Sperm Binding Ability Research Articles

Supplementation of spermidine enhances the quality of postovulatory aged porcine oocytes

BackgroundSpermidine (SPD) is an intermediate compound in the polyamine metabolism which takes critical part in a variety of cellular processes. In particular, it has been reported to exert anti-aging effects, suppress the age-related diseases, and extend lifespan across species. However, whether it has the favorable influence on the quality of postovulatory aged oocytes remains elusive.MethodsImmunostaining and fluorescence intensity measurement were used to evaluate the effects of postovulatory aging and SPD supplementation on the oocyte fragmentation, spindle/chromosome structure, actin polymerization, dynamics of cortical granules (CGs) and ovastacin, mitochondrial distribution and function, as well as autophagy levels. In addition, in vitro sperm binding assay and in vitro fertilization (IVF) experiment were applied to assess the impacts of postovulatory aging and SPD supplementation on the sperm binding ability and fertilization capacity of oocytes.ResultsHere, we showed that supplementation of SPD during postovulatory aging could relieve the deterioration of porcine oocytes. Specifically, we found that postovulatory aging impaired the oocyte quality by damaging the morphological integrity of oocytes, maintenance of spindle/chromosome structure, and dynamics of actin cytoskeleton. Postovulatory aging also weakened the sperm binding ability and fertilization capacity of oocytes by compromising the distribution pattern of CGs and their content ovastacin. Notably, supplementation of SPD attenuated these defects in postovulatory aged porcine oocytes via strengthening mitochondrial function, eliminating excessive reactive oxygen species (ROS), inhibiting apoptosis, and enhancing autophagy levels.ConclusionAltogether, our findings demonstrate that SPD supplementation is a feasible approach to ameliorate the quality of postovulatory aged oocytes, which can be potentially applied to the human assisted reproductive technology (ART) and in vitro production of animal embryos.

Prothioconazole exposure disrupts oocyte maturation and fertilization by inducing mitochondrial dysfunction and apoptosis in mice

Prothioconazole (PTC), a novel broad-spectrum triazole fungicide, has attracted widespread concern due to its wide use and toxicological effects on non-target organisms. However, little is known about the impact of PTC on oocyte quality and female fertility, especially on oocyte maturation and fertilization. In the present study, we reported that PTC exposure affects the oocyte developmental competence and oocyte fertilization ability to weaken female fertility. Firstly, PTC compromises oocyte development ability by disrupting spindle morphology and chromosome alignment, as well as decreasing acetylation level of α-tubulin and disrupting kinetochore-microtubule attachments. In addition, PTC compromises oocyte fertilization ability by weakening the sperm binding ability and impairing the dynamics of Juno, Cortical granule and Ovastacin. Finally, single-cell transcriptome analysis revealed that PTC exposure has potentially toxic effects on oocyte development and fertilization, which is caused by the mitochondrial dysfunction and the occurrence of oxidative stress and apoptosis. In summary, our results indicated that PTC exposure had potentially toxic effects on female fertility and led to poor oocyte quality in female mice.

Butylparaben weakens female fertility via causing oocyte meiotic arrest and fertilization failure in mice

Butylparaben is an ubiquitous environmental endocrine disruptor, that is commonly used in cosmetics and personal care product due to its anti-microbial properties. Butylparaben has been shown to cause developmental toxicity, endocrine and metabolic disorders and immune diseases. However, little is known about the impact on female fertility, especially oocyte quality. In the present study, we reported that butylparaben influenced female fertility by showing the disturbed oocyte meiotic capacity and fertilization potential. Specifically, butylparaben results in the oocyte maturation arrest by impairing spindle/chromosome structure and microtubule stability. Besides, butylparaben results in fertilization failure by impairing the dynamics of Juno and ovastacin and the sperm binding ability. Last, single-cell transcriptome analysis showed that butylparaben-induced oocyte deterioration was caused by mitochondrial dysfunction, which led to the accumulation of ROS and occurrence of apoptosis. Collectively, our study indicates that mitochondrial dysfunction and redox perturbation is the major cause of the weakened female fertility expoesd to butylparaben.

Short-term preservation of canine sperm-binding ability and other metrics using the INRA-96 in comparison to Tris-egg yolk extender.

The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA-96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA-96 extender as an alternative for the short-term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh and chilled-rewarmed samples, as well as the sperm-binding ability using the perivitelline membrane of the chicken egg as an indicator of the fertilizing capacity of the preserved semen. A total of 18 ejaculates from 9 French bulldogs (two ejaculates per dog) were collected and divided into two aliquots that were diluted in Tris-egg yolk 20% (control) or INRA-96 to a final concentration of 100 × 106 sperm/mL. Samples were refrigerated in a biological incubator at 5°C and evaluated at 0, 24 and 48 h time points. Comparing the two treatments after 48 h of refrigeration, both extenders showed similar values (p < .5) for the majority of kinetic parameters, with the INRA-96 group promoting a total motility of 88.1 ± 2.9%. In addition, the morphology, integrity and functionality of the plasma membrane were preserved above 70% in this group. Dilution with INRA-96 also provided a significantly higher amount of sperm bound (256.2 ± 21.1) to the perivitelline membrane of the egg yolk compared to the sperm-binding rates (p < .05) achieved at the use of Tris-egg yolk (215.2 ± 21 bound spermatozoa) at 48 h. Our study proved similar functional properties of dog sperm cells treated with INRA-96 in comparison to commonly used home-made Tris-based extender during short-time storage.

Melatonin improves the ability of spermatozoa to bind with oocytes in the mouse.

Melatonin is a powerful antioxidant regulating various biological functions, including alleviating male reproductive damage under pathological conditions. Here, we aim to analyse the effect of melatonin on normal male reproduction in mice. Male mice received an intraperitoneal injection of melatonin (10mg/kg body weight) for 35 consecutive days. The testis and epididymis morphology, and epididymal sperm parameters were examined. PCNA, HSPA2, SYCP3, ZO-1 and CYP11A1 expressions in epididymis or testis were detected by immunohistochemistry or Western blotting. Male fertility was determined by in vivo and in vitro fertilisation (IVF) experiments. The differentially expressed sperm proteins were identified by proteomics. No visible structural changes and oxidative damage in the testis and epididymis, and no significant side effects on testis weight, testosterone levels, sperm motility, and sperm morphology were observed in the melatonin-treatment group compared with the control group. Spermatogenesis-related molecules of PCNA, SYCP3, ZO-1, and CYP11A1 showed no significant differences in melatonin-treated testis. However, PCNA and HSPA2 increased their expressions in the epididymal initial segments in the melatonin-treatment group. Normal sperm fertilisation, two-cell and blastocyst development were observed in the melatonin-treated group, but melatonin significantly enhanced the sperm binding ability characterised as more sperm binding to one oocyte (control 7.2±1.3 versus melatonin 11.8±1.5). Sperm proteomics demonstrated that melatonin treatment enhanced the biological process of cell adhesion in sperm. This study suggests that melatonin can promote sperm maturation and sperm function, providing important information for further research on the physiological function and protective effect of melatonin in male reproduction.

Effect of Detergents Based on Sodium Dodecyl Sulfate on Functional Metrics of Frozen-Thawed Collared Peccary (Pecari tajacu) Semen.

We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen-thawed sperm. Semen aliquots from ten individuals were diluted in a Tris-egg yolk-glycerol extender alone or with 0.5% Equex STM® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris-yolk-glycerol extender optimized the freeze-thaw recovery of peccary semen.

Velvet antler water extract protects porcine oocytes from lipopolysaccharide-induced meiotic defects.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

Effects of capacitating media and incubation time on collared peccary sperm quality, acrosome reaction, and binding ability

Context Several assisted reproduction techniques have been proposed for collared peccaries due to the interest in its conservation. However, there is little information about the requirements for sperm capacitation, an initial step in in vitro fertilisation. Aims We aimed to determine the optimal conditions for collared peccary sperm capacitation by comparing Tyrode’s albumin lactate pyruvate (TALP) or the same media plus heparin, caffeine, or their combination at different exposure times (1, 3, and 6 h). Methods The samples were evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, morphology, DNA integrity, reactive oxygen species (ROS) expression levels, capacitation, and sperm binding ability using swine oocytes. Key results Samples incubated with caffeine or heparin had a higher percentage of capacitated spermatozoa. The maximum percentage of capacitation was achieved after 3 h of incubation with either agent. Moreover, spermatozoa subjected to heparin capacitation showed better motility than when subjected to caffeine, and lower ROS expression levels after 1 h. No differences were observed among incubation times for the binding ability. Conclusions In summary, collared peccary spermatozoa can be capacitated with caffeine or heparin; however, heparin better maintains sperm motility and ROS expression levels. The co-incubation of gametes in a medium with heparin for 3 h could be efficient for in vitro fertilisation in collared peccaries. Implications This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries.

Alpha-lipoic acid supplementation restores the meiotic competency and fertilization capacity of porcine oocytes induced by arsenite.

Arsenite is known as a well-known endocrine disrupting chemicals, and reported to be associated with an increased incidence of negative health effects, including reproductive disorders and dysfunction of the endocrine system. However, it still lacks of the research regarding the beneficial effects of ALA on arsenite exposed oocytes, and the underlying mechanisms have not been determined. Here, we report that supplementation of alpha-lipoic acid (ALA), a strong antioxidant naturally present in all cells of the humans, is able to restore the declined meiotic competency and fertilization capacity of porcine oocytes induced by arsenite. Notably, ALA recovers the defective nuclear and cytoplasmic maturation of porcine oocytes caused by arsenite exposure, including the impaired spindle formation and actin polymerization, the defective mitochondrion integrity and cortical granules distribution. Also, ALA recovers the compromised sperm binding ability to maintain the fertilization potential of arsenite-exposed oocytes. Importantly, ALA suppresses the oxidative stress by reducing the levels of ROS and inhibits the occurrence of DNA damage along with apoptosis. Above all, we provide a new perspective for the application of ALA in effectively preventing the declined oocyte quality induced by environmental EDCs.

294 Awardee Talk: Porcine Sperm Storage in the Oviduct Before Fertilization: A Sweet Spot

Abstract Sperm storage in the female reproductive tract from sperm deposition until ovulation is conserved among a wide variety of animals allowing some asynchrony between mating and ovulation. Mammals, reptiles, birds, amphibians, and insects are among those that store sperm from a day or two to as long as a decade. We have studied the molecular basis of how sperm are retained and then released from the reservoirs in the female tract to fertilize oocytes, primarily in swine. Previous research by others implicated various oviduct glycans as receptors to retain sperm in the lower oviduct, the isthmus, but the complete structures of the glycans that bound sperm had not been identified. We used an array of hundreds of glycans to identify those that bound sperm and identified several glycans with sperm binding ability. Further inspection revealed that all sperm binding glycans contained at least one of two motifs, either a branched 6-sialylated structure or a Lewis X trisaccharide. Profiling all glycans produced by the isthmus showed that these motifs were abundant among the complex N-linked glycans of the isthmus. Further, they were found on the apical surface of the isthmic epithelium where they are available to bind sperm. These glycans suppressed the normal increase in intracellular free Ca2+ in sperm during capacitation and, when immobilized on beads, lengthened sperm lifespan, mimicking the effect of sperm of binding to the oviduct. Binding to oviduct glycans alters sperm metabolism in specific ways to prolong sperm viability. Sperm are released from immobilized glycans by progesterone, which induces hyperactivated motility, and by capacitation, which reduces the binding affinity of capacitated sperm for oviduct glycans. This information may be used to improve sperm lifespan in vivo or enhance sperm storage outside of the female tract.

Investigating the need for antibiotic supplementation to the extender used for semen cryopreservation in collared peccaries

The objective was to investigate the effects of semen freezing extender supplementation with antibiotics on bacterial load of semen samples, sperm functional and morphological metrics in the collared peccary. Fresh ejaculates from 10 males were extended in Tris-egg yolk-glycerol supplemented or not (control) with gentamicin (70 μg/mL) streptomycin-penicillin (SP; 1 mg/mL−1000 IU/mL) or and cryopreserved in liquid nitrogen. Bacterial load, sperm motility patterns, morphology, membrane functionality and integrity, mitochondrial activity, chromatin integrity and sperm-binding ability were evaluated in fresh and frozen-thawed samples. Regardless of the use of antibiotics, the sole cryopreservation provoked a significant decrease (P < 0.05) in bacterial load compared to fresh samples (from average values > 1 x 106 CFU/mL to <0.4 × 106 CFU/mL). Post-thawing sperm kinetic parameters were not affected by the absence or presence of different antibiotics, except for beat cross frequency that was significantly (P < 0.05) impaired by SP supplementation compared to the group without antibiotics. After thawing, sperm morphology, membrane functionality and integrity, and mitochondrial activity were also not affected by the presence or absence of antibiotics; however, a significant decrease was observed in the group without antibiotics (P < 0.05) in comparison to fresh samples. Regarding sperm-binding ability, there were no differences among the different groups. While collared peccary semen could be efficiently cryopreserved in the absence of antibiotics in the extender, the use of both gentamicin or the streptomycin-penicillin combination is recommended as effective antibiotic supplementation for a further control of bacterial loads without affecting sperm parameters.

Octocrylene exposure impairs mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

One of organic ultraviolet (UV) filters, Octocrylene (OCL), is mainly used in various cosmetic products, which is being frequently detected in soil, sediment, aquatic systems and food chain. There is evidence confirmed the reproductive toxicity of OCL in Japanese medaka. However, less was known about the effects of OCL exposure on oocyte quality. Here, we investigated the impacts of OCL on mouse oocyte maturation and quality by exposing oocytes to OCL in vitro at concentrations of 8, 22, 30, 40 and 50 nM. The results showed that OCL markedly reduced mouse oocyte germinal vesicle breakdown (GVBD) at 50 nM and polar body extrusion (PBE) rates at 40 and 50 nM. OCL exposure further disrupted spindle assembly and chromosome alignment, finally inducing aneuploid. Mitochondrial function was also damaged by OCL exposure, leading to ROS overproduction and apoptosis in oocytes. Moreover, OCL treatment impaired the distribution of cortical granules and sperm binding ability of oocytes. In summary, these data demonstrated that OCL could disturb the oocyte meiotic maturation and reduce oocyte quality.

Resveratrol addition to freezing equine semen extender influence the number of sperm bound to bovine oviduct explants

During semen freezing, approximately 90% of the seminal plasma is discarded, resulting in higher sperm vulnerability to lipid peroxidation. Thus, the addition of antioxidants, such as resveratrol, to equine semen freezing extenders may increase the viability of cryopreserved sperm. Further, non- capacitated sperm usually bind to the isthmus region of the oviduct, which can be a way to identify the sperm able to fertilize the oocyte. The aim of the present study was to evaluate the binding capacity of cryopreserved equine sperm to bovine oviduct explants. To prepare the oviduct explants, the reproductive system of slaughtered cows was transported in saline solution at 35°C to the laboratory. After sectioning the uterus-tubal junction ipsilateral to the ovaries without a corpus luteum, the isthmus region was pressed towards the uterus and its contents were expelled in a Petri dish with culture medium. These oviduct content with the medium was deposited in 14 ml tube decanted and after five minutes it was disaggregated with a needle and syringe. After repeating this process three times, the contents were deposited in Petri dishes and cultured for 24 hours in an incubator in atmosphere with 5% CO 2 . The explants were distributed in six treatments according to the resveratrol concentration added to the INRA 96 freezing extender. Semen from sixstallions were frozen in the INRA96 (control, no resveratrol) and with 5, 10, 50, 100 and 150 μM resveratrol. The six treated semen samples were co-incubated with the explants for a min. Then, the explants were washed with a medium to withdraw the loosened sperm and placed on glass slide and covered with a coverslip. The number of sperm bound per nm of oviduct was observed with microscopy and analysed with the ImageJ software. For the statistical analysis, mean and standard error were calculated and when the data did not have a normal distribution, logarithmic transformation was performed, and the log means were evaluated with variance analysis and compared with Duncan's test. The probability of P<0.05 was considered significant. The addition of 5μM resveratrol to the equine semen freezing extender increased the number of sperm bound explants (64.6 ± 1.1 sperm/nm explant), while 50μM resveratrol reduced (21.4 ± 1.1 sperm/nm of explant) compared to the other treatments (control: 48.1 ± 1.0, 10 μM: 56.2 ± 1.0, 100 μM: 46.8 ± 1.0 and 150 μM resveratrol: 53.7 ± 1.1 sperm/nm of explant). In conclusion, the addition of 5 μM resveratrol in frozen equine semen extender was improved equine sperm binding ability to oviduct explants.

An ex-vivo assessment of differential sperm transport in the female reproductive tract between high and low fertility bulls

Despite passing all quality control checks at animal breeding centres, bulls with apparently normal semen quality can yield unacceptably low field fertility rates. This study took an ex-vivo approach to assess if bulls of divergent field fertility differ in the ability of their spermatozoa to interact with the female reproductive tract and its secretions. Six high and six low fertility Holstein Friesian bulls (+4.0 ± 0.2 and −15.7 ± 3.13, respectively; adjusted mean fertility ± s.e.m. mean of the bull population was 0) were selected from a population of 840 bulls with >500 field inseminations per bull. Thawed spermatozoa from each bull were analysed across a range of in vitro assays to assess their ability to transverse the female reproductive tract including; motility and kinematic parameters using computer-assisted sperm analysis, viability, membrane fluidity and acrosomal integrity using flow cytometry as well as mucus penetration tests, rheotactic behaviour and sperm binding ability to the oviductal epithelium. While there was no significant difference between high and low fertility bulls in most of the sperm motility, kinematic and sperm functional parameters (namely, motility, average path velocity, linearity, straightness, amplitude of lateral head movement), viability, membrane fluidity or acrosome intactness, high fertility bulls had higher curvilinear velocity compared to the low fertility group (P < 0.05) and a higher straight-line velocity was observed although it did not reach statistical significance (P = 0.08). There was no difference between treatment groups in the ability of spermatozoa to penetrate periovulatory cervical mucus or in their rheotactic response (P > 0.05). Interestingly, there was a positive correlation between the straight-line velocity of spermatozoa and their rheotactic response (r = 0.45, P < 0.001) and further linear regression analysis indicated 18.9% of the variance in sperm rheotaxis was accounted for by straight line velocity. A higher number of spermatozoa from the high fertility group compared to the low fertility group bound to oviductal explants (15.1 ± 0.98 and 12.5 ± 0.76, respectively; mean ± s.e.m; P < 0.05). In conclusion, the differences in the kinematics of sperm motility and ability to bind to oviductal explants between high and low fertility bulls were modest and are unlikely to explain the inherent differences in fertility between these cohorts of bulls.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility.Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10mg/kg/day/body weight through their drinking water continuously for 4weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

Melatonin rescues the reproductive toxicity of low-dose glyphosate-based herbicide during mouse oocyte maturation via the GPER signaling pathway.

Glyphosate-based herbicides (GBHs) are a group of widely used broad-spectrum agricultural pesticides. Due to the recalcitrance of GBH, it has been found in food and environment as a contaminant, posing a threat to public health. The health risks associated with GBH have been indicated by reporting acute toxicity data (an acute exposure of GBH at a 0.5% dose), which primarily discuss toxicity in relation to accidental high-rate exposure. Currently, there is little information regarding the toxicity of GBH at environmentally relevant levels. In this study, we used mature mouse oocytes to study the toxic effects of low-dose GBH exposure in vitro (0.00001%-0.00025%) and in vivo (0.0005%, orally administered through daily drinking water) during meiotic maturation. GBH exposure led to meiotic maturation failure with spindle defects and chromosome misalignment. In addition, GBH treatment severely reduced sperm-binding ability and disrupted early embryo cleavage. Moreover, GBH exposure significantly increased the reactive oxygen species (ROS) levels and apoptotic rates. Evidence indicates that such effects in GBH-exposed oocytes are likely due to overexpression of the G-protein estrogen receptor (GPER/GPR30). Remarkably, we found that melatonin administration elicited significant protection against GBH-induced oocyte deterioration via preserving the expression of GPR30, along with activation of its downstream signaling event (pERK/ERK). Taken together, these results revealed that low-dose glyphosate has a certain adverse effect on oocyte maturation and early embryo cleavage, and highlight the protective roles of melatonin.

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.

The combination treatment of cholesterol‐loaded methyl‐β‐cyclodextrin and methyl‐β‐cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-β-cyclodextrin (CLC; 0, 10, 15, 20mM) for 45min before vitrification and methyl-β-cyclodextrin (MβCD; 0, 2.25, 4.25, 6.25mM) for 45min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15mM CLC+4.25mM MβCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-β-cyclodextrin and methyl-β-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen

ABSTRACT We evaluated the effect of reducing free calcium in the cryopreservation medium, using the calcium chelator ethylene diamine tetracetic acid (EDTA) at 0.3% and 0.5% concentrations. Three male mixed breed dogs were subjected to semen collection by digital manipulation (n=16). Each ejaculate was divided in three aliquots, and each one was diluted in TRIS-glucose-egg yolk extender with 6% glycerol and 0.5% Equex STM Paste® (TGE, control); and added with 0.3% EDTA (EDTA 0.3) or 0.5% EDTA (EDTA 0.5). Calcium concentration reduced in EDTA 0.3 and all the calcium ions were chelated in EDTA 0.5. The EDTA addition did not affect sperm morphology or plasma membrane integrity; however, by removing all free calcium (EDTA 0.5), the sperm motility reduced (64.7% in TGE and 45% in EDTA 0.5; p&lt;0.05). Acrosome integrity and sperm binding ability were not improved by calcium chelation. The failure to prevent the premature AR may explain why sperm longevity was not affected by calcium removal. Thus, the partial or complete calcium removal, through EDTA addition, is not able to prevent acrosomal damage or premature acrosomal reaction, and therefore does not improve the dog sperm binding ability.

Glutathione alleviates the cadmium exposure-caused porcine oocyte meiotic defects via eliminating the excessive ROS

Heavy metal cadmium (Cd) is a widespread environmental contaminant with a potential toxicity that might adversely influence the health of experimental animals and humans. It has been known that Cd might accumulate in vertebrates for many years and thus leads to the hepatic and renal toxicity. Additionally, Cd concentration in the ovary increases with age and is highly related to the reproductive hazard. However, the underlying mechanisms regarding how Cd affects the female reproductive system especially the oocyte quality have not yet fully defined. Here, we reported that Cd exposure led to the defective nuclear maturation of oocytes via the impairment of cytoskeleton assembly, displaying the aberrant spindle organization, chromosome alignment and actin polymerization. In the meantime, Cd exposure caused the impaired cytoplasmic maturation by showing the disrupted dynamics of mitochondrial integrity and cortical granules, and thereby resulting in the compromised sperm binding ability and fertilization capacity of oocytes. More importantly, we found that glutathione (GSH) supplementation was able to recover the meiotic failure induced by Cd exposure through suppressing the excessive ROS level, DNA damage accumulation and apoptotic incidence. Taken together, our findings demonstrate that Cd exposure has the adverse effects on the oocyte meiotic maturation as well as subsequent fertilization, and provide a potential effective strategy to improve the quality of Cd-exposed oocytes.