Sperm Head Morphology Research Articles

Chromosome 17 translocation affects sperm morphology: Two case studies and literature review.

We present two cases of infertile males with teratozoospermia stemming from chromosome 17 translocation. The patients present karyotypes that have not been previously reported. Genes located on breakpoints (17p11.2, 9q31, and 11p15) were analysed to find the probable mechanism affecting sperm morphology. Our results suggest that ALKBH5, TOP3A, and LLGL1 interactions may be an underlying cause of abnormal sperm head morphology. Translocation of chromosome 17 occurred in conjunction with chromosome 9 and chromosome 11 translocation in the two cases, resulting in oligozoospermia and asthenozoospermia, respectively. These abnormal phenotypes may involve meiosis- and motility-related genes such as LDHC, DNHD1, UBQLN3, and NUP98. Translocation is thus a risk factor for sperm morphological abnormalities and motility deficiency. The interaction network of 22 genes on breakpoints suggests that they contribute to spermatogenesis as a group. In conclusion, this study highlighted the importance of investigating genes linked to sperm morphology, together with chromosome 17 translocation and reproductive risks. For patients interested in screening before a future pregnancy, we recommend preimplantation genetic diagnosis to reduce the risk of karyotypically unbalanced foetuses and birth defects.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Ensembled Deep Learning for the Classification of Human Sperm Head Morphology

Infertility is a growing global health concern, with male factor infertility contributing to half of all cases. Semen analysis is crucial to infertility diagnostics. However, sperm morphology assessment, as a routine part of analysis, is still performed manually and is thus highly subjective. Here, a stacked ensemble of convolutional neural networks (CNNs) is presented for automated classification of human sperm head morphology. By combining traditional CNN models with modern residual and densely connected architectures using a multi‐class meta‐classifier, classification rate improvements of 2.7% (to 98.2%) and 2.3% (to 63.3%) on the HuSHeM and SCIAN‐MorphoSpermGS (SCIAN) datasets, respectively, are achieved. This considerable improvement in prediction performance is achieved as the meta‐classifier improves upon the individual classification rates of the base models by ≈8.5%. The ensembled deep learning model is a powerful step toward an automated sperm morphology analysis, providing new opportunities to standardize clinical practice and reduce treatment costs to improve patient treatment.

Juvenile exposure and adult risk assessment with single versus repeated exposure of melphalan in the germ cells of male SD rat: Deciphering the molecular mechanisms

Melphalan significantly contributes to the increase in childhood cancer survival rate. It acts as a gonadotoxic agent and leads to testes damage, dysbalance in gonadal hormones, and impairment in the germ cell proliferation. Therefore, it might be a potent threat to male fertility in individuals who have undergone melphalan treatment during childhood cancer. However, the molecular mechanisms of melphalan-induced gonadal damage are not yet fully explored and they need to be investigated to determine the benefit-risk profile. In the present study, juvenile male SD rats were subjected to single and intermittent cycles of melphalan exposure in a dose-dependent (0.375, 0.75 and 1.5 mg/kg) manner. Methods of end-points evaluations were quantification of micronuclei formation in peripheral blood, sperm count, sperm motility and head morphology, sperm and testicular DNA damage, histological studies in testes, oxidative/nitrosative stress parameters. A single cycle of exposure at high dose (1.5 mg/kg) produced significant effect on micronuclei formation only after the first week of exposure, whereas failed to produce significant effect at the end of the sixth week. Intermittent cycles of exposure at the dose of 1.5 mg/kg produced significant alterations in all the parameters (micronuclei in peripheral blood, testes and epididymides weight and length, MDA, GSH and nitrite levels, sperm count and motility, sperm head morphology, testicular and sperm DNA damage, protein expression in testes and histological parameters). So, time of exposure as well as the amount of exposure (total dosage administered) is critical in determining the magnitude of the damage in germ cell risk assessment.

X-linked palindromic gene families 4930567H17Rik and Mageb5 are dispensable for male mouse fertility

Mammalian sex chromosomes are enriched for large, nearly-identical, palindromic sequences harboring genes expressed predominately in testicular germ cells. Discerning if individual palindrome-associated gene families are essential for male reproduction is difficult due to challenges in disrupting all copies of a gene family. Here we generate precise, independent, deletions to assess the reproductive roles of two X-linked palindromic gene families with spermatid-predominant expression, 4930567H17Rik and Mageb5. Sequence analyses reveals mouse 4930567H17Rik and Mageb5 are orthologs of human HSFX3 and MAGEB5, respectively, where 4930567H17Rik/HSFX3 is harbored in a palindrome in humans and mice, while Mageb5 is not. Additional sequence analyses show 4930567H17Rik and HSFX3 are rapidly diverging in rodents and primates, respectively. Mice lacking either 4930567H17Rik or Mageb5 gene families do not have detectable defects in male fertility, fecundity, spermatogenesis, or in gene regulation, but do show differences in sperm head morphology, suggesting a potential role in sperm function. We conclude that while all palindrome-associated gene families are not essential for male fertility, large palindromes influence the evolution of their associated gene families.

A Preliminary Study on the Evaluation of Human Sperm Head Morphology with a Domestic Digital Holographic Image System.

The head of sperm was imaged with domestic digital holographic microscopy (DHM), and then the quantitative three-dimensional size information of normal sperm and teratozoospermic sperm was compared and analyzed. DHM sperm imaging and repeated quantitative evaluation were used to determine the morphology of the sperm head in two patients with teratozoospermia and four volunteers with normal semen parameters. Sixty and 139 sperm of teratozoospermia patients and normal people were photographed by digital hologram, respectively. The differences in head height and width were compared and statistically analyzed. The sperm head height of the teratozoospermia group was 3.06 ± 1.66μm, which was significantly lower than that of the normal sperm group (4.54 ± 1.60μm, p < 0.01), but there was no significant difference in the head width between the two groups. Compared with the traditional two-dimensional optical microscope observation method, the DHM system can provide three-dimensional quantitative information for the sperm head and thus may help in the comprehensive clinical evaluation of the sperm head structure.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

PurposeSpermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1.MethodsTo elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry.ResultThe Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid‐pachytene spermatocytes to the early spermatids.Conclusion Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non‐obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

Sperm morphology analysis by using the fusion of two-stage fine-tuned deep networks

Traditional analysis of sperm morphology refers to the process of extracting and analyzing spatial-based (e.g., sperm eccentricity and sperm area) and/or transform based features (e.g., wavelet decomposition, Fourier analysis and descriptors) from microscopic images, with the ultimate goal being the acquisition of predictive models. Deep learning methods, on the other hand, are capable of learning the features that best distinguish normal and abnormal sperm pixels and can be learned in an end-to-end manner. Application of deep learning in microscopic imaging provides automatized discovery of sperm morphology features as well as exploration of the hierarchy and interaction lying behind the deep features obtained from sperm shapes. In the proposed study, two deep learning networks, which have already been trained by using benchmark image data sets, were employed to classify raw sperm patches by using high level spatial features comprising extreme distinctive ability. Subsequently, a decision level ensemble learning scheme was applied to the predictions of employed deep-nets by using a soft-voting rule. The experiments were carried on three public data sets named as the Sperm Morphology Image Data Set (SMIDS), Human Sperm Head Morphology Set (HuSHeM) and SCIAN-Morpho for performance validation. Additional to classical transfer of pre-trained network weights followed by fine-tuning, which was tested in both data sets, a two-stage sequential fine-tuning procedure was applied to HuSHeM and SCIAN-Morpho resulting in increased accuracy. The results point out that the learning model, in which the predictions of single-stage fine-tuned deep-nets are fused by using soft-voting, performs well for SMIDS, HuSHeM and SCIAN-Morpho reaching 90.87%, 88.89% and 72.08% accuracy values respectively. Besides, the two-stages fine-tuning approach increases the HuSHeM and SCIAN-Morpho performance up to 92.1% and 73.2% without any manual intervention in contrast to competitors employing manual cropping, rotation and biased augmentation steps.

The social shape of sperm: using an integrative machine-learning approach to examine sperm ultrastructure and collective motility.

Sperm is one of the most morphologically diverse cell types in nature, yet they also exhibit remarkable behavioural variation, including the formation of collective groups of cells that swim together for motility or transport through the female reproductive tract. Here, we take advantage of natural variation in sperm traits observed across Peromyscus mice to test the hypothesis that the morphology of the sperm head influences their sperm aggregation behaviour. Using both manual and automated morphometric approaches to quantify their complex shapes, and then statistical modelling and machine learning to analyse their features, we show that the aspect ratio of the sperm head is the most distinguishing morphological trait and statistically associates with collective sperm movements obtained from in vitro observations. We then successfully use neural network analysis to predict the size of sperm aggregates from sperm head morphology and show that species with relatively wider sperm heads form larger aggregates, which is consistent with the theoretical prediction that an adhesive region around the equatorial region of the sperm head mediates these unique gametic interactions. Together these findings advance our understanding of how even subtle variation in sperm design can drive differences in sperm function and performance.

CONCURRENT ULTRASTRUCTURAL ANALYSIS AND GENOMIC ASSESSMENT OF SPERMATOZOA TO ACCURATELY DIAGNOSE RARE AND SEVERE FORMS OF MALE INFERTILITY

In men with ART failure, sperm DNA sequencing with ultrastructural analysis can identify specific sperm defects and guide ART treatment. In the past 2 years, 22 consenting men who failed IVF cycles due to severe astheno-/teratozoospermia were recruited; specimens from 3 fertile men were used as controls. To assess ultrastructure by transmission electron microscopy (TEM), pelleted spermatozoa were fixed, embedded, sectioned to 100-nm slides by ultramicrotome, and viewed with an electron microscope (JEOL-1400) at 300,000X magnification. Sperm head morphology, fibrous sheath intactness, and axonemal/periaxonemal structure were examined on at least 100 spermatozoa to diagnose specific sperm organelle defects. DNA was extracted and amplified from at least 500 spermatozoa for next-generation sequencing (NGS) to identify mutations contributing to the ultrastructural abnormalities. Four types of sperm ultrastructural defects were identified: globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One case of combined globozoospermia and DFS and one case of combined DFS and proximal centriole defect were identified. In 13 globozoospermic cases, 97–100% of the spermatozoa displayed complete spherical heads with the absence of acrosomes, perinuclear theca deformities, and dispersed chromatin. Microtubular arrangement of centrosome and flagellum was conserved. NGS identified deletions (DPY19L, PICK1, SPATA16) directly responsible for the globozoospermic phenotype. Based on the findings, assisted oocyte activation was performed for 7 couples with an oocyte-activating agent, boosting fertilization from 5.7% to 40.2% (p<0.00001), resulting in 5 pregnancies and 4 deliveries. In patients with DFS (n=6), flagellar deformities were observed in 90–100% of spermatozoa with mitochondria disorganization, disarranged microtubules, and cytoplasmic remnant enveloping coiled flagella, indicating incomplete late spermiogenesis. Normal acrosomes and nuclei were seen. NGS identified deletions (AKAP4, SPAG16, CATSPER1) involved in flagellar development and function. For patients with proximal centriole defects (n=2), the sperm nuclei and fibrous sheath appeared normal. Approximately 90% of proximal centrioles exhibited microtubular disorganization, confirmed by an ODF2 mutation identified by NGS. For a patient with PCD, chaotic flagellar microtubular arrangement and the absence of outer dynein arms prevailed in 90% of the axonemal cross-sections examined, confirmed by a DNAH5 gene deletion. Nonetheless, a few motile spermatozoa were identified and used for ICSI, yielding 80% fertilization and the delivery of a healthy twin. Concurrent ultrastructural and genomic analysis can pinpoint sperm organelle disruption and lead to successful ART cycles.

Genotoxic activity of betel nut on germinal cell in Sarcoma 180 ascites tumour bearing male mice

The genotoxicity of the ethanolic extract of betel nut was evaluated using sarcoma 180 tumour bearing mouse considering sperm motility, sperm viability, biochemical estimation of fructose in seminal fluid and sperm head morphology assays. Sperm head morphology was studied by H-E staining and Toluidine blue staining method. But Toluidine blue staining method is a reliable method to evaluate the DNA damage of sperms. Ethanolic BNE (betel nut extract) can suppress the percentage of sperm motility, sperm viability and seminal fructose level. In addition, it can also enhance the percentage of DNA damaged sperms. Moreover, histological sections of testes have been studied in control and BNE treated sarcoma 180 tumour bearing mice to highlight the potential toxic effect of BNE. The significant decreasing rate of seminal fructose concentration, sperm motility as well as viability and increasing rate of sperm head abnormality in different doses of treated series may be as a result of different toxic alkaloid ingredients present in BNE. Therefore, the results showed the potential of the BNE to induce different types of germ cell abnormalities in tumour bearing male mice.

Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in male infertility treatment. Currently, sperm selection for ICSI in human assisted reproductive technology (ART) is subjective, based on a visual assessment by the operator. Therefore, it is desirable to develop methods that can objectively provide an accurate assessment of the shape and size of sperm heads that use low-magnification microscopy available in most standard fertility clinics. Recent studies have shown a correlation between sperm head size and shape and chromosomal abnormalities, and fertilization rate, and various attempts have been made to establish automated computer-based measurement of the sperm head itself. For example, a dictionary-learning technique and a deep-learning-based method have both been developed. Recently, an automatic algorithm was reported that detects sperm head malformations in real time for selection of the best sperm for ICSI. These data suggest that a real-time sperm selection system for use in ICSI is necessary. Moreover, these systems should incorporate inverted microscopes (×400-600 magnification) but not the fluorescence microscopy techniques often used for a dictionary-learning technique and a deep-learning-based method. These advances are expected to improve future success rates of ARTs. In this review, we summarize recent reports on the assessment of sperm head shape, size, and acrosome status in relation to fertility, and propose further improvements that can be made to the ARTs used in infertility treatments.

Intraspecific variation in testis organisation and sperm head morphology of the delicate mouse (

The delicate mouse (Pseudomys delicatulus) ranges from Queensland to northern Western Australia. In this study the morphology of the cellular organisation of the testis and cauda epididymal spermatozoa are compared. Individuals from the mainland of the Northern Territory and Western Australia invariably have only 60–70% of the testes composed of seminiferous tubules with the interstitial tissue between the tubules containing abundant lipid rich Leydig cells, whereas the cauda epididymal sperm have highly polymorphic, often pear-shaped, heads and basally attached tails. In individuals from Queensland the seminiferous tubules make up approximately 80% of the testis, whereas in the cauda epididymides the sperm populations are generally less variable and have bilaterally flattened heads with the tail attached to the lower concave surface. These differences in the morphology of the delicate mouse testis and spermatozoa in these two geographic regions suggest differences in intensity of intermale sperm competition, with individuals from northern Western Australia and the mainland of the Northern Territory exhibiting monogamy whereas those from Queensland may exhibit some degree of intermale sperm competition and hence possibly have a polyandrous or promiscuous mating system. These findings support the suggestion that P. delicatulus, as currently recognised, contains at least one cryptic species.

Fertilization modes drive the evolution of sperm traits in Baikal sculpins

AbstractBaikal sculpins are a unique group of fishes, including both externally and internally fertilizing species. Sperm traits, such as morphology and motility, are predicted to undergo adaptive changes due to species fertilization modes. However, studies exploring such traits in Baikal sculpins are lacking. Here, we conducted a field study and investigated sperm traits of three sculpins with external fertilization (Cottocomephorus grewingkii, Leocottus kesslerii and Paracottus knerii) and one sculpin with internal fertilization (Comephorus dybowskii). Sperm head morphology was spherical in both types of fertilizing species; however, the head cross‐sectional area of the sperm of the internal fertilizer was smaller than that of external fertilizers. This result implies that sperm head morphology might be adapted to swim in a viscous ovarian fluid. The internal fertilizer, C. dybowskii, had longer and faster‐swimming sperms with longer motility duration than the external fertilizers, perhaps due to different sperm competition levels. Sperms of the internal fertilizer were only motile in isotonic solutions, whereas sperms of the external fertilizers were motile in lake waters, suggesting that sperm motilities are specialized according to their external/internal environments. To our knowledge, this is the first study to demonstrate sperm traits of Baikal sculpins, which may have evolved adaptively along with changing fertilization modes.

CFAP43-mediated intra-manchette transport is required for sperm head shaping and flagella formation.

Mutation in CFAP43 leads to severe asthenozoospermia and multiple morphological abnormalities of the sperm flagellum (MMAF) in both human and mouse. Previous studies have shown that disruption of intra-manchette transport (IMT) caused failure of flagellum assembly and sperm head shaping. In a previous study, therefore, we postulated that disruption of IMT may contribute to the failure of sperm flagellum formation and result in MMAF, however the mechanisms underlying these defects are still poorly understood. Cfap43-deficient mice were studied here to reveal the cellular mechanisms of abnormal sperm head morphology and MMAF. Depletion of Cfap43 led to abnormal spermiogenesis and caused MMAF, sperm head abnormality and oligozoospermia. Furthermore, both abnormal manchette and disorganized ectoplasmic specialization (ES) could be observed at the elongated spermatids in Cfap43-deficient mice. Therefore, our findings demonstrated that, in mice, CFAP43-mediated IMT is essential for sperm head shaping and sperm flagellum formation.

Zinc deficient diet increases the toxicity of bisphenol A in rat testis.

Zinc (Zn) plays an important role in maintaining the process of spermatogenesis and reproductive health. Bisphenol A (BPA), an endocrine disrupting chemical is known to be a reproductive toxicant in different animal models. The present study was designed to study the effect of two of the utmost determinative factors (Zn deficient condition and influence of toxicant BPA) on germ cell growth and overall male reproductive health in the testis, epididymis, and sperm using (a) biochemical, (b) antioxidant, (c) cellular damage, (d) apoptosis, and (e) protein expression measurements. Rats were divided into Control (normal feed and water), BPA (100 mg/kg/d), zinc deficient diet (ZDD; fed with ZDD), and BPA + ZDD for 8 weeks. Body and organ weights, sperm motility and counts, and sperm head morphology were evaluated. The histology of testes, epididymides, and prostate wasinvestigated. Testicular deoxyribonucleic acid (DNA) damage was evaluated by Halo and Comet assay, apoptosis of sperm and testes were quantified by TUNEL assay. Serum protein electrophoretic patterns and testicular protein expressions such as Nrf-2, catalase, PCNA, and Keap1 were analyzed by Western blot analysis. The results showed that BPA significantly increased the testicular, epididymal, and prostrate toxicity in dietary Zn deficient condition due to testicular hypozincemia, hypogonadism, increased cellular and DNA damage, apoptosis, as well as perturbations in protein expression.

Acute reproductive toxicology after intratesticular injection of silver nanoparticles (AgNPs) in Wistar rats

This study aimed to evaluate the effects of an intratesticular injection of silver nanoparticles (AgNPs) on reproductive parameters and health of rats, and to evaluate the AgNPs biodistribution in order to develop a nanotechnological contraceptive agent for male animals. Treated animals received 220 μL of AgNPs solution (0.46 µg-Ag/ml) in each testicle and were euthanized: seven, 14, 28, and 56 days after injection. A significant decrease (p < 0.05) in the percentage of motile sperm in D7 (8.8%) was observed, comparing to the control (73.3%), D14 (86.0%), D28 (68.2%), and D56 (90.0%) groups. D7 group also presented a decrease (p < 0.05) in the percentage of normal spermatozoa. Additionally, D7 group showed an increase (p < 0.05) in abnormal midpiece and sperm head morphology compared to the Control group. Seminiferous tubules presented all germline cell types and spermatozoa for all groups. However, D7 group did not present spermatozoa in the epididymis, whereas some spermatozoa and cellular debris were visible in D14 and D28 groups. All animals presented hematological parameters, creatinine, and alanine aminotransferase values within the normal limits for Wistar rats. The percentage of silver found in the liver was always higher than in the other organs analyzed. A pioneering mathematical model is proposed, from which the half-life time of silver in the liver (17 days), spleen (23 days), lungs (30 days), and kidneys (35 days) was extracted. In conclusion, some acute and severe toxic effects were observed in sperm cells following intratesticular injection of AgNPs, although these effects were reversible. No adverse effects to general animal health were observed.

Automated sperm morphology analysis approach using a directional masking technique

Sperm Morphology is the key step in the assessment of sperm quality. Due to the effect of misleading human factors in manual assessments, computer-based techniques should be employed in the analysis. In this study, a computation framework including multi-stage cascade connected preprocessing techniques, region based descriptor features, and non-linear kernel SVM based learning is proposed for the classification of any stained sperm images for the assessment of the morphology. The proposed framework was evaluated on two sperm morphology datasets: the Human Sperm Head Morphology dataset (HuSHeM) and Sperm Morphology Image Data Set (SMIDS). The results indicate that cascading the preprocessing techniques used in the proposed framework, such as wavelet based local adaptive de-noising, modified overlapping group shrinkage, image gradient, and automatic directional masking, increased the classification accuracy by 10% and 5% for the HuSHeM and SMIDS, respectively. The proposed framework results in better overall accuracy than most state-of-the-art methods, while having significant advantages, such as eliminating the exhaustive manual orientation and cropping operations of the competitors with reasonable rates of consumption of time and source.

Deep Learning-Based Morphological Classification of Human Sperm Heads.

Human infertility is considered as a serious disease of the reproductive system that affects more than 10% of couples across the globe and over 30% of the reported cases are related to men. The crucial step in the assessment of male infertility and subfertility is semen analysis that strongly depends on the sperm head morphology, i.e., the shape and size of the head of a spermatozoon. However, in medical diagnosis, the morphology of the sperm head is determined manually, and heavily depends on the expertise of the clinician. Moreover, this assessment as well as the morphological classification of human sperm heads are laborious and non-repeatable, and there is also a high degree of inter and intra-laboratory variability in the results. In order to overcome these problems, we propose a specialized convolutional neural network (CNN) architecture to accurately classify human sperm heads based on sperm images. It is carefully designed with several layers, and multiple filter sizes, but fewer filters and parameters to improve efficiency and effectiveness. It is demonstrated that our proposed architecture outperforms state-of-the-art methods, exhibiting 88% recall on the SCIAN dataset in the total agreement setting and 95% recall on the HuSHeM dataset for the classification of human sperm heads. Our proposed method shows the potential of deep learning to surpass embryologists in terms of reliability, throughput, and accuracy.

Effect of different concentrations of resveratrol on the quality and in vitro fertilizing ability of ram semen stored at 5 °C for up to 168 h

The aim of the study was to investigate the effects of different concentrations of resveratrol on head morphology, motility characteristics, oxidative state and in vitro fertility of cooled ram spermatozoa. Pooled semen from three Najdi rams was diluted with Triladyl® having different concentrations of resveratrol, zero (control), 200 μM (45.65 μg/mL) and 400 μM (91.30 μg/mL) resveratrol, then stored at 5 °C for 168 h. The head morphometric, sperm kinematic parameters, Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and in vitro fertilizing capability of ram spermatozoa were evaluated after 24, 72, 120 and 168 h of cooling storage. The total motility (TM) of the sperm with resveratrol at 200 μM and 400 μM was significantly (p ≤ 0.05) higher than that in the control group at 72 and 120 h of cooling storage. On the other hand, the progressive motility (PM) of the sperm with resveratrol at 200 μM and 400 μM was significantly (p ≤ 0.05) higher than that in the control group at 168 h of cooling storage period. After 168 h of cooling storage, significantly higher straightness (STR) was observed in 400 μM group than two other groups and in 200 μM group than the control group. Both resveratrol groups had higher linearity (LIN) than control one at 120 and 168 h of cooling storage. The length, width and area of sperm head were lower (P ≤ 0.05) in the control compared to the other treatment groups after 120 and 168 h of storage. There was a significant increase in superoxide dismutase (SOD) concentration in the two resveratrol groups compared with the control one over the seven days of cooling storage and the same result was found in the reduced glutathione (GSH) concentration at 24, 72, and 168 h of storage. There was a significant (p ≤ 0.05) decrease in malondialdehyde (MDA) concentration in the 400 μM resveratrol group than that in two other groups over the seven days of storage period. Cleavage and blastocyst rates following in vitro fertilization were significantly higher (p ≤ 0.05) in 400 μM resveratrol than other groups at 72 h for cooling storage period. In conclusion, addition of resveratrol in the extender can protect sperm head morphology, improve kinematic parameters and in vitro fertility, and reduce oxidative stress of ram spermatozoa during liquid storage at 5 °C.