Magnetic-Assisted Control of Eggs and Embryos via Zona Pellucida-Linked Nanoparticles.

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature-like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature-competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof-of-concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets.

Impact of inclusion of post-spermatic ejaculate fraction in boar seminal doses on sperm metabolism, quality, and interaction with uterine fluid.

Boar ejaculate is composed of sperm cells and seminal plasma (SP) and is emitted in different fractions (pre-sperm fraction; spermatic-rich fraction; intermediate fraction; post-spermatic fraction), with different composition of SP and volume, which could influence the sperm quality during seminal doses preparation, conservation, and interaction with the female reproductive tract. In artificial insemination (AI) centers, seminal doses are usually prepared with the spermatic-rich and intermediate fractions, but the inclusion of other ejaculate fractions, although controversial, is beginning to be applied. The objective was to evaluate the synergic effect of accumulative ejaculated fractions on sperm functionality during seminal doses preparation, throughout storage and after incubation with uterine fluid (UF). For this purpose, a total of 57 ejaculates were collected, and the following experimental groups were prepared (n = 19 per group): (F1) spermatic-rich fraction; (F2) F1 plus intermediate fraction; (F3) F2 plus post-spermatic fraction. Each group was stored for 5 days at ∼16 °C, and the following parameters were evaluated: sperm metabolism of pure and diluted semen (day 1), sperm quality parameters (days 1, 3, 5), thermal-resistance test (TRT) and incubation with uterine fluid (UF) (day 5). Sperm metabolic rates between accumulative ejaculate fractions from pure and diluted semen did not show differences. Also, sperm quality parameters were not affected by the ejaculate fraction during storage. However, sperm subjected to TRT showed similar results except for progressive motility, which was better in F2 and F3 than F1. When sperm were incubated with UF, the quality decreased in each group, but sperm from F2 and F3 were less affected than those from F1. In conclusion, the post-spermatic fraction can be included in seminal doses for their use in AI-centers, with functionality of sperm of different SP origins not being impaired throughout the storage, and responding better to thermal and UF stress. However, further research in AI-centers is necessary to test the sperm behaviour under presented conditions.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.