Recovery of sperm after epididymal refrigeration from domestic cats using ACP-117c and Tris extenders / Recuperação de espermatozoides após refrigeração do epidídimo de gatos domésticos utilizando os diluidores ACP-117c e Tris

We aimed to compare fresh sperm and sperm cooled to 4ºC that had been recovered from the epididymides of cats using powdered coconut water (ACP-117c) and Tris extenders. Sixty epididymides were divided into 6 groups: 10 fresh epididymides were recovered using Tris (T0h); 10 were kept at 4°C/2h and recovered using Tris (T2h); 10 were kept at 4°C/4h and recovered using Tris (T4h); 10 fresh were recovered using ACP-117c (A0h); 10 were kept at 4°C/2h and recovered using ACP-117c (A2h), and 10 were kept at 4°C/4h and recovered using ACP-117c (A4h). The testis-epididymis complexes (TEC) control were not cooled. The others were cooled at 4°C for 2 or 4h. The epididymis was separated and the sperm was recovered by the modified flotation method. Sperm kinetic parameters were evaluated by a computer-system analysis, and vigor, viability, concentration, membrane function and morphology of the sperm were assessed under a light microscope. The progressive motility with ACP-117c declined after 2h of cooling, but did not differ between fresh and 4h. The vigor and membrane function were higher in A4h than A0h. The vigor at T2h and T4h were decreased compared to T0h. T0h was higher than A0h for vigor and sperm membrane function. However, after 4h of cooling, ACP-117c maintained a higher percentage of living cells. Feline epididymal sperm quality can be maintained to the degree necessary for artificial breeding programs following cooling and ACP-117c may be successfully used to recover cat sperm that have been cooled for up to 4h.(AU)

Objetivou-se comparar a qualidade de espermatozoides recuperados a fresco e após refrigeração a 4ºC do epidídimo de gatos domésticos utilizando-se os diluidores ACP-117c e Tris. Sessenta epidídimos foram distribuídos em seis grupos: 10 epidídimos a fresco com o Tris (T0h), 10 a 4°C/2h e recuperados com Tris (T2h), 10 a 4°C/4h e recuperados com Tris (T4h), 10 epidídimos a fresco com o ACP-117c (A0h), 10 a 4 °C/2h e recuperados com ACP-117c (A2h), 10 a 4°C/4h e recuperados com ACP-117c (A4h). Os complexos testículo-epidídimo (CTE) do controle não foram refrigerados. Os outros foram refrigerados a 4°C durante duas e quatro horas. Os epidídimos foram separados das demais estruturas, e os espermatozoides recuperados pela técnica de flutuação modificada. Os parâmetros cinéticos foram avaliados em um sistema computadorizado, e o vigor, a viabilidade, a concentração, a funcionalidade de membrana e a morfologia celular foram avaliados em microscopia de luz. A motilidade progressiva com ACP-117c declinou após duas horas de refrigeração, mas não diferiu entre a recuperação a fresco e após refrigeração por quatro horas. Vigor e integridade funcional da membrana celular foram significativamente superiores no grupo A4h em comparação ao A0h. O vigor espermático em T2h e T4h reduziu significativamente em comparação com T0h. T0h foi significativamente superior ao A0h quanto aos parâmetros de vigor e integridade funcional da membrana espermática, entretanto, após quatro horas de refrigeração, o ACP-117c apresentou um maior percentual de células vivas. Os espermatozoides epididimários de felinos domésticos conseguem manter a qualidade necessária para serem utilizados em programas de reprodução artificial após serem refrigerados e recuperados por meio da técnica de flutuação modificada, e o diluidor ACP-117c pode ser utilizado com sucesso para recuperação de células espermáticas refrigeradas de gatos por até quatro horas.(AU)

High insulin concentrations promote the in vitro growth and viability of canine preantral follicles.

To determine whether the effects of different concentrations of insulin on the development of canine preantral follicles in vitro were associated or not with FSH, secondary follicles were isolated and cultured. In Experiment 1, follicles were cultured in the following media: modified minimum essential medium (CtrlMEM) alone; CtrlMEM plus 5 ng mL⁻¹ insulin (Ins5ng); CtrlMEM plus 10 ng mL⁻¹ insulin (Ins10ng); and CtrlMEM plus 10 µg mL⁻¹ insulin. In Experiment 2, follicles were cultured in the same media but in the presence of sequential FSH (i.e. CtrlFSH, Ins5ngF, Ins10ngF and 10µgF, respectively). Increasing concentrations of FSH (100, 500 and 1000 ng mL⁻¹) were added sequentially to the culture medium on Days 0, 6 and 12 of culture. Viability were assessed at the end of culture and follicular diameter and the antrum formation rate at four time points (Days 0, 6, 12 and 18). In Experiment 1, the high insulin concentration significantly increased follicular viability (P<0.05). In contrast, in Experiment 2, viability was not affected by the inclusion of insulin. In addition, viability was significantly better in follicles cultured in CtrlFSH (P<0.05). The diameter of follicles in the high-insulin group in Experiment 1 and high-insulin plus FSH group in Experiment 2 was superior to other groups tested. In experiment 2, the Ins10µg and Ins10µgF groups exhibited significantly higher antrum formation rates than the other groups. In conclusion, in the absence of FSH, high concentrations of insulin have beneficial effects on follicular viability. However, to promote the growth of canine preantral follicles in vitro, it is recommended that a combination of insulin and FSH be added to the medium.

Eight-cell parthenotes originated from in vitro grown sheep preantral follicles.

We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles >110 µm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P < .05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P > .05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.

Intravaginal artificial insemination in bitches using frozen/thawed semen after dilution in powdered coconut water (ACP-106c).

The aim of this study was to evaluate powdered coconut water extender (ACP-106c; ACP Serviços Tecnológicos Ltda, ACP Biotecnologia, Fortaleza, Ceará, Brazil) as a diluent for freezing dog semen and the fertility after vaginal insemination of semen frozen therein. Ten ejaculates were collected from five dogs, evaluated fresh, diluted in ACP-106c, 10% egg yolk and 6% glycerol, cooled and frozen. In the first phase of the study, straws with frozen semen were thawed and immediately subjected to the same analysis as the fresh semen and, in addition, to Computer-Assisted Semen Analysis (CASA). In phase 2, 10 bitches that had been subjected to natural breeding during a preceding oestrous cycle were vaginally inseminated with thawed semen that had been re-diluted in ACP-106c. After thawing, a mean of 77% sperm motility was obtained through subjective analysis and 77.3% through CASA. Following artificial insemination, a 60% pregnancy rate was observed, resulting in a 50% parturition rate and a mean litter size of 3.4 (SEM 0.6), with 47.1% males and 52.9% females. ACP-106c can be successfully used for freezing canine semen, and vaginal deposition of such semen yields similar pregnancy rates to those reported in other studies.

The effect of LIF in the absence or presence of FSH on the in vitro development of isolated caprine preantral follicles.

We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 µm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.

Vascular endothelial growth factor-A(165) (VEGF-A(165)) stimulates the in vitro development and oocyte competence of goat preantral follicles.

The aim of this study was to evaluate the effect of vascular endothelial growth factor-A(165) (VEGF-A(165)) on the in vitro development of goat secondary preantral follicles. Preantral follicles (≥150 µm in diameter) were isolated from the ovaries of adult mixed-breed goats and individually cultured for 18 days in αMEM in the absence (control) or presence of VEGF-A(165) at concentrations of 10 ng/ml (VEGF10) and 100 ng/ml (VEGF100). Analyses of follicular survival, diameter, antrum formation and rate of daily growth were performed every 6 days. At the end of the culture period, morphologically normal oocytes (≥110 µm in diameter) were taken for in vitro maturation (IVM). The results demonstrated that all follicles presented oocytes and granulosa cells that were morphologically normal and after labeling with calcein-AM, high rates of oocyte viability were observed in all treatments. The follicular diameter and the growth rate achieved in the presence of VEGF10 were higher than those of the control. Both treatments with VEGF-A(165) showed higher rates of oocyte recovery for IVM when compared with the control. Moreover, only the addition of VEGF-A(165) permitted oocytes grown in vitro to reach metaphase II. Thus, the addition of VEGF-A(165) to the culture medium improves the development of goat preantral follicles cultured in vitro, allowing the production of mature oocytes.

Moment of addition of LH to the culture medium improves in vitro survival and development of secondary goat pre-antral follicles.

The present study investigated the effects of time of addition of luteinizing hormone (LH) to culture medium on the in vitro development of caprine pre-antral follicles. Pre-antral follicles (≥ 150 µm) were isolated from fragments of the goat ovarian cortex and individually cultured for 18 days in the absence (control) or presence of 100 ng/ml LH, added on days 0, 6 or 12 of culture. Follicular development was assessed based on antral cavity formation, increased follicular diameter as well as follicular and fully grown oocyte (>110 µm) viability. The results showed that after 18 days of culture, the percentage of surviving follicles in the control treatment was significantly lower when compared to other treatments (p < 0.05). There were no significant differences in antrum formation, follicular diameter and oocyte viability. The addition of LH at D6 of culture significantly increased the rates of oocytes ≥ 110 µm and the resumption of meiosis (p < 0.05). In contrast, when LH was added at the onset of culture, only germinal vesicle oocytes were obtained. In conclusion, the moment of addition of LH to the culture medium affects the performance of in vitro culture of caprine pre-antral follicles. The addition of LH to the medium from day 6 of culture onward improved the rates of follicular survival, as well as the ability of oocytes to resume meiosis. However, prolonged exposure to LH (addition at the onset of culture onward) showed detrimental effects for the meiotic resumption.