Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

The expression spectrum of yak epididymal epithelial cells reveals the functional diversity of caput, corpus and cauda regions.

Sperm undergo a series of changes in the epididymis region before acquiring the ability to move and fertilize, and the identification of genes expressed in a region-specific manner in the epididymis provides a valuable insight into functional differences between regions. We collected epididymal tissue from three yaks and cultured epithelial cells from the caput, corpus and cauda regions of the yak epididymis using the tissue block method. RNA sequencing analysis (RNA-seq) technology was used to detect gene expression in yak epididymal caput, corpus and cauda epithelial cells. The results showed that the DEGs were highest in the caput vs. corpus comparison, and lowest in the corpus vs. cauda comparison. Six DEGs were verified by real-time fluorescence quantitative PCR (qRT-PCR), consistent with transcriptome sequencing results. The significantly enriched DNA replication pathway in the caput vs. corpus was coordinated with cell proliferation, while upregulated DEGs such as POLD1 and MCM4 were found in the DNA replication pathway. The AMPK signaling pathway was found significantly enriched in the caput vs cauda, suggesting its involvement in sperm maturation and capacitation. The TGF beta signaling pathway was screened in the corpus vs cauda and is crucial for mammalian reproductive regulation. Upregulated DEGs (TGFB3, INHBA, INHBB) are involved in the TGF beta signaling pathway. This study provides a reference for culturing yak epididymal epithelial cells in vitro, and elucidates the transcriptional profiles of epithelial cells in different segments of the epididymis, revealing the regulatory and functional differences between different segments, providing basic data for exploring the molecular mechanism of yak sperm maturation and improving the reproductive capacity of high-altitude mammals.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Association between embryo morphokinetic development and intracytoplasmic sperm injection with epididymal sperm via time-lapse imaging.

The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Lower developmental potential of rat zygotes produced by ooplasmic injection of testicular spermatozoa versus cauda epididymal spermatozoa.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

Erratum: Lower developmental potential of rat zygotes produced by ooplasmic injection of testicular spermatozoa versus cauda epididymal spermatozoa.

J Reprod Dev, Vol. 70, No. 4, p. 255 Table 2 have been corrected. For the bottom of Survived (%) row, the Survived value, which read "69", should be replaced with "86".

Stability of the cytosine methylome during post-testicular sperm maturation in mouse.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

A simple method for repeated in vivo sperm collection from laboratory mice.

PURPOSE: Mouse spermatozoa for archiving laboratory mice or for in vitro fertilization (IVF) are routinely obtained from the cauda epididymis of adult males sacrificed for this purpose. To avoid the death of the donor, we tested whether a precisely timed interruption of the mating act could be used for repeated sperm collection from laboratory mice. METHODS: Sperm donors (B6D2F1) were mated with a receptive female, and mating behavior was observed. The stud was separated from the female 1-2 s after the onset of the ejaculatory shudder. The ejected copulatory plug with the yellowish viscous ejaculate was carefully removed from the penile cup. RESULTS: A total of 80 ejaculates were successfully obtained from 100 ejaculations. The latency to first mount was 1.1 ± 1.1 min (mean ± SD) and to ejaculation 8.1 ± 4.7 min. The average number of mounts to ejaculation was 10.5 ± 5.8, and the mean number of spermatozoa per collected ejaculate was 1.86 ± 1.05 × 106. An average fertilization rate of 76% was observed after IVF. CONCLUSIONS: Separating the stud from the female just before ejaculation is feasible, easy to learn, and requires no special equipment. The sperm count of collected ejaculates is lower than natural ejaculations, but higher than previous in vivo sperm collection methods achieved. We recommend this simple sperm collection method in mice, especially when the donor cannot be sacrificed and/or repeated sperm collection from the same animal is required for experimental purposes.

Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia.

Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

Polarized cellular mechano-response system for maintaining radial size in developing epithelial tubes.

Size control in biological tissues involves multicellular communication via mechanical forces during development. Although fundamental cellular behaviours in response to mechanical stimuli underlie size maintenance during morphogenetic processes, the mechanisms underpinning the cellular mechano-response system that maintains size along an axis of a polarized tissue remain elusive. Here, we show how the diameter of an epithelial tube is maintained during murine epididymal development by combining quantitative imaging, mechanical perturbation and mathematical modelling. We found that epithelial cells counteract compressive forces caused by cell division exclusively along the circumferential axis of the tube to produce polarized contractile forces, eventually leading to an oriented cell rearrangement. Moreover, a mathematical model that includes the polarized mechano-responsive regime explains how the diameter of proliferating tubes is maintained. Our findings pave the way for an improved understanding of the cellular response to mechanical forces that involves collective multicellular behaviours for organizing diverse tissue morphologies.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

Novel role of proton-secreting epithelial cells in sperm maturation and mucosal immunity.

Epithelial cells are immune sensors and mediators that constitute the first line of defense against infections. Using the epididymis, a model for studying tubular organs, we uncovered a novel and unexpected role for professional proton-secreting 'clear cells' in sperm maturation and immune defense. The epididymal epithelium participates in the maturation of spermatozoa via the establishment of an acidic milieu and transfer of proteins to sperm cells, a poorly characterized process. We show that proton-secreting clear cells express mRNA transcripts and proteins that are acquired by maturing sperm, and that they establish close interactions with luminal spermatozoa via newly described 'nanotubes'. Mechanistic studies show that injection of bacterial antigens in vivo induces chemokine expression in clear cells, followed by macrophage recruitment into the organ. Injection of an inflammatory intermediate mediator (IFN-γ) increased Cxcl10 expression in clear cells, revealing their participation as sensors and mediators of inflammation. The functional diversity adopted by clear cells might represent a generalized phenomenon by which similar epithelial cells decode signals, communicate with neighbors and mediate mucosal immunity, depending on their precise location within an organ.

Testosterone regulation on quiescin sulfhydryl oxidase 2 synthesis in the epididymis.

The epididymis is an androgen-responsive organ, whose structure and functions are modulated by the coordination between androgen and epididymal cues. Highly regulated molecular interaction within the epididymis is required to support viable sperm development necessary for subsequent fertilization. In the present study, we extended our earlier findings on a promising epididymal protein, quiescin sulfhydryl oxidase 2 (QSOX2), and demonstrated a positive correlation between testosterone and QSOX2 protein synthesis through the use of loss- and restore-of-function animal models. Moreover, based on transcriptomic analyses and 2D culture system, we determined that an additional polarized effect of glutamate is indispensable for the regulatory action of testosterone on QSOX2 synthesis. In conclusion, we propose noncanonical testosterone signaling supports epididymal QSOX2 protein synthesis, providing a novel perspective on the regulation of sperm maturation within the epididymis.

Postnatal cadmium administration affects the presence and distribution of carbohydrates in the sperm membrane during maturation in the epididymis in adult Wistar rats.

Cadmium (Cd) is a heavy metal related to a decrease in sperm parameters. The transit of spermatozoa through the epididymis is necessary to generate changes in the sperm membrane, such as the assembly of various carbohydrates that are added to the spermatazoan's surface to prepare it for successful fertilisation of the oocyte. No studies have yet analysed whether Cd alters the presence and distribution of these carbohydrates. We aimed to evaluate the changes induced by Cd in the distribution pattern of N-acetylglucosamine, sialic acid, mannose and fucose on the sperm membrane in the epididymis (e.g. caput, corpus, cauda) and if it alters the epididymal epithelium. Male Wistar pups were treated with Cd doses (0.125, 0.25 and 0.5mg/kg) on postnatal days 1-49. At postnatal day 90, they were humanely killed, sperm samples were obtained from the epididymis and tissue samples were taken for histological analysis. Cd concentrations in the blood and epididymis increased in proportion to the dose administered and decreased the serum testosterone levels and sperm quality. Histological analysis revealed alterations in the epithelium in all Cd-treated groups. Cd altered the distribution patterns of carbohydrates and fluorescence indices. All these alterations affected the structure and functioning of sperm.

Susceptibility of epididymal sperm against reactive oxygen species in dogs.

In order to add information to physiology of sperm maturation and help to underline future antioxidant supplementation treatment to epididymal sperm, the aim of this study was to evaluate susceptibility of caput, corpus and cauda epididymal sperm to different reactive oxygen species (ROS) in dogs. Epididymal sperm was separately collected from each segment (caput, corpus and cauda) and subjected to 4 different ROS-challenges: superoxide anion, hydrogen peroxide, hydroxyl radical, malondialdehyde (MDA) or maintained as control. After 30 min of incubation in each ROS, sperm was evaluated for sperm kinetics, plasma and acrossomal membrane integrity, mitochondrial activity and resistance to oxidative stress. Decreased total and progressive sperm motility and rapid velocity at epididymal corpus occurred after exposure to hydrogen peroxide, hydroxyl radical and MDA. However, for cauda epididymis, hydrogen peroxide and malondialdehyde promoted higher deleterious effect regarding sperm motility and velocity. Only at cauda epididymis MDA decreased sperm mitochondrial activity index and no kinetics alterations (motility or velocity) occurred after exposure to superoxide anion in corpus and cauda epididymis. In conclusion, corpus and cauda epididymal sperm are highly susceptible to deleterious effect of hydrogen peroxide, malondialdehyde and hydroxyl radical. In addition, epididymal canine sperm is relatively resistant to superoxide anion damage.

Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm.

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece-principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1-/- mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Evaluation of the Freezability of the Bovine Epididymis Tail Sperm with the Addition of Antioxidants.

BACKGROUND: The cryopreservation and recovery of epididymis tail sperm is an important biotechnology dependent on the composition of the freezing medium. OBJETIVE: To evaluate the effect of melatonin, added to commercial freezing medium extender, on the kinetics and viability of bovine epididymis tail sperm. MATERIAL AND METHODS: Five routines were performed, each consisting of eight epididymis and the structures were sliced onto a glass plate containing a commercial diluting medium for Botubov. The samples were divided into four groups, with 80 x 106 spermatozoa per mL. Group 1: samples diluted in Botubov. Group 2: samples centrifuged (600 g, 10 min), and the pellet re-suspended in Botubov. Group 3, samples diluted in Botubov containing 100 pM melatonin. Group 4: samples centrifuged (600 g, 10 min) and the pellet resuspended in Botubov with 100 pM melatonin. The samples were transferred to 0.5 mL straws at 40 x 106 viable spermatozoa, stabilized at 5º C for 4 h, transferred to liquid nitrogen vapour for 20 min, dipped in liquid nitrogen and stored in a cryogenic cylinder. After thawing (46ºC, 15s), sperm kinetics and viability parameters were evaluated. RESULTS: There was no difference in the parameters of total motility (MT, %), progressive motility (MP, %), progressive linear velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), linearity (LIN, %), spermatozoa with rapid movement (RAP, %) and level of intact plasma membranes and acrosome (IPMA, %) among the groups studied. However, a difference was observed between the routines performed. CONCLUSION: The protocol for freezing bovine epididymis tail sperm is applicable; however, there is an influence of the epididymis used, for the best efficacy of this biotechnology.