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.

Participation of apoptotic markers in the process of maturation and elimination of spermatozoa in the epididymis of the Corynorhinus mexicanus bat.

The Corynorhinus mexicanus bat is characterized by a specific form of reproductive asynchrony between males and females. After mating, some sperm remain in the male's epididymis, the organ where the sperm had matured. It has not yet been determined if apoptotic markers participate in the process of the maturation and/or elimination of these cells, so studying this topic is essential for our understanding of this species. Male bats were collected during three stages: Before mating; during the Mating phase; After mating and the final phase, which we call, Storage. Their epididymides were removed, weighed and measured. Sperm were extracted and the following sperm parameters were evaluated: active caspases, phosphatidylserine externalization, and mitochondrial membrane potential. Sperm from the testes enter the epididymis during Before mating, causing the organ to grow. During Mating phase, spermatozoa present a large amount of active caspases with externalization of phosphatidyl serine, even while still alive. This suggests that these two markers could participate in maturation and elimination, respectively.

One-Step In Vitro Generation of ETV2-Null Pig Embryos.

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations.

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.