Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility.

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.

A simple method for selection of cumulus-oocyte complexes from bovine ovaries by sedimentation with percoll.

Selection of bovine cumulus-oocyte complexes (COCs) for in vitro embryo production (IVP) is generally based on the morphological characteristics of the cumulus cells surrounding the oocytes and the ooplasm under microscopic observation. The purpose of this study was to examine a simple method for selection of COCs by sedimentation with Percoll solutions. COCs were aspirated from ovaries derived from a local slaughterhouse, and the COCs were classified by the morphology of their cumulus cell layers, as follows: Class A, compact and thick; Class B, compact but thin; Class C, partially denuded and thin; and Class D, denuded. Percoll solutions were prepared by diluting Percoll to 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30% solutions, respectively. COCs were placed on the surface of the Percoll solution for 3 min, and the precipitated COCs were transferred to stepwise high density solution. The percentage of Percoll solution just before buoyancy was considered to the specific sedimentary value of the COC and oocyte. The mean sedimentary value of Class A COCs was higher than those of the other classes (P<0.01). The mean sedimentary values of denuded oocytes from Classes A and B were higher than those from Classes C and D (P<0.01). Our results show that sedimentation of COCs and denuded oocytes was generally related to the morphological quality of the COCs, although the sedimentary values ranged widely for one class of COCs and oocytes. The Percoll method can be used for simple selection of COCs.

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.