Expression of sperm microRNAs related to bull fertility: A systematic review.

In this study we proposed to address the following question: "Are there differentially expressed sperm microRNAs related to fertility in bulls?". A systematic review of scientific literature until November 2022 was performed, in accordance with PRISMA guidelines. The main outcome was differentially expressed sperm microRNA from bulls with low versus high fertility profiles identified by using different methods such as field fertility evaluation and sperm laboratory analysis. Were identified 786 documents, of which 13 were selected for qualitative analysis. A total of 182 unique differentially expressed miRNAs were identified, among these, 49 miRNAs were found in common between at least two studies. It is believed that from these 49 miRNAs, it is possible that miRNAs such as miR-10a, -10b, -103, -15b, -122, -125b, -126-5p, -151-5p, -193a-5p, -196a, -27a-5p and -99b could be potential universal biomarkers to assess the reproductive potential of males.

Birthweight leads to seminal and testicular morphofunctional commitment in sexually mature boars.

Given the importance of males as semen donors for artificial insemination (AI) and the high incidence of low birthweight piglets at commercial farms, the impact of birthweight on fertility in boars deserves special attention. The aim of this study was to evaluate testicular morphofunctional parameters and semen characteristics in different birthweight boars. Forty littermate males were selected at birth and divided into two experimental groups, according to birthweight: high (HW, birthweight ranging from 1.80 to 2.15 kg, n = 20) and low (LW, birthweight ranging from 0.75 to 1.10 kg, n = 20). At 170 days of age, a sub-set of 24 littermate boars (n = 12 HW and n = 12 LW) were randomly selected for semen collection, which was performed once a week, at a 15-day interval, during five weeks. At 300 days of age, boars were orchiectomized, and the testis processed for histological and molecular analyses. The HW group was heavier and presented larger testes compared to LW animals (P < 0.05). Despite that, birthweight did not significantly affect semen volume or sperm quality parameters (concentration, motility, vigor or morphology), although LW boars produced 38.2% fewer total sperm and 24% lower semen concentration, leading to 36.8% less inseminating doses. The histomorphometrical evaluation showed that seminiferous tubules diameter and germinal epithelium height were similar between experimental groups. However, LW boars presented shorter seminiferous tubules and, consequently, fewer Sertoli cells per testis (P 0.05). Even though plasma testosterone levels were equivalent in both birthweight groups, LW testis presented less androgen receptors (P < 0.05). Additionally, birthweight was positively correlated with total seminiferous tubule length and number of Sertoli cells (P < 0.01), and with body and testis weights (P < 0.01). Taken together, even though adult LW boars showed no evident semen pathologies or spermatogenesis commitment, mature HW males have the potential to produce more spermatozoa, consequently more semen doses per ejaculate, being more valuable to an industry that relies on AI.

Docosahexaenoic acid and phenazine ethosulfate are not efficient lipid modulators for porcine in vitro maturation systems.

The high lipid content in porcine oocytes impairs in vitro embryo production (IVP). Here, we evaluated the influence of two different lipid modulators during in vitro maturation (IVM) on the embryo development and the lipid content of oocytes and embryos. In Experiment I, oocytes were exposed to 50 µM docosahexaenoic acid (DHA) with (+) or without (-) the presence of porcine follicular fluid (pFF). In Experiment II, phenazine ethosulfate (PES) was added during IVM at two concentrations (0.5 and 0.05 µM). The pFF- with 50 µM DHA treatment impaired nuclear maturation, cleavage and blastocyst rates (p < 0.05). Oocytes in pFF- media accumulated less lipids (p < 0.05). The addition of 0.5 µ M PES reduced all development rates (p < 0.05) and resulted in higher lipid content for oocytes and embryos. Only 0.05 µM PES oocytes matured similarly to the control (p > 0.05), although embryo development and embryo lipid content was similar to 0.5 µM PES oocytes (p > 0.05). Thus, 50 µM DHA supplementation in the IVM medium without pFF impaired oocyte maturation and embryo development rates without interfering in oocyte lipid content even in the presence of pFF. Maturation with PES neither favored porcine embryo development nor reduced their lipid content.

Capillary electroporation affects the expression of miRNA-122-5p from bull sperm cells.

Sperm-mediated gene transfer (SMGT) has a potential application in the generation of transgenic animals. Capillary electroporation consists of the application of electrical pulses, resulting in an increased transfection rate. Little is known about the impacts of the transfection of exogenous DNA on sperm epigenetics. MicroRNAs are epigenetic factors that are related to sperm motility. MiRNA-122-5p regulates genes that influence motility, and consequently, the fertilizing potential of sperm. Therefore, we aimed at identifying whether epigenetic factors such as microRNAs could be altered after DNA transfection, using the capillary electroporation technique. In this study, bull sperm was electroporated using voltages of 600 V, 1500 V, and 0 V (control group), with or without exogenous DNA. Parameters of sperm quality were analyzed using CASA and flow cytometry, and expression of the miRNA-122-5p was analyzed using RT-qPCR. It was observed that electroporation increased the internalization of exogenous DNA (P < 0.05), but did not impair the mitochondrial activity (P > 0.05). It reduced sperm motility (P < 0.05). The expression of miRNA-122-5p was upregulated in sperm electroporated at 1500 V, and the presence of exogenous DNA did not affect its expression. Thus, we can conclude that electroporation influences the expression of miRNA-122-5p from bull sperm cells.

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Melatonin delivery by nanocapsules during in vitro bovine oocyte maturation decreased the reactive oxygen species of oocytes and embryos.

In this work, a promising approach to increase the advantageous properties of melatonin through its encapsulation into lipid-core nanocapsules (LNC) was examined. Oocytes were treated during in vitro maturation with non-encapsulated melatonin (Mel), melatonin-loaded lipid-core nanocapsules (Mel-LNC), and unloaded LNC. Cytotoxicity, meiotic maturation rate, development to the blastocyst stage, reactive oxygen species (ROS) and glutathione levels, mean cell number and apoptotic cell/blastocyst, and mRNA quantification were evaluated. Both Mel and Mel-LNC enhanced in vitro embryo production, however, Mel-LNC proved to be more effective at decreasing ROS levels and the apoptotic cell number/blastocyst, increasing the cleavage and blastocyst rates, up-regulating the GPX1 and SOD2 genes, and down-regulating the CASP3 and BAX genes. Mel-LNC could penetrate into oocytes and remain inside the cells until they reach the blastocyst stage. In conclusion, when melatonin was encapsulated in LNC and applied during in vitro oocyte maturation, some quality aspects of the blastocysts were improved.

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation.

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.