outras pesquisas indicam que a prolactina, e as enzimas arginina esterase, fosfatase ácida e fosfatase alcalina poderiam ser utilizadas com sucesso como biomarcadores de doenças reprodutivas. Assim, esta revisão de literatura objetiva abordar aspectos relacionados às proteínas do plasma seminal canino, suas influências sobre a fertilidade, e sua importância como biomarcadores de doenças reprodutivas.