ABSTRACT
Microfluidic systems are on the rise in several studies that evaluate reproductive cells. However, the material used for manufacturing can still be considered relatively expensive. The objective of this study was to develop a new microfluidic device, using a modified polydimethylsiloxane ((PDMS) Silpuran®), test its viability and carry out a selection of bovine sperm. Sperm was collected from epididymis (n = 10) and evaluated at different incubation times (60 min, 120 min, 180 min) to assess polydimethylsiloxane toxicity, where a tube was used as a control and the microfluidic device as treatment. An additional ten epididymis were used for the sperm selection test, which utilized four types of solutions: in vitro maturation medium (IVM) with and without oocyte, progesterone and saline solution (SS). The Percoll gradient was used as a control and the microfluidic device as treatment. The kinetic parameters of sperm were evaluated using the computer-assisted semen analysis (CASA). Morphology was performed with Bengal Rose, the integrity, and viability of the sperm using the hypoosmotic test and fluorescent microscopy probes, respectively. Mann-Whitney test was used in the first experiment, Kruskall-Wallis variance analysis tests with post hoc and Student-Newman-Keuls used in the second experiment. Regarding the non-toxic effects, most motility parameters demonstrated the superiority of the microfluidic device compared to the control. In the second experiment, the sperm showed equivalence between the microfluidic device and the Percoll gradient Silpuran® PDMS was not toxic to the cells and can be efficient for selecting bovine sperm, achieving better results in a medium for IVM with or without oocytes.
Subject(s)
Epididymis , Sperm Motility , Animals , Cattle , Dimethylpolysiloxanes , Humans , Male , Microfluidics , SpermatozoaABSTRACT
Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.
ABSTRACT
Ovarian xenografting is an auxiliary reproductive technique that allows the conservation of germplasm of high-value livestock and endangered species. The use of exogenous gonadotropins assists in developing xenografted tissues and obtaining viable follicles for in vitro embryo production; however, this use has not been reported in the xenografting of cats' (Felis catus) ovaries with C57BL/6 female SCID mice (Mus musculus) as recipients. Therefore, the aim of this study was to evaluate the responses of queens' ovaries to eCG when grafted into C57BL/6 female SCID mice. Ovarian cortex fragments from queens were grafted under the kidney capsule of 15 C57BL/6 SCID mice after bilateral ovariectomy. After 45 days, the recipients were divided into two groups: those that did not receive hormone induction (eCG-), which were euthanized at the time of induction, and those that received hormonal induction (eCG+), which were euthanized 48 hours later. All the tissues collected were histologically processed. The proportions of the different ovarian follicles were compared by chi-square test. The morphology of the follicles was compared between the experimental groups by Tukey (primordial, primary, and secondary follicles) and Kruskal-Wallis (antral follicles) tests. Macroscopically, we observed a few antral follicles that were over 1 mm in size in grafts treated with eCG. Microscopically, follicles of all categories were observed in the grafts, and all had normal morphology for the species studied. However, larger primordial and primary follicles were observed in the eCG+ transplants than those in the eCG- transplants. There was a decrease in primordial follicles and an increase in the other follicles, particularly in the antral follicles of the eCG+ group, a phenomenon that we propose to term "follicular right shift". Luteinized follicles were also observed in grafts treated with eCG. Therefore, treatment with eCG is effective for follicular development but does not provoke a good superovulatory response, so the correct application time should be identified. Other protocols should be tested, to obtain viable follicles that can be used for in vitro embryo production.
