Generation of Porcine and Rainbow Trout 3D Intestinal Models and Their Use to Investigate Astaxanthin Effects In Vitro.

Astaxanthin (AST) is a natural compound derived from shellfish, microorganisms, and algae, with several healthy properties. For this reason, it is widely used in the diet of humans and animals, such as pigs, broilers, and fish, where its addition is related to its pigmenting properties. Moreover, AST's ability to reduce free radicals and protect cells from oxidative damage finds application during the weaning period, when piglets are exposed to several stressors. To better elucidate the mechanisms involved, here we generate ad hoc pig and rainbow trout in vitro platforms able to mimic the intestinal mucosa. The morphology is validated through histological and molecular analysis, while functional properties of the newly generated intestinal barriers, both in porcine and rainbow trout models, are demonstrated by measuring trans-epithelial electrical resistance and analyzing permeability with fluorescein isothiocyanate-dextran. Exposure to AST induced a significant upregulation of antioxidative stress markers and a reduction in the transcription of inflammation-related interleukins. Altogether, the present findings demonstrate AST's ability to interact with the molecular pathways controlling oxidative stress and inflammation both in the porcine and rainbow trout species and suggest AST's positive role in prevention and health.

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts.

PURPOSE: This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. METHODS: A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. RESULTS: Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. CONCLUSION: We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

3D ECM-Based Scaffolds Boost Young Cell Secretome-Derived EV Rejuvenating Effects in Senescent Cells.

Aging is a complex, multifaceted degenerative process characterized by a progressive accumulation of macroscopic and microscopic modifications that cause a gradual decline of physiological functions. During the last few years, strategies to ease and counteract senescence or even rejuvenate cells and tissues were proposed. Here we investigate whether young cell secretome-derived extracellular vesicles (EVs) ameliorate the cellular and physiological hallmarks of aging in senescent cells. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli, directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after exposure to young cell secretome. The results obtained demonstrate that young cells release EVs endowed with the ability to counteract aging. In addition, comparison between young and aged cell secretomes shows a significantly higher miR-200 content in EVs produced using fibroblasts isolated from young donors. The effect exerted by young cell secretome-derived EVs is transient, but can be stabilized using a young ECM microenvironment. This finding indicates a synergistic interaction occurring among molecular effectors and ECM-derived stimuli that cooperate to control a unique program, driving the cell clock. The model described in this paper may represent a useful tool to finely dissect the complex regulations and multiple biochemical and biomechanical cues driving cellular biological age.

The Role of Genistein in Mammalian Reproduction.

Genistein is a natural compound belonging to flavonoids, having antioxidant, anti-inflammatory, and anti-neoplastic properties. Genistein is considered a phytoestrogen. As such, genistein can bind estrogen receptors (ERα and ERß), although with a lower affinity than that of estradiol. Despite considerable work, the effects of genistein are not well established yet. This review aims to clarify the role of genistein on female and male reproductive functions in mammals. In females, at a high dose, genistein diminishes the ovarian activity regulating several pathway molecules, such as topoisomerase isoform I and II, protein tyrosine kinases (v-src, Mek-4, ABL, PKC, Syk, EGFR, FGFR), ABC, CFTR, Glut1, Glut4, 5α-reductase, PPAR-γ, mitogen-activated protein kinase A, protein histidine kinase, and recently circulating RNA-miRNA. The effect of genistein on pregnancy is still controversial. In males, genistein exerts an estrogenic effect by inducing testosterone biosynthesis. The interaction of genistein with both natural and synthetic endocrine disruptors has a negative effect on testis function. The positive effect of genistein on sperm quality is still in debate. In conclusion, genistein has a potentially beneficial effect on the mechanisms regulating the reproduction of females and males. However, this is dependent on the dose, the species, the route, and the time of administration.

Seven days ex vivo perfusion of whole ewe ovaries with follicular maturation and oocyte retrieval: towards the development of an alternative fertility preservation method.

Fertility preservation methods for prepubertal women about to undergo gonadotoxic chemo and/or radiation therapy are limited. Therefore, the aim of this study was to investigate the feasibility to develop an alternative fertility preservation method based on an ex vivo perfusion platform for whole ewe ovaries. Thirteen ewe ovaries were divided into two groups (group 1 and 2) that were perfused in a bioreactor for up to 7days. Group 1 (n =3) were stimulated with human menopausal gonadotropin (hMG) administered in single daily dose, while group 2 (n =10) were stimulated continuously for 24h. The perfused ovaries in group 1 showed no significant differences in follicular density, sub-follicular morphology and oocyte quality after ischaemia and after ex vivo perfusion compared with non-perfused control ovaries. The perfused ovaries in group 2 showed a significant decrease in the follicular reserve and oocyte quality compared with the control group. In total, 16 GV-MI oocytes were retrieved from both groups. This study describes for the first time the ex vivo maintenance of viable follicles of ewe ovaries with oocyte integrity and the retrieval of oocytes after ex vivo hormonal perfusion with two different protocols for up to 7days.

Current Advances in 3D Tissue and Organ Reconstruction.

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.

Rho Signaling-Directed YAP/TAZ Regulation Encourages 3D Spheroid Colony Formation and Boosts Plasticity of Parthenogenetic Stem Cells.

Cell proliferation, apoptosis and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs) that are able to proliferate maintaining pluripotency and, at the same time, to give rise to all populations belonging to the three germ layers, in response to specific stimuli. ESCs are, therefore, considered a well-established in vitro model to study the complexity of these processes. In this perspective, we previously generated parthenogenetic embryonic stem cells (ParthESC), that showed many features and regulatory pathways common to bi-parental ESCs. However, we observed that mono-parental cells demonstrate a high ability to form outgrowths and generate 3D spheroid colonies, which are distinctive signs of high-plasticity. Furthermore, preliminary evidence obtained by WTA, revealed the presence of several differentially expressed genes belonging to the Rho and Hippo signaling pathways. In the present study, we compare bi-parental ESCs and ParthESC and analyze by Real-Time PCR the differentially expressed genes. We demonstrate up-regulation of the Rho signaling pathway and an increased expression of YAP and TAZ in ParthESC. We also show that YAP remains in a dephosphorylated form. This allows its nuclear translocation and its direct binding to TEADs and SMADs, that are up-regulated in ParthESC. Altogether, these complex regulatory interactions result in overexpression of pluripotency related genes, in a global DNA hypomethylation and a histone-dependent chromatin high permissive state that may account for ParthESC high potency, possibly related to their exclusive maternal origin.

Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering.

PURPOSE: To develop a new protocol for whole-ovary decellularization for the production of a 3D bioscaffold suitable for in vitro/ex vivo studies and for the reconstruction of a bioengineered ovary. METHODS: Porcine ovaries were subjected to the decellularization process (DECELL; n = 20) that involved a freeze-thaw cycle, followed by sequential incubations in 0.5% SDS for 3 h, 1% Triton X-100 for 9 h, and 2% deoxycholate for 12 h. Untreated ovaries were used as a control (CTR; n = 6). Both groups were analyzed to evaluate cell and DNA removal as well as ECM preservation. DECELL bioscaffolds were assessed for cytotoxicity and cell homing ability. RESULTS: DECELL ovaries maintained shape and homogeneity without any deformation, while their color turned from red to white. Histological staining and DNA quantification confirmed a decrease of 98.11% in DNA content, compared with the native tissue (CTR). Histochemical assessments demonstrated the preservation of intact ECM microarchitecture after the decellularization process. This was also confirmed by quantitative analysis of collagen, elastin, and GAG contents. DECELL bioscaffold showed no cytotoxic effects in co-culture and, when re-seeded with homologous fibroblasts, encouraged a rapid cell adhesion and migration, with repopulating cells increasing in number and aggregating in cluster-like structures, consistent with its ability to sustain cell adherence, proliferation, and differentiation. CONCLUSION: The protocol described allows for the generation of a 3D bioscaffold that may constitute a suitable model for ex vivo culture of ovarian cells and follicles, as well as a promising tool for the reconstruction of a bioengineered ovary.

The 3D Pattern of the Rainbow Trout (Oncorhynchus mykiss) Enterocytes and Intestinal Stem Cells.

We previously showed that, according to the frequency and distribution of specific cell types, the rainbow trout (RT) intestinal mucosa can be divided in two regions that form a complex nonlinear three-dimensional (3D) pattern and have a different renewal rate. This work had two aims. First, we investigated whether the unusual distribution of cell populations reflects a similar distribution of functional activities. To this end, we determined the protein expression pattern of three well-defined enterocytes functional markers: peptide transporter 1 (PepT1), sodium-glucose/galactose transporter 1 (SGLT-1), and fatty-acid-binding protein 2 (Fabp2). Second, we characterized the structure of RT intestinal stem-cell (ISC) niche and determined whether the different proliferative is accompanied by a different organization and/or extension of the stem-cell population. We studied the expression and localization of well-characterized mammal ISC markers: LGR5, HOPX, SOX9, NOTCH1, DLL1, and WNT3A. Our results indicate that morphological similarity is associated with similar function only between the first portion of the mid-intestine and the apical part of the complex folds in the second portion. Mammal ISC markers are all expressed in RT, but their localization is completely different, suggesting also substantial functional differences. Lastly, higher renewal rates are supported by a more abundant ISC population.

The Role of Resveratrol in Mammalian Reproduction.

Resveratrol is one of the most investigated natural polyphenolic compounds and is contained in more than 70 types of plants and in red wine. The widespread interest in this polyphenol derives from its antioxidant, anti-inflammatory and anti-aging properties. Several studies have established that resveratrol regulates animal reproduction. However, the mechanisms of action and the potential therapeutic effects are still unclear. This review aims to clarify the role of resveratrol in male and female reproductive functions, with a focus on animals of veterinary interest. In females, resveratrol has been considered as a phytoestrogen due to its capacity to modulate ovarian function and steroidogenesis via sirtuins, SIRT1 in particular. Resveratrol has also been used to enhance aged oocyte quality and as a gametes cryo-protectant with mainly antioxidant and anti-apoptotic effects. In males, resveratrol enhances testes function and spermatogenesis through activation of the AMPK pathway. Furthermore, resveratrol has been supplemented to semen extenders, improving the preservation of sperm quality. In conclusion, resveratrol has potentially beneficial effects for ameliorating ovarian and testes function.

Safety and Efficacy of Epigenetically Converted Human Fibroblasts Into Insulin-Secreting Cells: A Preclinical Study.

Type 1 Diabetes Mellitus (T1DM) is a chronic disease that leads to loss of insulin secreting ß-cells, causing high levels of blood glucose. Exogenous insulin administration is not sufficient to mimic the normal function of ß-cells and, consequently, diabetes mellitus often progresses and can lead to major chronic complications and morbidity. The physiological control of glucose levels can only be restored by replacing the ß-cell mass.We recently developed a new strategy that allows for epigenetic conversion of dermal fibroblasts into insulin-secreting cells (EpiCC), using a brief exposure to the demethylating agent 5-aza-cytidine (5-aza-CR), followed by a pancreatic induction protocol. This method has notable advantages compared to the alternative available procedures and may represent a promising tool for clinical translation as a therapy for T1DM. However, a thought evaluation of its therapeutic safety and efficacy is mandatory to support preclinical studies based on EpiCC treatment.We here report the data obtained using human fibroblasts isolated from diabetic and healthy individuals, belonging the two genders. EpiCC were injected into 650 diabetic severe combined immunodeficiency (SCID) mice and demonstrated to be able to restore and maintain glycemic levels within the physiological range. Cells had the ability to self-regulate and not to cause hypoglycemia, when transplanted in healthy animals. Efficacy tests showed that EpiCC successfully re-established normoglycemia in diabetic mice, using a dose range that appeared clinically relevant to the concentration 0.6 × 106 EpiCC. Necropsy and histopathological investigations demonstrated the absence of malignant transformation and cell migration to organs and lymph nodes.The present preclinical study demonstrates safety and efficacy of human EpiCC in diabetic mice and supports the use of epigenetic converted cells for regenerative medicine of diabetes mellitus.

Methylation mechanisms and biomechanical effectors controlling cell fate.

Mammalian development and cell fate specification are controlled by multiple regulatory mechanisms that interact in a coordinated way to ensure proper regulation of gene expression and spatial restriction, allowing cells to adopt distinct differentiation traits and a terminal phenotype. For example, cell potency is modulated by changes in methylation that are under the control of methyltransferases and ten-eleven translocation (TET) enzymes, which establish or erase a phenotype-specific methylation pattern during embryo development and mesenchymal to epithelial transition (MET). Cell plasticity is also responsive to extracellular factors, such as small molecules that interact with cell fate definition and induce a transient pluripotent state that allows the direct conversion of an adult mature cell into another differentiated cell type. In addition, cell-secreted vesicles emerge as powerful effectors, capable of modifying cell function and phenotype and delivering different signals, such as octamer-binding transcription factor-4 (Oct4) and SRY (sex determining region Y)-box 2 (Sox2) mRNAs (implicated in the preservation of pluripotency), thus triggering epigenetic changes in the recipient cells. In parallel, mechanical properties of the cellular microenvironment and three-dimensional rearrangement can affect both cell potency and differentiation through marked effects on cytoskeletal remodelling and with the involvement of specific mechanosensing-related pathways.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries.

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n=6), not perfused and fixed; Groups SM72 and SM72-FSH (n=6 each), perfused with a simple medium for 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.

The differentiated state of mature cells of adult organisms is achieved and maintained through the epigenetic regulation of gene expression, which consists of several mechanisms including DNA methylation. The advent of induced pluripotent stem cell technology enabled the conversion of adult cells into any other cell type passing through a stable pluripotency state. However, indefinite pluripotency is unphysiological, inherently labile, and makes cells prone to culture-induced alterations. The direct conversion of one cell type to another without an intermediate pluripotent stage is also possible but, at present, requires the viral transfection of appropriate transcription factors, limiting its therapeutic potential. The aim of this study was to investigate whether it is possible to achieve the direct conversion of an adult cell by exposing it to a demethylating agent immediately followed by differentiating culture conditions. Adult human skin fibroblasts were exposed for 18 h to the DNA methyltransferase inhibitor 5-azacytidine, followed by a three-step protocol for the induction of endocrine pancreatic differentiation that lasted 36 d. At the end of this treatment, 35 ± 8.9% fibroblasts became pancreatic converted cells that acquired an epithelial morphology, produced insulin, and then released the hormone in response to a physiological glucose challenge in vitro. Furthermore, pancreatic converted cells were able to protect recipient mice against streptozotocin-induced diabetes, restoring a physiological response to glucose tolerance tests. This work shows that it is possible to convert adult fibroblasts into insulin-secreting cells, avoiding both a stable pluripotent stage and any transgenic modification.

Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts.

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues.

In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200's ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor's cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms.

Sperm fertilizing ability in vitro influences bovine blastocyst miRNA content.

MicroRNAs (miRNAs) are small highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through post-transcriptional regulation of gene expression. During development, miRNAs play a key role in driving embryo patterning and morphogenesis in a specific and stage-dependent manner. Here, we investigated whether sperm from bulls with different fertilizing ability in vitro influence blastocyst quality and miRNA content. Results demonstrate that blastocysts obtained using sperm from high fertility sires (H group) display significantly greater cleavage and blastocyst development as well as greater transcript abundance in blastocysts for the developmental competence markers CDX2, KRT8, NANOG, OCT4, PLAC8, PTGS2, SOX17, and SOX2, compared to blastocysts generated using sperm from low fertility sires (L group). In parallel, high throughput deep sequencing and differential expression studies revealed that H blastocysts exhibit a greater miRNA content compared to L blastocysts, with hsa-miR-4755-5p and hsa-miR-548d-3p uniquely detected in the H group, and greater abundance of hsa-miR-1225-3p in the H group. Gene ontology (GO) and KEGG pathway analyses indicated that the 3 differentially expressed miRNAs identified are involved in the regulation of many biological mechanisms with a key role in aspects of early embryo development, including transcriptional regulation, cellular biosynthesis, nucleic acid metabolism, cellular differentiation, apoptosis, cytoskeleton remodeling, cell-to-cell interactions, and endocytosis. Overall, our results indicate that sperm fertilizing ability influences blastocyst developmental ability and miRNA content. In addition, we demonstrate an association between blastocyst quality and miRNA content, thus suggesting the possibility to score miRNA expression as biomarkers for improved routine embryo selection technologies to support assisted reproductive efforts.

Use of assisted reproductive technologies (ARTs) to shorten the generational interval in ruminants: current status and perspectives.

The challenges posed by climate change and increasing world population are stimulating renewed efforts for improving the sustainability of animal production. To meet such challenges, the contribution of genomic selection approaches, in combination with assisted reproductive technologies (ARTs), to spreading and preserving animal genetics is essential. The largest increase in genetic gain can be achieved by shortening the generation interval. This review provides an overview of the current status and progress of advanced ARTs that could be applied to reduce the generation time in both female and male of domestic ruminants. In females, the use of juvenile in vitro embryo transfer (JIVET) enables to generate offspring after the transfer of in vitro produced embryos derived from oocytes of prepubertal genetically superior donors reducing the generational interval and acceleration genetic gain. The current challenge is increasing in vitro embryo production (IVEP) from prepubertal derived oocytes which is still low and variable. The two main factors limiting IVEP success are the intrinsic quality of prepubertal oocytes and the culture systems for in vitro maturation (IVM). In males, advancements in ARTs are providing new strategies to in vitro propagate spermatogonia and differentiate them into mature sperm or even to recapitulate the whole process of spermatogenesis from embryonic stem cells. Moreover, the successful use of immature cells, such as round spermatids, for intracytoplasmic injection (ROSI) and IVEP could allow to complete the entire process in few months. However, these approaches have been successfully applied to human and mouse whereas only a few studies have been published in ruminants and results are still controversial. This is also dependent on the efficiency of ROSI that is limited by the current isolation and selection protocols of round spermatids. In conclusion, the current efforts for improving these reproductive methodologies could lead toward a significant reduction of the generational interval in livestock animals that could have a considerable impact on agriculture sustainability.