Search

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high-efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen-thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen-thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen-thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen-thawed boar sperm., (© 2018 Japanese Society of Animal Science.)

Sperm cryopreservation in goats has been a challenge for many years due to the detrimental effects of seminal plasma enzymes produced by the bulbo-urethral glands which catalyse the hydrolysis of lecithins in egg yolk to fatty acids and lysolecithins which are deleterious to spermatozoa. This fact implies to carry out additional processing steps during sperm cryopreservation for seminal plasma removal triggering different sperm responses which may affect sperm functionality. The objective of the present study was to determine specific sperm subpopulation responses in different handling steps during the cryopreservation process by using functional sperm kinematic descriptors in caprine ejaculates. Buck ejaculates (n = 40) were analysed for sperm concentration, viability, morphology and acrosome integrity. Moreover, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system after five different handling steps (fresh sperm, 1st washing, 2nd washing, cooling and frozen-thawed sperm) during a standard cryopreservation protocol for goat semen. The results were analysed using Principal Component Analysis (PCA) and multivariate clustering procedures to establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st and 4th steps, four sperm kinematic subpopulations were observed explaining more than 75% of the variance. Based on velocity and linearity parameters and the subpopulations disclosed, the kinematic response varies among processing steps modifying sperm movement trajectories in a subpopulation-specific and handling step-dependent manner (p < 0.001). The predominant motile subpopulation in freshly ejaculated buck sperm had very fast velocity characteristics and a non-linear trajectory (41.1%). Washing buck sperm twice altered the subpopulation structure as well as cooling which resulted in a dramatic reduction in sperm velocities (p < 0.01). Frozen-thawed spermatozoa showed similar characteristics to cooled sperm except there was a further increase in linearity with a large proportion of sperm attributed to new slow, linear cluster (32.5%). In conclusion, this study confirms the variability and heterogeneity of goat sperm kinematic patterns throughout the cryopreservation process and suggests that the predominant motility pattern (assayed in vitro via CASA) of high quality spermatozoa might be typified by high speed and a non-linear trajectory. The relationships among the number and distribution of sperm subpopulations and the different handling steps were particularlly relevant, specially after the cooling and the post-thawing steps, when effects derived from these critical handling steps were evident and altered drastically the sperm motion patterns., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Study Question: Could a more detailed evaluation of marmoset spermatogonial morphology, kinetics and niches using high-resolution light microscopy (HRLM) lead to new findings?, Summary Answer: Three subtypes of marmoset undifferentiated spermatogonia, which were not evenly distributed in terms of number and position along the basal membrane, and an extra premeiotic cell division not present in humans were identified using HRLM., What Is Known Already: The seminiferous epithelium cycle (SEC) of marmosets is divided into nine stages when based on the acrosome system, and several spermatogenic stages can usually be recognized within the same tubular cross-section. Three spermatogonial generations have been previously described in marmosets: types Adark, Apale and B spermatogonia., Study Design, Size, Duration: Testes from five adult Callithrix penicillata were fixed by glutaraldehyde perfusion via the cardiac route and embedded in Araldite plastic resin for HRLM evaluation. Semi-thin sections (1 μm) were analyzed morphologically and morphometrically to evaluate spermatogonial morphology and kinetics (number, mitosis and apoptosis), spermatogenesis efficiency and the spermatogonial niche., Participants/materials, Setting, Methods: Shape and nuclear diameter, the presence and distribution of heterochromatin, the granularity of the euchromatin, as well as the number, morphology and degree of nucleolar compaction were observed for morphological characterization. Kinetics analyses were performed for all spermatogonial subtypes and preleptotene spermatocytes, and their mitosis and apoptosis indexes determined across all SEC stages. Spermatogenesis parameters (mitotic, meiotic, Sertoli cell workload and general spermatogenesis efficiency) were determined through the counting of Adark and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and Sertoli cells at stage IV of the SEC., Main Results and the Role of Chance: This is the first time that a study in marmosets demonstrates: the existence of a new spermatogonial generation (B2); the presence of two subtypes of Adark spermatogonia with (AdVac) and without (AdNoVac) nuclear rarefaction zones; the peculiar behavior of AdVac spermatogonia across the stages of the SEC, suggesting that they are quiescent stem spermatogonia; and that AdVac spermatogonia are located close to areas in which blood vessels, Leydig cells and macrophages are concentrated, suggesting a niche area for these cells., Large Scale Data: Not applicable., Limitations, Reasons for Caution: The C. penicillata spermatogonial kinetics evaluated here consider spermatogonial number across the SEC and their mitotic and apoptotic figures identified in HRLM sections. Therefore, caution is required when comparing absolute values between species. Although morphometric evaluation has suggested that AdVac spermatogonia are stem cells, a functional proof of this is still missing. It is known that parameters of the spermatogenic process in C. penicillata have similarities with those of the common marmoset C. jacchus, however, a detailed study of spermatogonial morphology, kinetics and niche has not yet been performed in C. jacchus, and a full comparison of the two species is not possible., Wider Implications of the Findings: Our findings in C. penicillata contribute to a better understanding of the spermatogonial behavior and spermatogenesis efficiency in non-human primates. Given the phylogenetic closeness of the marmoset to the human species, similar processes might occur in humans. Therefore, marmosets may be an excellent model for studies regarding human testicular biology, fertility and related disorders., Study Funding/competing Interest(s): Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest.

Purpose: The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model., Methods: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods., Results: Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption., Conclusions: These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris-glucose-egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm., (© 2018 Blackwell Verlag GmbH.)

Study Question: What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility?, Summary Answer: While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function., What Is Known Already: CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4., Study Design, Size, Duration: In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization., Participants/materials, Setting, Methods: Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype., Main Results and the Role of Chance: While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis., Limitations Reasons for Caution: Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated., Wider Implications of the Findings: The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility., Large Scale Data: Not applicable., Study Funding and Competing Interest(s): This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

Purpose: Spermatozoa undergo critical changes in structure and function during the epididymal transit. Our previous studies in the domestic cat demonstrated that incidence of cenexin-a key protein involved in the centrosomal maturation-progressively increases in sperm cells from caput to cauda epididymidis. The objectives of the study were to (1) characterize mechanisms involved in transferring key factors-using the cenexin as a marker-between the epididymis and maturing sperm cells and (2) demonstrate the impact of such mechanisms on the acquisition of functional properties by spermatozoa., Methods: Epididymides were dissected from adult cat testes to assess the presence and localization of cenexin in testicular tissues and each epididymal segment (caput, corpus, and cauda) via immunofluorescence, Western blot, and mass spectrometry., Results: Results showed that tissues, luminal fluid, and isolated epididymosomes from each segment contained cenexin. Co-incubation of immature sperm cells for 3 h with luminal fluid or epididymosomes followed by immunostaining revealed that percentages of sperm cells containing cenexin significantly increased in samples co-incubated with epididymosome suspensions. Additionally, epididymosome co-incubation with immature spermatozoa resulted in sustained motility compared to untreated spermatozoa while there was no significant effect on acrosome integrity., Conclusions: Taken together, these results suggest that epididymosomes play a critical role in epididymal sperm maturation and could be ideal vehicles to assist in the enhancement or suppression of male fertility.

Male infertility is a major and growing problem and, in most cases, the specific root cause is unknown. Here we show that the transcription factor SOX30 plays a critical role in mouse spermatogenesis. Sox30-null mice are healthy and females are fertile, but males are sterile. In the absence of Sox30 meiosis initiates normally in both sexes but, in males, germ cell development arrests during the post-meiotic round spermatid period. In the mutant testis, acrosome and axoneme development are aberrant, multinucleated germ cells (symplasts) form and round spermatids unable to process beyond step 3 of spermiogenesis. No elongated spermatids nor spermatozoa are produced. Thus, Sox30 represents a rare example of a gene for which loss of function results in a complete arrest of spermatogenesis at the onset of spermiogenesis. Our results suggest that SOX30 mutations may underlie some instances of unexplained non-obstructive azoospermia in humans.

The SpermVital ® technology comprises embedding of spermatozoa within an alginate gel to facilitate release of sperm cells over a prolonged period in utero after AI. The aim of this study was to examine whether the survival time of spermatozoa is extended when applying this immobilization technology in combination with cryopreservation. Sperm cell survival (acrosome and plasma membrane integrity) was studied in vitro for 48 hr at physiological temperature. One dose of SpermVital ® (SV) semen was compared with single doses of Biladyl ® (B) processed semen as well as double doses of B (B double). B double was obtained by adding a second B dose the following day, thereby mimicking double AI. Furthermore, reproductive performance applying single early timed AI (TAI) with SV following oestrus synchronization was studied in a field trial. Double insemination (TAI on two consecutive days) with B semen served as control. Number of acrosome-intact live sperm cells decreased over time in vitro for all treatments (p < .05). There was no difference between SV sperm cell survival and B double after 24 hr (p > .05). However, after 48 hr, SV sperm cell survival was higher than B double (p < .05). Moreover, multivariate analysis showed that the outcome of single early TAI with SV was not significantly different from B double (p > .05). Likelihood of pregnancy and calving in the heifer group was higher than in the cow group (p < .05). These results imply that spermatozoa immobilized in alginate gel have prolonged survival., (© 2017 Blackwell Verlag GmbH.)

A trial was conducted to check effect of adding gum Arabic (GA) instead of egg yolk (EY) as a cryoprotectant for stallion sperm. Two experiments were designed; experiment I tested adding 3 levels of nonheated GA (i.e., 3, 6 and 9 g/100 mL diluents) in HF-20 extender. However, in experiment II the same levels were tested except that GA was heated at 80 °C for 60 min. HF-20 containing 10% of EY was used as control. In experiment I, sperm frozen in HF-20 containing nonheated GA exhibited lower percentages of motile sperm, progressively motile sperm and sperm with intact plasma membranes, vitality rate, and acrosome integrity after cooling or after deep freezing. Frozen semen in HF-20 containing 3-6% of preheated GA in experiment II maintained sperm motility at 46-50% and elevated progressive motility at 27%. The semen diluted in preheated GA (6%) and frozen exhibited a fertility rate of 40% (2/5). A similar fertility rate (40%) was found in the control semen (i.e. 10%) compared to those that were inseminated with frozen semen in preheated 3% GA (20%, 1/5). These results suggest that preheated GA could be used as an alternative cryoprotectant for cryopreserving stallion sperm., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study examines the impact of taurine on the viability, morphology and acrosome integrity of rabbit spermatozoa in vitro. Semen samples, obtained from four to five sexually mature and healthy New Zealand White rabbits, were pooled in heterospermic semen sample. This was divided and treated with taurine in a concentration of 0 (control), 1.5, 7, 12.5, 50 mM to a final concentration of 10 8  spermatozoa/ml. The samples were then incubated at 37°C for 4 hr. A combination of fluorescent probes SYBR-14/propidium iodide/PNA-Alexa Fluor 647 was used to assess spermatozoa viability and acrosome integrity on a flow cytometer. The sperm morphology was evaluated under a light microscope following fixation in 1.5% paraformaldehyde. The experiment was repeated three times. According to the obtained results, the spermatozoa neither could have benefit from immediate taurine treatment, nor had they after 4-hr incubation with respect to viability and acrosome integrity. Taurine did not initially alter the total and acrosome morphology of treated spermatozoa nor has it by 4 hr upon treatment. In conclusion, taurine may have no protective effect on the viability, morphology and acrosome integrity of short-term stored rabbit spermatozoa., (© 2017 Blackwell Verlag GmbH.)

The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the 'spermatozoidal activator factor' (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN 2 ] for 10 min, plunging in LN 2 ; FR2, programmable medium, +4°C to -15°C at 3°C min -1 , from -15 to -80°C at 10°C min -1 and final holding for 1 min at -80°C, plunging in LN 2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to -20°C at 10°C min -1 , from -20°C to -100°C at 30°C min -1 , final holding for 1 min at -100°C and plunging in LN 2 ) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curved line velocity (μm s -1 ), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa., (© 2016 Blackwell Verlag GmbH.)

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen-thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen-thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post-thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post-thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI., (© 2017 Japanese Society of Animal Science.)

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

This study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein-Friesian ejaculates (n=10 bulls) were split across four treatments and processed: (1) NS fresh at 3×10 6 spermatozoa, (2) X-SS frozen at 2×10 6 spermatozoa, (3) X-SS fresh at 2×10 6 spermatozoa and (4) X-SS fresh at 1×10 6 spermatozoa. NS frozen controls of 20×10 6 spermatozoa per straw were sourced from previously frozen ejaculates (n=3 bulls). Experiment 2: Aberdeen Angus ejaculates (n=4 bulls) were split across four treatments and processed as: (1) NS fresh 3×10 6 spermatozoa, (2) Y-SS fresh at 1×10 6 spermatozoa, (3) Y-SS fresh at 2×10 6 spermatozoa and (4) X-SS fresh at 2×10 6 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0=day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P<0.001), NS frozen treatments had the greatest PLM (P<0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P<0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P<0.01).

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

In order to study the function of kinesin-14 motor protein KIFC1 during spermatogenesis of Procambarus clarkii, the full length of kifc1 was cloned from testes cDNA using Rapid-Amplification of cDNA Ends (RACE). The deduced KIFC1 protein sequence showed the highest similarity between Procambarus clarkii and Eriocheir senensis (similarity rate as 64%). According to the results of in situ hybridization (ISH), the kifc1 mRNA was gathered in the acrosome location above nucleus in the mid- and late-stage spermatids. Immunofluorescence results were partly consistent with the ISH in middle spermatids, while in the late spermatids the KIFC1 was distributed around the nucleus which had large deformation and formed four to six nuclear arms. In the mature sperm, KIFC1 and microtubules were distributed around the sperm, playing a role in maintaining the sperm morphology and normal function. Overexpression of P. clarkii kifc1 in GC1 cells for 24 hours resulted in disorganization of microtubules which changed the cell morphology from circular and spherical into fusiform. In addition, the overexpression also resulted in triple centrosomes during mitosis which eventually led to cell apoptosis. RNAi experiments showed that decreased KIFC1 protein levels resulted in total inhibition of spermatogenesis, with only mature sperm found in the RNAi-testis, implying an indispensable role of KIFC1 during P. clarkii spermiogenesis.

Reasons for Performing Study: The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially., Objectives: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation., Study Design: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares., Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 10 6 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted to 25 or 111 × 10 6 sperm/ml (-SP25 and -SP111). Sperm were stored for 18 h at 10 to 15°C and -SP25 and +SP25 treatments were centrifuged and resuspended to 111 × 10 6 sperm/ml. Sperm were incubated under H33342 staining conditions and motility, viability and acrosome integrity assessed. Semen was collected from stallions (n = 4), liquid stored at 10-15°C for up to 5 h and sperm either cryopreserved directly, sex-sorted and cryopreserved, or sex-sorted and returned to liquid storage until insemination. Low-dose hysteroscopic insemination was performed in 23 mares randomly allocated to the semen preparation group and pregnancy determined following embryo flushing on Day 9 after ovulation, or via transrectal ultrasonography on Day 14 after ovulation., Results: Skimmed milk was superior to I-BSA in maintaining motility, viability and acrosome integrity. Seminal plasma removal did not affect the parameters measured at the concentrations examined. Conception rates did not differ significantly between the groups, although a high incidence of pregnancy loss was observed in both the cryopreserved groups., Conclusions: While the conception rates achieved are among the highest yet reported for sex-sorted, cryopreserved stallion sperm, the high incidence of pregnancy loss suggests that the development of the resulting embryos was significantly impaired by the sperm processing treatments., (© 2016 EVJ Ltd.)

Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein. Among the tissues examined, CCDC62 was found to be expressed at the highest level in mouse testis by reverse transcriptase-PCR (RT-PCR) and Western blot analysis. With immunofluorescent staining, we demonstrated that CCDC62 was expressed in the cytoplasm and the developing acrosome in the spematids of mouse testis, and was specifically localized at the acrosome in mature sperm. The complementation analysis by mating repro29/+ mice with Ccdc62 -/- mice (generated by CRISPR-Cas9 strategy) further provided genetic proof that the infertility of repro29/repro29 mice was caused by Ccdc62 mutation. Finally, it was found that intracellular colocalization and interaction of CCDC62 and Golgi-associated PDZ and coiled-coil motif-containing protein may be important for acrosome formation. Taken together, this study identified a nonsense mutation in Ccdc62, which directly results in male infertility in repro29/repro29 mice., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 μM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 μM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 μM and Res 2 mM) significantly increases the total efficiency of fertilization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1 -/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis., (© 2017. Published by The Company of Biologists Ltd.)

Research into an optimal cryoprotectant, its concentration and equilibration time underlies the successful cryopreservation of dromedary camel spermatozoa. This study assessed the cryo-efficiency of different cryoprotectants, their concentration and equilibration time and any interactions. In experiment 1, semen samples (n = 4 males; 2 ejaculates/male) were frozen using Green Buffer containing one of four cryoprotectants (3% glycerol, ethylene glycol, methyl formamide, dimethyl sulfoxide) and using 4 equilibration times (10 min, 0.5, 1 and 2 h). Glycerol and ethylene glycol provided the best motility recovery rates and different equilibration times were not significant for any cryoprotectant nor were any interactions noted. However different equilibration times were pertinent for improved kinematic parameters BCF and VSL. In experiment 2, glycerol and ethylene glycol were evaluated at 4 concentrations (1.5, 3, 6, 9%) with 0.5 h equilibration (n = 4 males, 3 ejaculates/male). Sperm motility recoveries, kinematics and acrosome status were assessed. Higher values for LIN and STR were found with ethylene glycol. At 0 and 1 h post thaw 3 and 6% of either cryoprotectant resulted in better motility values than 1.5%. Acrosome integrity was compromised at 9% cryoprotectant. There were interactions between cryoprotectant and concentration in total motility at 0 and 1 h. For glycerol, total motility recoveries were best at 3-9%; for ethylene glycol 1.5-6% were best at 0 h and 3-6% at 1 h. In conclusion, 3-6% glycerol or ethylene glycol offered the best cryoprotection for camel sperm while different equilibration times were not critical., (Copyright © 2016. Published by Elsevier Inc.)

Several species of cervids are currently classified as threatened or endangered due to a rapid decline in their populations. Sperm cryopreservation, in association with assisted reproductive technologies, can find application for the conservation of endangered cervids. In cases of unsuccessful sperm retrieval through other means prior to the death of the animal, adult testis is the only source of sperm. Recovery of viable sperm from adult testes depends on the effective preservation of testicular tissues through optimization of cryopreservation protocols. The present study evaluated combinations of 10% dimethyl sulfoxide (DMSO) with 0% or 80% fetal bovine serum (FBS) and 20% DMSO with 0 or 20% FBS for the cryopreservation of testicular tissues of three adult cervids using uncontrolled slow freezing protocol. The cryopreserved testis was compared to chilled tissue without cryoprotectants. Results revealed that testicular tissues of barking deer cryopreserved in 20% DMSO (D20) had all the analyzed 7 parameters (number of TNP1-, PRM2 and acrosin-expressing cells/tubule and, the number of viable, morphologically normal, acrosome intact, Annexin V-negative sperm) comparable to the chilled testis. However, testicular tissues of sambhar and hog deer cryopreserved only in D20S20 had 5 of 7 parameters comparable to the chilled testis. In conclusion, D20 is acceptable for cryopreservation of barking deer and D20S20 for sambar and hog deer testes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ram sperm are subjected to extreme oxidative stress during their preservation at -196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4-5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10 6 spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze-thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05). In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The impact of thyroid hormone (TH) disorders on male reproductive biology has been a controversial issue for many years. Recently, we reported that hypothyroid male rats have a disruption of the seminiferous epithelium, which may compromise spermatogenesis. To improve the understanding of the reproductive pathogenesis of hypothyroidism and hyperthyroidism, male Wistar rats that developed these dysfunctions in adulthood were used as an experimental model. We evaluated the sperm production, reserves, transit time, morphology, and functionality (acrosome integrity, plasma membrane integrity, and mitochondrial activity), and the testicular expression of the TH receptors (Thra1 and Thra2, Thrb1, and Thrb2), deiodinases (Dio2 and Dio3), and the Mct8 transporter (Slc16a2) were assessed by reverse transcription followed by real-time quantitative PCR (RT-qPCR). The results were evaluated statistically by ANOVA and Tukey HSD test (P < 0.05). Hypothyroidism decreased the total and daily sperm productions and increased the sperm transit time through the epididymis, while the sperm functionality was reduced in both thyroid dysfunctions. Regarding the modulation of gene expression in the testis, hypothyroidism increased the expression of Thra1 and decreased the expression of Dio3, and hyperthyroidism increased the expression of Slc16a2. The observed alterations in spermatic production and function and in the expression of the TH receptor, deiodinase, and the TH transporter are suggestive of TH participation in spermatogenesis in adulthood.

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Of the 934 lizard species evaluated by the International Union for the Conservation of Nature (IUCN), at least one-third is threatened with extinction. However, there are no reports of semen cryopreservation efforts for lizards. Invasive Argentine black and white tegus were captured in the Florida Everglades, and sperm was collected postmortem. Initial motility score (IMS; % motile × speed of progression 2 × 100), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded. Sperm was diluted in TEST-yolk buffer with a final glycerol or dimethylsulfoxide (DMSO)concentration of 8%, 12%, or 16%, and frozen at 0.3 °C, 1.0 °C, or 6.3 °C/min. At thaw, all variables were expressed as the percentage of initial (%IMS, %IPL, and %IAC). The 0.3 °C freeze rate was more successful than 1.0 °C and 6.3 °C/min in preserving %IMS and %IPL. DMSO preserved %IMS, %IPL, and %IAC better than glycerol. To determine the best overall cryopreservation protocol, a sperm quality index was calculated, giving equal weight to each of the three indicators of cryosurvival. Because there were significant interactions between freeze rate and cryoprotectant concentration, each treatment was compared with all others. The sperm quality index analysis revealed that tegu sperm frozen at 0.3 °C/min with 12% DMSO exhibited the highest postthaw viability compared with all other treatments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effects of different concentrations of glutathione in Modena on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 5, 10, 15 mmol/L) of glutathione. Sperm motility, effective survival period, plasma membrane integrity, acrosome integrity, total antioxidant capacity (T-AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H 2 O 2 ) content were measured and analyzed. The results showed that Modena supplemented with 1, 5 and 10 mmol/L glutathione improved sperm motility, effective survival period, plasma membrane integrity and T-AOC, and decreased MDA content and H 2 O 2 content. Meanwhile, the semen sample diluted with Modena containing 1 mmol/L glutathione achieved optimum effect, and effective survival period was 6.1 days. After 5 days preservation, sperm motility, plasma membrane integrity and T-AOC of the group treated with 1 mmol/L glutathione were all higher than that of other groups. Meanwhile, MDA content and H 2 O 2 content were lower than that of other groups. In conclusion, Modena supplemented with glutathione decreased the oxidative stress and improved the quality of boar semen during liquid storage at 17°C, and 1 mmol/L concentration was the optimum concentration. © 2016 Japanese Society of Animal Science., (© 2016 Japanese Society of Animal Science.)

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

Identifying inducers of sperm acrosomal exocytosis (AE) to understand sperm functionality is important for both mechanistic and clinical studies in mammalian fertilization. Epifluorescence microscopy methods, while reproducible, are laborious and incompatible for high throughput screening. Flow cytometry methods are ideal for quantitative measurements on large numbers of samples, yet typically rely on the use of lectins that can interfere with physiologic AE-inducers. Here, we present an optimized triple stain flow cytometric method that is suitable for high-throughput screening of AE activation by glycopolymers. SYTO-17 and propidium iodide (PI) were used to differentiate cells based on their membrane integrity or viability, and membrane impermeable soybean trypsin inhibitor (SBTI) was used to monitor acrosome exocytosis. The SBTI/PI/SYTO-17 combination provides a positive screen for viability and AE of live sperm cells with minimal noise or false positives. A scattering gate enables the use of samples that may be contaminated with non-cellular aggregates, e.g., cryopreservation agents. This assay format enabled detailed analysis of glycopolymer dose response curves. We found that fucose polymer has a narrow effective dose range (EC50 = 1.6 μM; IC50 = 13.5 μM); whereas mannose polymer and β-N-acetylglucosamine polymer have broader effective dose ranges (EC50 = 1.2 μM and 3.4 μM, respectively). These results highlight the importance of testing inducers over a large concentration range in small increments for accurate comparison., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present work aimed to assess the effect of equilibration time on post-thaw motility parameters of canine sperm frozen in three extenders: 6% low-density lipoproteins (LDL), 6% liposomes (LIPO), and 40% egg yolk plasma (EYP). A second experiment is aimed at evaluating the functional integrity of canine spermatozoa frozen in the three extenders at the best equilibration time found in the experiment one. In the first experiment, 20 ejaculates harvested from 7 dogs, were frozen in three extenders (LDL, LIPO, and EYP) after four equilibration times (30min, 1h, 3h, and 6h). The semen was evaluated after thawing using an image analyser (HT-IVOS 14.0). The 6h equilibration time gave better results of motility and progressive motility in the three studied extenders. (LDL: 58.9% vs. 42.7%; LIPO: 54.4% vs. 31.9%; EYP: 55.4% vs 40.5% for motility 6 vs. 1h). In the second experiment, 10 ejaculates taken from 6 dogs were frozen under the same conditions as the previous experiment, after 6h equilibration time. The integrity parameters of the spermatozoal membrane (hypo-osmotic swelling test, and SYBR14/propidium Iodide staining), acrosome (FITC-Pisium sativum Aglutinin staining), and DNA (acridine orange staining) were evaluated at three different stages: post-dilution (T0), post-equilibration, and post-thawing. Post-thaw results were as follows: membrane integrity (HOSt: 62;6% vs 58% vs 64.4%; SYBR14/IP: 63.6% vs 57.9% vs 64.8%); acrosome integrity (FITC-PSA: 79.4% vs 74% vs 76.2%) and DNA integrity (Acridine-orange: 98.9% vs 98.5% vs 98.7%) respectively for LDL vs. LIPO vs. EYP. No significant difference existed between the extenders tested; thus 6%LIPO and 40%EYP could be good candidates for replacement of 6%LDL in the protection of canine sperm during the freeze-thaw process without altering motility and integrity parameters., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm(2) and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection., (© 2015 Blackwell Verlag GmbH.)

Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2(35-149) and huZP2(39-154) peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2(Rescue) zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2(Rescue) eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice., (Copyright © 2016, American Association for the Advancement of Science.)

We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

The aim of this study was to compare the in vitro and in vivo efficiency of different concentrations (0, 10 and 20 mM) of reduced glutathione supplemented to the extender for canine semen cryopreservation. Six normospermic dogs were used and each ejaculate was divided in 3 experimental groups, according to GSH concentration (GSH-0, GSH-10 and GSH-20 Groups). After thawing, samples were evaluated by sperm motility by computer-assisted sperm analysis (CASA), flow cytometric evaluation of plasma and acrosome membrane integrity, mitochondrial membrane potential and activity, chromatin susceptibility to acid-induced denaturation, and measurement of spontaneous and induced production of thiobarbituric acid reactive substances (TBARS). In vivo tests were carried out with GSH-0 and GSH-10 groups, for which six bitches were inseminated with semen cryopreserved in extender without GSH or containing 10 mM GSH. Intrauterine insemination was performed by cervical catheterization on the 5th and 6th days after the LH surge, detected by serum progesterone and LH assays. In the CASA evaluation, GSH-20 group had the lowest total and progressive motility and lower percentage of sperm with rapid and slow speed. Groups treated with glutathione showed lower percentage of acrosome damage, but higher percentage of plasma membrane injury. GSH-20 group had higher percentage of sperm with low mitochondrial activity and higher concentration of induced TBARS. Both groups (GSH-0 and GSH-10) had positive pregnancies. In conclusion, 20 mM GSH supplementation to canine cryopreservation extender promoted sperm damage, especially to mitochondrial activity. However, addition of 10 mM GSH resulted in acrosome protection, preserving fertility rate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Molecular biomarkers that can assess sperm acrosome status are very useful for evaluating sperm quality in the field of assisted reproductive technology. In this review, we introduce and discuss the localization and function of acrosomal proteins that have been well studied. Journal databases were searched using keywords, including "human acrosome", "localization", "fertilization-related protein", "acrosomal membrane", "acrosomal matrix", "acrosome reaction", "knockout mouse", and "acrosome marker".

Equine spermatozoa from the cauda epididymis were previously collected and frozen, and the fertility was assessed. Most studies were performed on healthy stallions that had undergone routine castration or on the epididymis collected at the abattoir, but there are no studies on the quality of epididymal semen in subjects which have died from colic or which underwent intensive care. The present study was designed to verify whether a severe illness could affect epididymal semen quality and freezability in the stallion. Therefore, epididymal semen characteristics during the freezing process in stallions which had died from colic and in healthy stallions submitted to elective castration were compared. Five stallions that had died from colic (ill stallions [ISs]) and seven stallions that had undergone elective castration (healthy stallions) were castrated, and cauda epididymis spermatozoa were collected and processed. Sperm quality was tested after collection, after washing procedures, at the end of the equilibration (5 °C for 75 minutes), and after freezing/thawing. Sperm quality was measured by objective motility characteristics, membrane and acrosome integrity, and mitochondrial activity. After collection, sperm in ISs showed low kinetic parameters (total motility: 17.3 ± 3%, progressive motility: 6 ± 1%, average path velocity: 57.4 ± 35.4 μm/s, straightness: 74.2%) compared with healthy stallions (total motility: 90.8 ± 3.7%, progressive motility: 70 ± 4%, average path velocity: 118.1 ± 12.6 μm/s, straightness: 82.4%) but demonstrated similar membrane and acrosome integrity (85 ± 2.8% vs. 87.6 ± 3.1%). Sperm kinetic parameters increased after washing procedures and cooling in ISs, reaching comparable values after equilibration (5 °C for 75 minutes) and freezing/thawing. The data reported in this study suggest that the quality of the equine epididymal spermatozoa cryopreserved in stallions that had died from colic was similar to that reported in epididymal sperm after elective castration and was also similar to the data reported in literature for cryopreserved equine semen., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Spermiogenesis is a complex process of differentiation and morphologic alteration, in which sperm acrosome formation is an important stage. Acrosome is an essential component of the sperm head, which develops in four distinct phases: Golgi, cap, acro- somal, and maturation, each supported by precise and orderly regulation of various genes. The regulatory genes which act on Golgi ap- paratus include GOPC, Hrb, SPATA16, PICK1, and CK2α', those involved in the cap phase are Fads2, syntaxin 2, Kdm3a, and UBR7, and participating in acrosomal and maturation phases are KIFC1, Rnf19a, and DPY19L2. The abnormalities of these genes may affect male fertility by influencing the connection of the nuclear dense lamina and acroplaxome with the nuclear membrane and then the fusion and transportation of vesicles. This review focuses on the genes involved in different phases of acrosome formation.

Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction.

Until recently, no special attention has been paid to the question of the site of mammalian sperm acrosome reaction (AR) in the female reproductive tract. Because AR is an essential process that enables the spermatozoon to fertilize, it is generally believed that it occurs at a specific step during sperm-egg interaction. It is generally thought that "the site of action coincides with the site of commitment." Thus, understanding the roles of AR and acrosomal substances is needed to gain insight into the site of the sperm commitment to undergo AR.

The aim of this study was to investigate whether there is a relationship of young boar testes size with semen traits and with productive traits. The dimensions (length, width and volume) of each testis and semen traits (semen volume, percentage of sperm with progressive motility, sperm concentration, total number of sperm in semen, percentage of sperm with normal acrosome, percentage of sperm with major and minor morphological defects, osmotic resistance test value and activity of aspartate aminotransferase in seminal plasma) were determined on 120 young boars aged 6 months. At 180 day of age, the boars backfat thickness and leanness (by ultrasonic apparatus) and body weight were also measured. The average daily gain was determined in the period from 70 to 180 days of age of the boars. The results showed that the sperm concentration, total number of sperm in semen and percentage of progressive motile sperm were a significantly positively correlated with width and volume of the left (p ≤ 0.01) and right testis (p ≤ 0.05) and with total volume of both testes (p ≤ 0.01). But the highest values of correlation coefficients were found between the width of the left testis and sperm concentration, total number of sperm in semen and percentage of progressive motile sperm. A correlations of dimensions (length and width) and volume of testes with other semen traits were very low and statistically non-significant. The volume of testes (left and right testis and total testes) was significantly positively correlated with body weight at 180 days of age and daily gain (p ≤ 0.01), but lower correlation coefficient was between left testis and daily gain (p ≤ 0.05), whereas correlations were low and non-significant with leanness and backfat thickness., (© 2015 Blackwell Verlag GmbH.)

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 μm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone., (© 2015 Blackwell Verlag GmbH.)