Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials.

Nowadays there is an increasing demand for assisted reproductive technologies due to the growth of infertility problems. Naturally, fertilization occurs in the oviduct, where the oviductal epithelial cells (OECs) secrete many molecules that affect the embryo's metabolism and protect it from oxidative stress. When the OECs are grown in 3D culture systems, they maintain a great part of their functional characteristics, making them an excellent model for in vitro fertilization (IVF) studies. In this work, we aimed to evaluate the suitability of different 3D-printing processes in conjunction with the corresponding set of commercially available biomaterials: extrusion-based processing using polylactic acid (PLA) and polycaprolactone (PCL) and stereolithography or digital-light processing using polyethylene-glycol-diacrylate (PEGDA) with different stiffness (PEGDA500, PEGDA200, PEGDA PhotoInk). All the 3D-printed scaffolds were used to support IVF process in a bovine embryo assay. Following fertilization, embryo development and quality were assessed in terms of cleavage, blastocyst rate at days 7 and 8, total cell number (TCN), inner cell mass/trophectoderm ratio (ICN/TE), and apoptotic cell ratio (ACR). We found a detrimental effect on cleavage and blastocyst rates when the IVF was performed on any medium conditioned by most of the materials available for digital-light processing (PEGDA200, PEGDA500). The observed negative effect could be possibly due to some leaked compound used to print and stabilize the scaffolds, which was not so evident however with PEGDA PhotoInk. On the other hand, all the extrusion-based processable materials did not cause any detrimental effect on cleavage or blastocyst rates. The principal component analysis reveals that embryos produced in presence of 3D-printed scaffolds produced via extrusion exhibit the highest similarity with the control embryos considering cleavage, blastocyst rates, TCN, ICN/TE and ACR per embryo. Conversely, all the photo-cross linkable materials or medium conditioned by PLA, lead to the highest dissimilarities. Since the use of PCL scaffolds, as well as its conditioned medium, bring to embryos that are more similar to the control group. Our results suggest that extrusion-based 3D printing of PCL could be the best option to be used for new IVF devices, possibly including the support of OECs, to enhance bovine embryo development.

Differences in Weight, Hierarchy, and Incidence of Lameness between Two Groups of Adult Pigs Derived from Assisted Reproductive Technologies.

The in vitro production (IVP) and subsequent transfer of embryos (ET) to recipient mothers is not yet an established reproductive technology in the pig industry, as it is in cattle. However, that the trade of IVP-cryopreserved pig embryos is expected to start in the next decades. Society and governments are increasingly aware of the repercussions that IVP could have for animal health, welfare, behavior, or food safety, but proven scientific information for this type of animal does not exist, since no colonies of pigs have been created to this end. We created a small one and studied the differences between 16 IVP-derived pigs and 14 pigs derived from artificial insemination (AI), at 3.5 years of age, conceived from the same boar, and housed and fed under the same conditions since they were born. Incidence of lameness, position in the herd hierarchy, weight, adenosine deaminase activity, and hematological and biochemical analytes were compared between the two groups of animals. The results showed that the IVP animals weighed more, occupied higher positions in the herd hierarchy, and had a lower incidence of lameness. Although genetic differences from the maternal line could explain some of these results, it is also possible that the IVP animals developed better adaptative abilities, but more studies with a higher number of animals are necessary to reach consistent conclusions.

The embryo culture media in the era of epigenetics: is it time to go back to nature?

This review is intended to draw attention to the importance of the culture media composition on the health of the embryos, fetuses, newborns, and adults derived from assisted reproductive technologies (ART). Although current research and industry trends are to use chemically defined media because of their suitability for manufacturing, commercialization, and regulatory purposes, compelling evidence indicates that those media fail to adequately account for the biological demands of early embryogenesis. Here, we list the main undesirable consequences of the ART described in the literature and results we and others have obtained over the past decade exploring an alternative and more natural way to support embryo growth in vitro: inclusion of endogenous reproductive fluids as additives in the ART culture media for pigs, cows, and humans. This review systematically assesses the pros and cons of using reproductive fluid additives, as well as the requirements to implement this approach in the future.

Sperm selection by rheotaxis improves sperm quality and early embryo development.

The objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates.

Involvement of nitric oxide during in vitro oocyte maturation, sperm capacitation and in vitro fertilization in pig.

The importance of porcine species for meat production is undeniable. Due to the genetic, anatomical, and physiological similarities with humans, from a biomedical point of view, pig is considered an ideal animal model for the study and development of new therapies for human diseases. The in vitro production (IVP) of porcine embryos has become widespread as a result of these qualities and there is significant demand for these embryos for research purposes. However, the efficiency of porcine embryo IVP remains very low, which hinders its use as a model for research. The high degree of polyspermic fertilization is the main problem that affects in vitro fertilization (IVF) in porcine species. Furthermore, oocyte in vitro maturation (IVM) is another important step that could be related to polyspermic fertilization and low embryo production. The presence of nitric oxide synthase (NOS), the enzyme that produces nitric oxide (NO), has been detected in the oviduct, the ovary, the oocyte and the sperm cell of porcine species. Its functions include regulating oviductal activity, ovulation, acquisition of meiotic competence, oocyte activation, sperm capacitation, and gamete interaction. Therefore, in this review, we summarize the current knowledge on the role of NO/NOS system in each of the steps that lead to the production of porcine embryos in an in vitro environment, i.e. IVM, sperm capacitation, IVF, and embryo culture. We also discuss the possible ways in which the NO/NOS system could be used to enhance IVP of porcine embryos.

Epididymal and ejaculated sperm functionality is regulated differently by periovulatory oviductal fluid in pigs.

BACKGROUND: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. OBJECTIVES: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. MATERIAL AND METHODS: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyrosine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status. RESULTS: The pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte. CONCLUSION: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa.

Delta and kappa opioid receptors on mouse sperm cells: Expression, localization and involvement on in vitro fertilization.

The endogenous opioid peptides have been reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with sentences around the harmful effect of opioids in male fertility but, actually, there is only one study regarding the real fertility potential of spermatozoa that have been exposed to mu specific opioids. The aim of the present study was to see if the modulation of delta (OPRD1) and kappa (OPRK1) opioid receptors in mouse sperm during capacitation was able to vary the embryo production after in vitro fertilization (IVF). The presence of OPRD1 and OPRK1 in mouse mature spermatozoa was analyzed by RT-PCR and immunofluorescence. Incubating the sperm with, on one hand, the delta specific agonist DPDPE and/or antagonist naltrindole, and, on the other hand, the kappa specific agonist U-50488 and antagonist nor-binaltorphimine, we analyzed the involvement of OPRD1 and OPRK1 on IVF and preimplantational embryo development. We verified the presence of OPRD1 and OPRK1 in mouse mature spermatozoa, not only at the mRNA level but also at protein level. Moreover, the sperm incubation with DPDPE, before the IVF, had an effect on the fertilization rate of sperm and reduced the number of reached blastocysts, which was reverted by naltrindole. Instead, the use of the kappa agonist U-50488 and the antagonist nor-binaltophimine did not have any effect on the amount and the quality of the achieved blastocysts. Although nowadays the pure delta or kappa opioid ligands are not used for the clinic, clinical trials are being conducted to be used in the near future, so it would be interesting to know if the modulation of these receptors in sperm would generate any consequence in relation to fertilization capacity.

The endocannabinoid system modulates the ovarian physiology and its activation can improve in vitro oocyte maturation.

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.

Mu opioid receptor expression and localisation in murine spermatozoa and its role in IVF.

The endogenous opioid peptides are reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with statements on the harmful effect of opioids on male fertility but, in fact, there are no studies regarding the real fertilisation potential of spermatozoa that have been exposed to opioids. The aim of the present study was to examine if modulation of mu opioid receptor (OPRM1) in murine spermatozoa during capacitation influenced embryo production after IVF. The presence of OPRM1 in murine mature spermatozoa was analysed by reverse transcription-polymerase chain reaction and immunofluorescence. We analysed the involvement of OPRM1 on IVF and pre-implantational embryo development by incubating the spermatozoa with the opioid agonist morphine and/or antagonist naloxone. We verified the presence of OPRM1 in murine mature spermatozoa, not only at the mRNA level but also the protein level. Moreover, incubation of the spermatozoa with morphine, before IVF, had an effect on the fertilisation rate of the spermatozoa and reduced the numbers of blastocysts, which was reversed by naloxone. Considering that opioids are widely used clinically, it is important to take into account their effect, via OPRM1, on the fertility of patients.

Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system.

PURPOSE: Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. METHODS: Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. RESULTS: Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. CONCLUSIONS: These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.

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.

Hyperactivated stallion spermatozoa fail to exhibit a rheotaxis-like behaviour, unlike other species.

The journey of spermatozoa through the female genital tract is facilitated by rheotaxis, or the cell's preference to swim against a flow, as well as thigmotaxis, the wall tracking behaviour, which guides them to the site of fertilisation. The aim of this study was to characterise the rheotactic and thigmotactic response of stallion sperm within a microfluidic channel. Stallion sperm rheotaxis was assessed within the microfluidic channel with regard to: (i) A range of flow velocities, (ii) Varying media viscosity and (iii) Sperm hyperactivation. Sperm distribution across the microfluidic channel was also studied and compared to human and ram sperm. Stallion sperm progressed furthest at a velocity range of 10-30 µm/s, with an optimum velocity of 20 µm/s. A flow viscosity of 2.5cP or greater reduced sperm rheotaxis (P < 0.05). Stallion sperm that were hyperactivated were unable to exhibit rheotaxis within the microfluidic channel, whereas, both hyperactivated human and ram sperm did exhibit positive rheotaxis under the same conditions. The number of sperm swimming near the microfluidic channel walls was higher than in the microfluidic channel centre (P < 0.05). This is the first study to illustrate that stallion sperm are rheotactically responsive and increasing viscosity reduces this response. We also demonstrated that sperm are predominantly inclined to swim along a surface and uniquely, hyperactivated stallion sperm are non-progressive and do not exhibit a rheotactic response unlike other species.

α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa.

The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (IVF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa-ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were co-incubated for 15 min. A significant increase in spermatozoa-ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa.

Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency.

OBJECTIVE: To determine optimal conditions for the inclusion of oviductal fluid (OF) in IVF protocols. DESIGN: Experimental prospective study. SETTING: Mammalian reproduction research laboratory. ANIMAL(S): Oviducts and ovaries from porcine females were collected at a slaughterhouse. A total of 30 oviducts and 1,285 oocytes were used. Boar-ejaculated spermatozoa were also used. INTERVENTION(S): In vitro-matured porcine oocytes were preincubated with OF collected from animals before or after ovulation and later fertilized in vitro. Zona pellucida digestion time in oocytes after preincubation in OF was assessed. Concentrations of E2 and P4 in OF were measured. IVF was performed, including within the culture media the E2 and P4 concentrations found in the preovulatory OF. The effect of preovulatory OF on IVF efficiency was compared between fresh and frozen-thawed spermatozoa. MAIN OUTCOME MEASURE(S): E2 and P4 concentrations in OF; penetration and monospermy rates; number of spermatozoa within the ooplasm and on the zona pellucida after IVF under different experimental conditions; zona pellucida resistance to protease digestion. RESULT(S): Preincubation of oocytes in OF collected before ovulation enhances IVF efficiency in the pig compared with OF collected after ovulation (29.58 ± 3.84 vs. 11.03 ± 2.69). When frozen-thawed spermatozoa are used for the IVF of these OF-treated oocytes, their fertilization ability increases compared with fresh semen. OF collected before and after ovulation shows significantly different concentrations of E2 (99.00 ± 8.72 vs. <10 pg/mL) and P4 (2.53 ± 0.66 vs. 12.27 ± 2.33 ng/mL), respectively. Addition of E2 and P4 at concentrations similar to those in the OF before ovulation partially simulates the effect of the fluid on IVF outcome. CONCLUSION(S): Preincubation of oocytes in OF collected before ovulation is a suitable protocol for increasing the efficiency of IVF with fresh semen in the pig model and could be a useful tool to increase the fertilization ability of frozen-thawed spermatozoa in other species. E2 concentrations in preovulatory OF are higher than those reported in blood serum at the same phase of the estrous cycle.