Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

The last decades of research have revealed that many other factors besides gamete genomes are able to determine the reproductive outcomes. Indeed, paternal factors have been observed to be capable of modulating multiple crucial features of the reproductive process, such as sperm physiology, the maternal environment and, even, the offspring health. These recent advances have been encompassed with the emergence of OMICS technologies, as they comprehensively characterise the molecular composition of biological systems. The present narrative review aimed to take a closer look at the potential of these technologies in the field of reproductive biology. This literature revision shows that most studies up to date have followed a non-targeted approach to screen mammalian seminal plasma (SP) and sperm metabolite composition through different metabolome platforms. These studies have proposed metabolites of multiple natures as potential in vivo fertility biomarkers. Yet, targeted approaches can be used to answer specific biological question, and their power is exemplified herein. For instance, metabolomic studies have uncovered not only that glycolysis is the main ATP energy source of pig sperm, but also that sperm metabolism can trigger DNA damage, hence compromise embryo development. In conclusion, this review shows the potential of both non-targeted and targeted metabolomics for the discovery of cell pathways that govern the reproductive process. Understanding these systems could help make progress in different areas, including livestock efficient breeding, the improvement of artificial reproductive technologies, and the development of biomarkers for infertility detection.

Livestock management is evolving into a new era, characterized by the analysis of vast quantities of data (Big Data) collected from both traditional breeding methods and new technologies such as sensors, automated monitoring system, and advanced analytics. Artificial intelligence (A-In), which refers to the capability of machines to mimic human intelligence, including subfields like machine learning and deep learning, is playing a pivotal role in this transformation. A wide array of A-In techniques, successfully employed in various industrial and scientific contexts, are now being integrated into mainstream livestock management practices. In the case of swine breeding, while traditional methods have yielded considerable success, the increasing amount of information requires the adoption of new technologies such as A-In to drive productivity, enhance animal welfare, and reduce environmental impact. Current findings suggest that these techniques have the potential to match or exceed the performance of traditional methods, often being more scalable in terms of efficiency and sustainability within the breeding industry. This review provides insights into the application of A-In in porcine breeding, from the perspectives of both sows (including welfare and reproductive management) and boars (including semen quality and health), and explores new approaches which are already being applied in other species.

The effects of important nutrients such as calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), selenium (Se), and zinc (Zn) have been investigated in relation to male fertility due to their roles in proper spermatogenesis, sperm maturation, motility, and optimal sperm function. An imbalance between these elements has been associated with several pathologic conditions and male reproductive issues. The purpose of this study was to determine the essential trace and electrolytes elements, such as Ca, Cu, Fe, Mg, Se, and Zn, in human biological samples (blood, serum, and semen) from patients with male infertility. This study used correlational analysis to determine the potential associations between these elements and male fertility. Imbalances in these elements have been linked to various pathological conditions and male reproductive issues. One hundred eighty referent male adults and two hundred twenty-nine patients diagnosed with subtypes of infertility were included in the study, divided into two age groups. Acid digestion was controlled using a microwave oven, and the essential trace elements and electrolytes in the oxidized biological samples were determined using atomic absorption spectrometry. Certified reference materials of blood and serum were used to validate the accuracy of the methodology. The results showed that the concentrations of Ca, Cu, Fe, Mg, Se, and Zn in the blood, serum, and seminal plasma of male adults in all age groups were higher than those in patients with different infertility phenotypes. Essential element deficiency in all biological fluid samples may significantly negatively affect human reproductive health and lead to male infertility. Through a multidimensional approach, our study sought to unravel the intricate biochemical signatures associated with OAT, providing insights that may shape the landscape of diagnostic and therapeutic strategies for male reproductive health.

Methamphetamine (METH), an amphetamine-type stimulant, has been extensively abused globally in the past decades. METH use causes great harm to the major systems of the human body. Specifically, METH has a negative impact on the hypothalamic- pituitary-testicular axis, testicular structure, sperm function, ovarian folliculogenesis, oocyte quality, embryo development, and newborns. However, the mechanisms underlying these toxic effects have not yet been fully described. This study reviews the evidence concerning the impact of METH on male and female reproduction in the context of the testis, sperm, ovaries, oocytes, reproductive hormones, embryo development, and newborns, discussing the potential pathophysiological mechanisms in the reproductive toxicity induced by METH.

The assessment of epigenetic profiles in sperm is sensitive to somatic cell contamination, which can influence methylation signals at gene promoters. This contamination is particularly problematic in the assessment of DNA methylation in samples with low sperm counts, where fractional amounts of somatic cell DNA can lead to significant shifts in measured methylation state. In this study, a new method of detecting possible somatic cell contamination is proposed through two multi-region bioinformatic models: a traditional differential methylation analysis and a machine learning logistic regression model. These models were trained on publicly available sperm (n = 489) and blood (n = 1029) DNA methylation array data and tested on a contamination set, wherein the sperm of four donors with normal sperm counts were run on a 450k methylation array with four permutations each, including pure blood, half blood and half sperm by DNA concentration, half blood and half sperm by cell count, and pure sperm (n = 16). The DMR and logistic regression model classified the contamination testing set with 100% and 94% accuracy, respectively. These new methods of detecting the effects of somatic cell contamination allow for more accurate differentiation between epigenetic profiles that contain a biological somatic-like shift and those that have somatic-like signatures because of contamination.

To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.

OBJECTIVE: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters.
METHODS: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318).
RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients.
CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane integrity, lipid peroxidation, and DNA fragmentation. The findings indicated that 25-100 μg/mL of silymarin significantly improved viability and mitochondrial membrane potential while reducing the stallion sperm lipid peroxidation, DNA fragmentation, and apoptosis compared with the control group (p < 0.05). Silymarin concentrations of 75 μg/mL and 100 μg/mL significantly increased progressive motility and plasma membrane integrity (p < 0.05). Based on our findings, it can be inferred that silymarin exhibited a dose-dependent enhancement in the frozen-thawed Arabian stallion sperm quality. The most favorable outcomes were observed when 100 μg/mL silymarin was used.