In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.(AU)