Flavia Regina Oliveira de Barros

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Effect of age on expression of spermatogonial markers in bovine testis and isolated cells

Spermatogonial stem cells (SSC) are the most undifferentiated germ cell present in adult male testes and, it is responsible to maintain the spermatogenesis. Age has a negative effect over stem cell, but the aging effect on SSC is not elucidated for bovine. The present study aim to evaluate the effect of age on the expression of undifferentiated spermatogonial markers in testis and in enriched testicular cells from prepubertal calves and adult bulls. In this matter, testicular parenchyma from calves (3-5 months) (n=5) and bulls with 3 years of age (n=5) were minced and, isolated cells were obtained after two enzymatic digestions. Differential platting was performed for two hours onto BSA coated dish. Cell viability was assessed by Trypan Blue solution exclusion method and testicular cells enriched for SSC was evaluated by expression of specific molecular markers by qRT-PCR (POU5F1, GDNF, CXCR4, UCHL1, ST3GAL, SELP, ICAM1 and ITGA6) and flow cytometry (GFRA1, CXCR4 and ITGA6). CXCR4 and UCHL1 expression was evaluated in fixated testes by immunohistochemistry. We observed that age just affected the expression of selective genes [SELP (Fold Change=5.61; p=0.0023) and UCHL1 (Fold Change=4.98; p=0.0127)]. By flow cytometry, age affected only the proportion of ITGA6+ cells (P<0.001), which was higher in prepubertal calves when compared to adult bulls. In situ, we observed an effect of age on the number of UCHL1+ (p=0.0006) and CXCR4+ (p=0.0139) cells per seminiferous tubule. At conclusion, age affects gene expression and the population of cells expressing specific spermatogonial markers in the bovine testis.

Spermatogonial Stem Cells and Animal Transgenesis

Spermatogonial stem cells (SSCs) are unipotent adult stem cells responsible for the maintenance of the spermatogenesis throughout the entire life of the male. We could say that the mammalian spermatogenesis is a classic adult stem cell-dependent process, sustained by self renewal and differentiation of SSCs. They are the only germline stem cells in adults. These cells can be found in the seminiferous tubule, lying near to the basement membrane. The SSC may choose to self-renewal or generate a daughter cell committed to differentiation. Studying SSCs provides a model to better understand adult stem cell biology and decipher the mechanisms that control SSC functions. It was reported that these cells hold the ability to colonize the seminiferous tubules after transplantation, restoring spermatogenesis. Besides the biomedical potential to perform studies of infertility in many species, SSCs present a promising application in biotechnology in the production of transgenic animals. This alternative route for transgenesis is of interest because a single male will generate by regular mate a variety of transgenic progenies. The production of a transgenic gonad can overcome the obstacles faced with the sperm-mediated gene transfer (SMGT) due to the high specialization of sperm. The use of SSC for transgenesis relies on targeting a much more undifferentiated germ cell and the potential permanent modification of the germ line. In this manner, this chapter aims to review the following topics regarding SSCs: (1) Mammalian spermatogenesis and SSCs; (2) Characterization of SSCs; (3) Isolation and in vitro culture of SSCs; (4) Transplantation of SSCs and animal transgenesis.

Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability

It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. Three homeopathic treatments composed by Pulsatila CH6, Pulsatila and Avena CH6, Avena CH6 and one control treatment (sucrose) were added to diluted boar semen, which were cooled for 24 or 48xa0h. Interestingly, no positive effect of homeopathic treatments was observed over semen viability. However, it was demonstrated that the 24xa0h of cooling storage provided more viable sperm cells when compared to the 48-h period. This effect of storage period on sperm viability was assessed by intact plasmatic membrane, intact acrosome and mitochondrial membrane potential evaluation.