Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro.

BACKGROUND: Currently, high polyspermy remains a significant obstacle to achieving optimal efficiency in in vitro fertilization (IVF) and in vitro embryo production (IVP) systems in pigs. Developing strategies that would prevent polyspermy is essential in overcoming this challenge and maximizing the potential of this reproductive biotechnology. Previous results have demonstrated that using boar spermatozoa subjected to a high-extension and reconcentration procedure and then cryopreserved resulted in significant improvements in IVF/IVP systems with high rates of monospermy and penetration. OBJECTIVE: The aim of the present study was to unveil the molecular mechanisms that may underlie the changes in fertilization patterns exhibited by highly extended and cryopreserved boar spermatozoa. MATERIALS AND METHODS: To achieve this goal, we used quantitative proteomic analysis (LC‒ESI‒MS/MS SWATH) to identify differentially abundant proteins (DAPs) between highly extended (HE) and conventionally (control; CT) cryopreserved boar spermatozoa. Prior to the analysis, we evaluated the in vitro post-thawing fertilizing ability of the sperm samples. The results demonstrated a remarkable improvement in monospermy and IVF efficiency when using HE spermatozoa in IVF compared with CT spermatozoa. RESULTS: At the proteomic level, the combination of high-extension and cryopreservation had a significant impact on the frozen-thawed sperm proteome. A total of 45 proteins (24 downregulated and 21 upregulated) were identified as DAPs (FC > 1 or ≤1; p < 0.05) when compared with CT spermatozoa. Some of these proteins were primarily linked to metabolic processes and the structural composition of sperm cells. The dysregulation of these proteins may have a direct or indirect effect on essential sperm functions and significantly affect spermatozoa-oocyte interaction and, therefore, the sperm fertilization profile under in vitro conditions. While these findings are promising, further research is necessary to comprehend how the disturbance of specific proteins affects sperm fertilization ability.

Oviduct fluid during IVF moderately modulates polyspermy in in vitro-produced goat embryos during the non-breeding season.

The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 106 cells/mL) for 18 h in SOF medium supplemented with 5 µg/mL of heparin, 4 µg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x106 sperm per mL is used.

Manipulation of bicarbonate concentration in sperm capacitation media improvesin vitro fertilisation output in porcine species.

BACKGROUND: The in vivo concentration of bicarbonate (HCO3 -), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3 - concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. RESULTS: Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3 -, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 - stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 - concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). CONCLUSIONS: Optimising HCO3 - concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 - in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.

High pre-freezing sperm dilution improves monospermy without affecting the penetration rate in porcine IVF.

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation.

Effect of porcine uterus as ex vivo model of fertilizing ability and gene expression pattern on blastocysts.

The success of in vitro embryo production demonstrates that the oviduct can be bypassed during early embryonic development. Using an ex vivo model of porcine uterus is one of the strategies used to investigate fertilization within the oviductal environment. In this study, in vitro-matured porcine oocytes (MII) were fertilized with 7.5 × 107, 15 × 107, or 30 × 107 sperm cells for 20 min in the oviduct of a porcine uterine ex vivo model. MII oocytes used for in vitro fertilization (IVF) served as control 1; those cultured in the oviduct of the ex vivo model for 20 min before IVF served as control 2. In present study, the penetration rate, polyspermy, and fertilization efficiency, and accumulated reactive oxygen species (ROS) levels in the treatment groups were significantly decreased compared to those in the control 1 group. During embryonic development, the cleavage rates in the treatment groups were significantly lower than those in the control groups. The cleavage rate in the 30 × 107 sperm cell-treated group was higher than that in the 7.5 × 107 sperm cell-treated group. The blastocyst formation rate in control 1 and 2, and 30 × 107 sperm cell-treated groups increased compared to that in the 7.5 and 15 × 107 sperm cell-treated groups. PCNA, HSP70.2, and GLUT1 were upregulated in the treatment groups and POU5F1, BAX, GPX1 were upregulated in the treatment and control 2 groups, compared to the control 1 group. These results suggest that an ex vivo model may decrease the penetration rate and fertilization efficiency by increasing the accumulated ROS levels and inducing the expression of apoptosis- and stress-related genes. However, the model improved the monospermy rate and expression of embryo developmental competence genes. This is the first study that evaluates the effect of an ex vivo model of porcine uterus on fertilization parameters, and the development of porcine embryos.

Pig in vitro fertilization: Where are we and where do we go?

The pig is an important livestock animal. Biotechnological interest in this species has increased due to its use, among others, in the generation of transgenic animals for use in biomedicine based on its greater physiological proximity to the human species than other large domestic animals. This development has paralleled an improvement in Assisted Reproduction Techniques (ART) used for this species. However, the ability to generate animals from embryos produced entirely in vitro is still limited and a wide margin for improvement remains. Here we review the procedures, additives, and devices used during pig in vitro fertilization (IVF), focusing on the main points of each step that have offered the best results in terms of increased efficiency of the system. The lack of standardized protocols and consensus on the parameters to be assessed makes it difficult to compare results across different studies, but some conclusions are drawn from the literature. We anticipate that new physiological protocols will advance the field of swine IVF, including induction of prefertilization ZP hardening with oviductal fluid, sperm preparation by swim-up method, increased viscosity through the addition of inert molecules or reproductive biofluids, and the incorporation of 3D devices. Here we also reflect on the need to expand the variables on which the efficiency of pig IVF is based, providing new parameters that should be considered to supply more objective and quantitative assessment of IVF additives and protocols.

Alpha-Linolenic Acid: It Contribute Regulation of Fertilization Capacity and Subsequent Development by Promoting of Cumulus Expansion during Maturation.

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and 100 µM ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in 50 µM ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by 50 µM ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and 50 µM ALA groups (p<0.05), especially, treatment of 50 µM ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and 50 µM ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 µM), higher sildenafil concentrations (25 and 250 µM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 µM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes / Pré-incubação dos espermatozoides suínos diminui polispermia e aumenta a produção embrionária em oócitos de baixa qualidade

ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.

RESUMO: A principal causa da baixa eficiência na PIV de embriões suínos é a elevada taxa de polispermia, que é exacerbada em oócitos de baixa qualidade. O experimento 1 avaliou o desenvolvimento embrionário de oócitos de baixa e alta qualidade. O experimento 2 avaliou a qualidade e o desenvolvimento embrionário de oócitos de baixa qualidade fecundados com sêmen pré-incubado por 0h (controle), 0,5h, 1h e 1,5h. O experimento 3 investigou a fecundação e as taxas de monospermia dos mesmos grupos do experimento 2. O experimento 4 avaliou o desenvolvimento embrionário, a densidade celular, a fecundação e as taxas de monospermia de oócitos de alta qualidade, fecundados com sêmen pre-incubado com o melhor tempo observado nos experimentos anteriores. As taxas de clivagem e de blastocistos foram submetidas ao teste de Qui-quadrado e os demais dados submetidos à ANOVA e teste de Tukey (P≤0,05). As taxas de clivagem (74,8 vs 51,7%) e de blastocistos (33,7 vs 9,8%) foram superiores nos oócitos de alta qualidade, comparados aos de baixa qualidade, não havendo diferenças na quantidade de células embrionárias. As taxas de fecundação (65,6 vs 79,5%) não foram influenciadas pelo tempo de pré-incubação. Todavia, a pré-incubação do sêmen por 1,5h aumentou a penetração monospérmica (53,3%) e a taxa de clivagem (92,5%), nos oócitos de baixa qualidade. As taxas de blastocisto aumentaram com sêmen pré-incubado por 1,5h, que foram ainda inferiores às obtidas dos oócitos de alta qualidade do grupo controle. Finalmente, a pré-incubação do sêmen não influencia na fecundação, na penetração monospérmica, no desenvolvimento embrionário, nem na quantidade de células embrionárias com oócitos de alta qualidade. Oócitos suínos de baixa qualidade produzem melhores taxas de desenvolvimento embrionário se fecundados in vitro com sêmen pré-incubado por 1,5 horas.