Evaluation of postnatal growth, hematology, telomere length and semen attributes of multiple clones and re-clone of superior buffalo breeding bulls.

The present study comprehensively evaluates the postnatal growth, hematology, telomere length, and semen attributes of multiple clones and re-clone derived from superior buffalo breeding bulls. To the best of our knowledge, we successfully produced multiple clones and a re-clone of an earlier cloned buffalo bull from an embryo developed from an adult bull's skin-derived cell for the first time. The cloned bulls' growth, blood hematology, plasma biochemistry, and telomere length were all shown to be normal at various stages of development. The bulls were used for semen production after being screened for testicular growth and training. Semen characteristics such as volume, concentration, and initial motility of fresh sperm as well as motility and kinetics characteristics such as straightness (STR), average lateral head displacement (ALH), and beat cross frequency (BCF) of frozen-thawed sperms of the cloned bulls were found to be similar to those of non-cloned bulls, including the donor bulls. Additionally, it was found that cloned bulls' functional sperm attributes, including acrosome intactness, mitochondrial membrane potential, and superoxide anion status, were comparable to those of non-cloned bulls. These characteristics are necessary for sperm to pass through the female reproductive system, penetrate the oocyte, and efficiently fertilize. Finally, this study adds to our understanding of the postnatal development, hematology, telomere length, and sperm characteristics of superior buffalo breeding bulls that have been cloned and re-cloned. The findings provide the groundwork for improving cloning practices, refining reproductive procedures, and optimizing the use of cloned genetic material in animal breeding and conservation.

Inhibition of phospholipase C reduces the capacitation of cryopreserved ovine sperm.

This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 µM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the "Computer-assisted Sperm Analysis" system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 µM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 µM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 µM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes.

The effects of boar seminal plasma extracellular vesicles on sperm fertility.

Extracellular vesicles (EVs) are abundant in body fluid and are critical in cell interaction. Seminal plasma contains numerous EVs which affecting sperm function via transferring regulatory cargoes to the sperm. However, the mechanism of seminal plasma extracellular vesicles (SP-EVs) is still not clear. The present study aimed to isolate the boar SP-EVs and explore its potential function, then identify the key protein involved in SP-EVs and sperms interaction, and elucidate mechanism of SP-EVs protein on sperms. Here, we successfully isolated and concentrated boar SP-EVs, the SP-EVs showed a typical vesicle structure under transmission electron microscopy, most of their diameters range between 50 and 200 nm and express EVs biomarkers CD9 and CD63. We proved that SP-EVs could inhibit sperm acrosome reaction and in vitro fertility. Through a data-independent acquisition analysis of protein profiles of noncapacitated sperms, normal capacitated sperms and SP-EVs treated capacitated sperms, we identified that EZRIN was one of the active proteins that participated in SP-EVs and sperms interaction. Furthermore, we tested that the inhibition of EZRIN could promote boar sperm fertility, which is in consistence with the function of SP-EVs. The results may facilitate future research of SP-EVs on sperm function and male infertility.

Efficient isolation and purification of spermatogonia, spermatocytes, and spermatids from mice, piglets, and adult boars using an optimized STA-PUT method.

Spermatogenesis is a delicate and complex biological process in which spermatogonial stem cells continue to proliferate and differentiate into mature spermatozoa, maintaining sperm production in male mammals throughout the lifetime. To study the molecular mechanism of spermatogenesis, researchers had to isolate different germ cell subpopulations for in vitro culture and characterization. However, due to the existence of several stages of germ cells and a variety of populations of somatic cells in the testis of male mammals, it is a challenge for us to obtain high-purity germ cell subpopulations for further research. Here, we optimized the STA-PUT device and successfully applied it to isolate and purify spermatogonia populations in piglets, and multiple germ cell populations at different developmental stages in testes of adult mice and boars. This work provides a simple platform for germ cell fractionation to facilitate the molecular mechanistic study of animal spermatogenesis in vitro.

Structural Analysis of Sperm Centrioles Using N-STORM.

A prominent technical barrier when imaging swimming sperm is capturing a singular sperm cell's head and tail position simultaneously at a high resolution to understand their relationship in different stages of the sperm tail beating cycle. This is due to the sperm's high beating frequency, rotational movement, and the large difference in diameter between the head and tail. These intricacies increase the complexity of determining the position of a dynamic subcellular structure in the sperm neck, such as the centriole. We have developed a way to obtain this information by snap freezing mobile sperm at different stages of the sperm tail beating cycle and then analyzing them with super-resolution microscopy. This method captures the position of both the sperm head and tail at the microscale and centriolar substructure details at the nanoscale. This chapter describes the detailed procedures for the selection, preparation, antibody staining, 3D N-STORM imaging, and image quantification of bovine spermatozoa.

Unveiling the impact of environmental microplastics on mussel spermatozoa: First evidence of prothymosin-α detection in invertebrate's male gametes.

Mytilus galloprovincialis mussels, like many other marine invertebrates, employ external fertilization as a mating strategy, exposing their gametes to various contaminants upon release into seawater. Environmental microplastics (EMP) are prevalent marine pollutants that pose a significant threat to aquatic biota. In this regard, our study aimed to investigate the potential effects of exposing mussels' male gametes to increasing concentrations of EMP (1, 10, 50, and 100 µg/l) collected from a Mediterranean sandy beach. We focused on assessing gamete quality by analysing physiological parameters such as viability, mitochondrial membrane potential, oxidative status, and motility. Additionally, we evaluated DNA integrity and activation of apoptosis. Furthermore, our study aimed to detect the presence of the prothymosin-α (PTMA) protein, which has never been previously investigated in invertebrate spermatozoa. Our data revealed that exposure of mussel spermatozoa to EMPs altered their oxidative status and mitochondrial membrane potential, induced a decrease in motility, DNA integrity, and an increased apoptotic occurrence, leading to a decline in overall viability. The localization of PTMA into the head and flagellum of spermatozoa further supported its presence and susceptibility to the effects of microplastics. These findings raise concerns about the reproductive capacity of mussels under environmental microplastic pollution and highlight potential long-term threats to population sustainability.

Toxic effects and potential mechanisms of zinc pyrithione (ZPT) exposure on sperm and testicular injury in zebrafish.

Zinc pyrithione (ZPT) is widely recognized for its beneficial properties as an antifouling, antibacterial, and antifungal agent. Despite its positive industrial contributions, ZPT has been proven to exhibit toxicity towards various ecosystems, particularly affecting marine life. However, there is still a dearth of comprehensive research on ZPT toxicity and its toxicological mechanism in reproductive systems of aquatic organisms. In our study, we conducted a thorough analysis and unveiled a multitude of abnormalities in zebrafish sperm and testicular tissue caused by ZPT exposure, including a dose-dependent diminishing of testosterone levels, various sperm deformities, decreased sperm concentration and motility, and ROS-induced testicular tissue DNA damage. In addition, our study suggested that ZPT-induced testicular damage is associated with heightened oxidative stress, apoptosis, and possible hyperpolarization of the mitochondrial membrane. Through RNA-seq analysis, a total of 409 DEGs associated with ZPT-induced testicular injury were identified, and the hub gene was determined using a protein-protein interaction network (PPI). The genes and pathways uncovered in this study point to potential mechanisms of ZPT exposure on sperm and testicular injury in zebrafish.

Exposure to PM2.5, seminal plasma metabolome, and semen quality among Chinese adult men: Association and potential mediation analyses.

Exposure to ambient fine particulate matter (PM2.5) has been linked to a decline in semen quality, but the underlying mechanisms for this association remain unclear. We aimed to examine whether specific metabolites act as mediators in the association between PM2.5 exposure and changes in semen quality. We conducted untargeted metabolomics analysis using LC-MS/MS platforms to identified seminal plasma metabolites associated with various semen quality parameters among 200 Chinese adult men. Additionally, we performed mediation analyses to examine the effects of the seminal plasma metabolites on the association between PM2.5 exposure and semen quality. We identified 140 differential metabolites between the normal and abnormal semen groups, involving two metabolic pathways: Alanine, aspartate and glutamate metabolism, and Aminoacyl-tRNA biosynthesis. We additionally identified 7 specific seminal plasma metabolites that were associated with discrepant metabolic networks related to semen quality. The mediation analysis revealed that D-Aspartate might play a mediating role in the adverse effects of ambient PM2.5 exposure on both total and progressive motility during spermatogenesis period (70-90 days before ejaculation), with a proportion of mediation up to 16% and 17%, respectively. Exposure to PM2.5 was associated with alterations in D-Aspartate levels, which might partially mediate the association between PM2.5 and reduced sperm motility.

Transgenerational epigenetic effects imposed by neonicotinoid thiacloprid exposure.

Neonicotinoids are a widely used class of insecticides that are being applied in agricultural fields. We examined the capacity of a neonicotinoid, thiacloprid (thia), to induce transgenerational effects in male mice. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5-E15.5 using different doses. Testis sections were used for morphology analysis, ELISAs for testosterone level analysis, RT-qPCR and RNA-seq for gene expression analysis, MEDIP-seq and MEDIP-qPCR techniques for DNA methylation analysis, and Western blot for a protein analysis. The number of meiotic double-strand breaks and the number of incomplete synapsed chromosomes were higher in the thia 6-treated group of F3 males. Genome-wide analysis of DNA methylation in spermatozoa revealed that differentially methylated regions were found in all three generations at the promoters of germ cell reprogramming responsive genes and many superenhancers that are normally active in embryonic stem cells, testis, and brain. DNA methylation changes induced by thia exposure during embryonic period are preserved through several generations at important master regulator regions.

Transcriptome analysis of meiotic and post-meiotic spermatogenic cells reveals the potential hub genes of aging on the decline of male fertility.

Genetic and epigenetic changes in sperm caused by male aging may be essential factors affecting semen parameters, but the effects and specific molecular mechanisms of aging on male reproduction have not been fully clarified. In this study, to explore the effect of aging on male fertility and seek the potential molecular etiology, we performed high-throughput RNA-sequencing in isolated spermatogenic cells, including pachytene spermatocytes (marked by the completion of chromosome synapsis) and round spermatids (produced by the separation of sister chromatids) from the elderly and the young men. Functional enrichment analysis of differentially expressed genes (DEGs) in round spermatids between the elderly and young showed that they were significantly enriched in gamete generation, spindle assembly, and cilium movement involved in cell motility. In addition, the expression levels of DEGs in round spermatids (post-meiotic cells) were found to be more susceptible to age. Furthermore, ten genes (AURKA, CCNB1, CDC20, CCNB2, KIF2C, KIAA0101, NR5A1, PLK1, PTTG1, RAD51AP1) were identified to be the hub genes involved in the regulation of sperm quality in the elderly through Protein-Protein Interaction (PPI) network construction and measuring semantic among GO terms and gene products. Our data provide aging-related molecular alterations in meiotic and post-meiotic spermatogenic cells, and the information gained from this study may explain the abnormal aging-related male fertility decline.

Environmentally relevant exposure to tetrabromobisphenol A induces reproductive toxicity via regulating glucose-6-phosphate 1-dehydrogenase and sperm activation in Caenorhabditis elegans.

Tetrabromobisphenol A (TBBPA), a ubiquitous brominated flame-retardant environmental pollutant, has been reported to cause reproductive toxicity by chronic exposure. However, the acute reproductive risk and mechanisms of TBBPA toxicity to individuals, especially at environmentally relevant levels, remains a topic of debate. In this study, Caenorhabditis elegans was used to investigate the reproductive toxicity of acute exposure to TBBPA at environmentally relevant doses. The reproductive end points (embryonic lethality ratio and brood size), oxidative stress, sperm activation, and molecular docking were evaluated. Results showed that, after 24 h of TBBPA treatment, even at the lowest concentration (1 µg/L), the embryonic lethality ratio of C. elegans increased significantly, from 1.63 % to 3.03 %. Furthermore, TBBPA induced oxidative stress with significantly increased expression of sod-3 in C. elegans, which further raised the level of reproductive toxicity through inhibiting the activation of sperm in nematodes. In addition, molecular docking suggested TBBPA might compete for the glucose-6-phosphate-binding site of glucose-6-phosphate 1-dehydrogenase, resulting in oxidative stress generation. Accordingly, our findings indicate that even acute exposure to environmental concentrations of TBBPA may induce reproductive toxicity through reducing sperm activation in nematodes.

The multifaceted role of extracellular ATP in sperm function: From spermatogenesis to fertilization.

Extracellular adenosine 5'-triphosphate (ATP) is a vital signaling molecule involved in various physiological processes within the body. In recent years, studies have revealed its significant role in male reproduction, particularly in sperm function. This review explores the multifaceted role of extracellular ATP in sperm function, from spermatogenesis to fertilization. We discuss the impact of extracellular ATP on spermatogenesis, sperm maturation and sperm-egg fusion, highlighting the complex regulatory mechanisms and potential clinical applications in the context of male infertility. By examining the latest research, we emphasize the crucial role of extracellular ATP in sperm function and propose future research directions to further.

Cerium oxide nanoparticles improve the post-thaw quality and in-vivo fertility of Beetal buck spermatozoa.

The motility, health quality, and membrane disorders of spermatozoa are adversely affected during the process of semen cryopreservation due to the over-production of reactive oxygen species (ROS). Cerium oxide nanoparticles (CeO2NPs) possess properties to scavenge ROS either by mimicking specific antioxidants or by enhancing the activities of antioxidant enzymes. Therefore, we aimed at evaluating the effects of adding the CeO2NPs in the TRIS-citrate-yolk extender on in-vitro antioxidant enzyme activities, spermatozoa quality attributes, and in-vivo fertility of post-thaw Beetal buck spermatozoa. The CeO2NPs were prepared and characterized (UV-spectrophotometry, FTIR, and XRD). Semen samples, collected from bucks (n = 5), were distributed into five aliquots and diluted in an extender containing increasing concentrations of nanoparticles (0 µg/ml, called the control group, 25 µg/mL, 50 µg/mL, 75 µg/mL, and 100 µg/mL). At post-thaw, spermatozoa were evaluated for the above-mentioned attributes and the pregnancy rate by inseminating Beetal does (n = 252). Results demonstrated that CeO2NPs mitigated the detrimental effects of cryopreservation as ROS production and lipid peroxidation were lower (P < 0.001) in the 25, 50, and 75 µg/mL CeO2NPs-added groups compared to the control and 100 µg/ml CeO2NPs-added group. The addition of 25 µg/mL CeO2NPs improved (P < 0.001) the activities of superoxide dismutase, catalase, and peroxidase and the concentration of reduced glutathione (P < 0.001) compared to the other groups. In terms of sperm kinematics and velocity parameters, the groups added with the 25 and 50 µg/mL CeO2NPs exhibited higher total motility (P < 0.001), sperm progressive motility (P = 0.003), and rapid velocity (P < 0.001). The group added with the 50 µg/mL CeO2NPs had the highest (P = 0.04) average path velocity. The groups added with the 25 and 50 µg/mL CeO2NPs also exhibited higher plasma membrane integrity (P = 0.003), acrosomal integrity, and viability (P < 0.001) compared to the control group. The DNA integrity was also higher (P < 0.001) in all the CeO2NPs-added groups. The pregnancy rate was higher (P = 0.003) in the 25 (51.92 %) and 50 µg/mL CeO2NPs (58.33 %) groups compared to the other groups. Conclusively, our findings suggest that the inclusion of cerium oxide nanoparticles in the TRIS-citrate-yolk freezing extender can reduce the occurrence of cryopreservation-induced damages to Beetal's buck spermatozoa and ultimately enhance the pregnancy rate in does.

The sperm structure of the Scraptiidae (Coleoptera; Tenebrionoidea).

The sperm ultrastructure of two members of the Scraptiidae Anaspis pulicaria and A. lurida was studied. The results confirm the general organization of the sperm in the superfamily Tenebrionoidea. The sperm bundles at the end of the spermiogenesis show the same peculiar antiparallel distribution at the two opposite poles of the germ cyst, observed in other Tenebrionoidea. The sperm have a bi-layered acrosome, a long cylindrical nucleus with two infoldings at its basal region, two elliptical equal mitochondrial derivatives and two triangular accessory bodies. The flagellar axoneme has the common 9 + 9 + 2 microtubular pattern that at the tail end results disorganized. All these sperm characteristics are quite similar to those found in Pythidae, a closely related family, according to molecular data.

PCB126 impairs human sperm functions by affecting post-translational modifications and mitochondrial functions.

Over the past few decades, there has been a consistent decline in semen quality across the globe, with environmental pollution being identified as the primary cause. Among the various contaminants present in the environment, persistent organic pollutants (POPs) have garnered significant attention due to their high toxicity, slow degradation, bio-accumulation, and long-range migration. PCBs, which include 210 congeners, are a crucial type of POPs that are known to have harmful effects on the environment and human health. Among the various PCB congeners, 3,3',4,4',5-pentachlorobiphenyl (PCB126) is a typical environmental endocrine-disrupting chemical that is widely distributed and has been associated with several health hazards. However, the impact and mechanism of PCB126 on human sperm function has not been fully elucidated. We aimed to investigate the effects of different concentrations of PCB126 (0.01, 0.1, 1, 10 µg/mL) on sperm motility, viability, hyperactivation, and acrosome reaction after incubation for different periods (1 and 2 h), delving deeper into the molecular mechanism of human sperm dysfunction caused by PCB126. First, we investigated the link between PCB126 treatment and the occurrence of protein modifications that are critical to sperm function regulation, such as tyrosine phosphorylation and lysine glutarylation. Second, we examined the potential impact of PCB126 on different parameters related to mitochondrial function, including reactive oxygen species, malondialdehyde levels, mitochondrial membrane potential, mitochondria respiration and adenosine triphosphate generation. Our findings indicate that exposure to environmental pollutants such as PCB126 in vitro may have a negative impact on human sperm functions by interfering with post-translational modifications and mitochondrial functions.

The Ancient Origin and Function of Germline Cysts.

Gamete production in most animal species is initiated within an evolutionarily ancient multicellular germline structure, the germline cyst, whose interconnected premeiotic cells synchronously develop from a single progenitor arising just downstream from a stem cell. Cysts in mice, Drosophila, and many other animals protect developing sperm, while in females, cysts generate nurse cells that guard sister oocytes from transposons (TEs) and help them grow and build a Balbiani body. However, the origin and extreme evolutionary conservation of germline cysts remains a mystery. We suggest that cysts arose in ancestral animals like Hydra and Planaria whose multipotent somatic and germline stem cells (neoblasts) express genes conserved in all animal germ cells and frequently begin differentiation in cysts. A syncytial state is proposed to help multipotent stem cell chromatin transition to an epigenetic state with heterochromatic domains suitable for TE repression and specialized function. Most modern animals now lack neoblasts but have retained stem cells and cysts in their early germlines, which continue to function using this ancient epigenetic strategy.

Reproductive hormones, organophosphate esters and semen quality: Exploring associations and mediation effects among men from an infertility clinic.

Accumulating evidence indicates that organophosphate esters (OPEs) exposure may affect semen quality. As a crucial factor in male reproduction, reproductive hormones might be linked organophosphate esters (OPEs) exposure and semen quality. This study aimed to explore the mediating role of reproductive hormones on the association between OPEs exposure and semen quality. Five serum reproductive hormones, semen quality, and 16 urinary OPE metabolites were measured among 491 reproductive-aged men from a reproductive center. The associations of urinary OPE metabolites with reproductive hormones and semen quality were assessed using multivariable linear regression models, and the mediating role of reproductive hormones was evaluated by mediation analyses. We found that follicle stimulating hormone (FSH) was positively associated with diphenyl phosphate (DPHP) that in turn was negatively associated with sperm total count. In addition, inverse associations were exhibited between serum FSH and sperm concentration, sperm total count, total motility, and progressive motility (all Ptrend <0.05). Mediation analysis further showed that FSH mediated 13.7% of the inverse association of DPHP and sperm total count. Although further investigations are required, our results suggest that FSH was an intermediate mechanism in the associations between OPEs exposure and impaired semen quality.

Exploring the molecular and toxicological mechanism associated with interactions between heavy metals and the reproductive system of Mytilus galloprovincialis.

A large number of heavy metals resulted toxic to the reproductive system, but invertebrate infertility has been poorly explored, and above all, there are limited molecular, cellular and toxicological studies. In the present work, we exposed Mytilus galloprovincialis to three individual metal chlorides (CuCl2 15 µM, CdCl2 1.5 µM, NiCl2 15 µM) and their mixture for 24 h, to evaluate the effects on the protamine-like proteins (PLs), sperm DNA and on their interaction in the formation of sperm chromatin. Under all exposure conditions, but particularly after exposure to the metals mix, relevant changes in the electrophoretic pattern, by AU-PAGE and SDS-PAGE, and in fluorescence spectroscopy measurements of PLs were shown. In addition, alterations in DNA binding of these proteins were observed by Electrophoretic Mobility Shift Assay (EMSA) and through their release from sperm nuclei. Moreover, there was evidence of increased accessibility of micrococcal nuclease to sperm chromatin, which was also confirmed by toluidine blue staining. Furthermore, morphological analyses indicated severe gonadal impairments which was also corroborated by increased PARP expression, by Western blotting, and sperm DNA fragmentation, by comet assay. Finally, we investigated the expression of stress genes, gst, hsp70 and mt10, in gonadal tissue. The latter investigations also showed that exposure to this metals mix was more harmful than exposure to the individual metals tested. The present results suggest that these metals and in particular their mixture could have a negative impact on the reproductive fitness of M. galloprovincialis. Based on these evidences, we propose a molecular mechanism.

Participation of WD repeat-containing protein 54 (WDR54) in rat sperm-oocyte fusion through interaction with both IZUMO1 and JUNO.

Fertilization is a complex process that depends on the fusion of the cell membrane of sperm with that of oocyte, and it involves sperm-oocyte recognition, binding, and fusion, which are mediated by multiple proteins. Among those proteins, IZUMO1 and its receptor JUNO have been identified as essential factors for sperm-oocyte recognition and fusion. However, the interaction between IZUMO1 and JUNO alone does not lead to cell membrane fusion, suggesting the involvement of additional proteins in sperm-oocyte membrane fusion. In this study, we have discovered that a protein called WDR54, which consists of WD-repeat modules, is located on the cell membrane of sperm, as well as on the cell membrane and in the cytoplasm of the oocyte. We have found that WDR54 is involved in sperm-oocyte fertilization. When sperm and oocyte were treated with anti-WDR54 ascites, the in vitro fertilization (IVF) rate significantly decreased. Furthermore, our research has shown that WDR54 interacts with both IZUMO1 and JUNO, and it colocalizes with IZUMO1 on the surface of the sperm head and with JUNO on the oocyte surface. Through structural analysis of the putative complexes of WDR54-IZUMO1 and WDR54-JUNO, we infer that these three proteins could form a complex of JUNO-WDR54-IZUMO1-JUNO (referred to as the "JWIJ complex") on the oocyte surface. Our findings suggest that WDR54 is an important factor involved in sperm-oocyte adhesion and fusion. This discovery provides new insight into the mechanisms of mammalian sperm-oocyte adhesion and fusion.

Effects of Nobiletin supplementation on the freezing diluent on porcine sperm cryo-survival and subsequent in vitro embryo development.

Nobiletin (NOB) is a bioflavonoid compound isolated from citrus fruit peels. The present study aimed to elucidate whether NOB facilitates the porcine sperm cryosurvival and embryo development after in vitro fertilization (IVF). To this end, spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 50, 100, 150, and 200 µM Nobiletin. The kinematic patterns of frozen-thawed (FT) sperm were assessed after 30 and 90 min incubation using a Sperm Class Analyzer (SCA). Viability, acrosome integrity, and mitochondrial membrane potential (MMP) were measured by fluorescence microscopy 30 min after thawing using SYBR-14/PI, PSA/FITC, and R123/PI, respectively. Lipid peroxidation was determined using MDA assay after incubation for 90 min. The addition of 100 µM and 150 µM NOB to the extender significantly improved sperm progressive motility, and acrosome integrity compared to the control group (P < 0.05). The proportion of viable spermatozoa was significantly higher in the 150 µM NOB group. MDA levels were less in 50 µM and 150 µM NOB treated groups compared to the control. In addition, IVF with FT sperm was used to assess the embryo developmental competence. Treatment with 150 µM NOB before cryopreservation increased the cleavage and blastocyst formation rates compared to the control group. Furthermore, the relative expression of POU5F1 and AMPK, genes related to pluripotency and cell differentiation were significantly upregulated in embryos resulting from NOB-treated sperm compared to the control group. These results suggest that Nobiletin is a functionally novel phytochemical to mitigate oxidative stress during the freezing-thawing of porcine spermatozoa as reflected by improved FT sperm quality and IVF outcome.