Sperm selection techniques in cattle: Microfilter device versus conventional methods.

Microfluidics and microfilter devices have been developed to mimic the characteristics of the female reproductive tract, minimizing the risk of sperm damage. This study aimed to compare the use of a microfilter device versus conventional methods for sperm selection used in in vitro fertilization (IVF). For selecting spermatozoa, the pooled samples were processed in a microfilter device, swim-up and mini-Percoll gradient. Kinematic and morphometric parameters, vitality and DNA damage were analysed before and after sperm selection. After selection, 10,000 motile spermatozoa per oocyte were used in IVF drops. Embryos were assessed at three (cleavage rate) and seven (blastocyst rate) days post-IVF. Results of sperm kinematic parameters including average path velocity, velocity straight line, curvilinear velocity, linearity, lateral head displacement with the microfilter device were superior to density gradient (p < 0.05), but similar to swim-up method. Likewise, sperm DNA damage was significantly reduced using the microfilter device and swim-up method. Regarding the total sperm recovery rate post selection, results with the microfilter device (17.64%) and mini-Percoll gradient (18.27%) were higher than with swim-up method (6.52%). However, the cleavage and blastocyst rates were the lowest using the microfilter device. In conclusion, sperm selection using the microfilter device and swim-up method can improve kinematic parameters, although the mini Percoll gradient was the most efficient method for embryo production.

PTEN inhibitor and kit ligand increase in vitro activation and survival of primordial follicles in alpaca.

In mammals, activation of primordial follicles to primary follicle is a progressive and highly regulated process. There is evidence in mice that phosphatase and tensin homologue deleted on Chromosome 10 (PTEN) silencing is an important negative regulator of phosphatidylinositol 3-kinase (PI3K), which initiates activation of dormant follicles. The objective of the study was to evaluate the effect of the addition of PTEN inhibitor (bpV(HOpic)) (10 µM) and/or Kit Ligand (KL) (100 ng/mL) on the in vitro activation and survival of alpaca primordial follicles. Ovarian cortical fragments from 11 adult alpacas were cultured for 24 h in tissue culture medium (α-MEM+ ) supplemented with KL and bpV or the association of both. Subsequently, each sample was processed by classical histology and follicular counting and classification were performed. The results obtained show a reduction (p < 0.05) of primordial follicles in more than 50% in follicular tissue cultured in vitro in α-MEM+ or supplemented with bpV and/or KL versus the control (not cultured). Further, >25% increase in primary follicles in follicular tissue cultured in vitro in α-MEM+ or supplemented with KL and/or bpV versus control. However, the follicular survival rate showed a decrease of 20% in the cultured tissues, except for the α-MEM+ supplemented with KL and bpV. In conclusion, supplementation of bpV (HOpic) (10 µM) and KL (100 ng/mL) increased the activation in vitro of primordial follicles and survival after in vitro culture of alpaca ovarian tissue.

Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue.

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.