Sperm Suspensions Research Articles

Изучение токсичности средств для мытья посуды с антибактериальным действием на различных экспериментальных моделях

Synthetic detergents (SD) with the declared antibacterial action for washing dishes can be a source of harmful effects on the human body. The main components of SD are surface-active substances (surfactants). With single and repeated contact, detergents can have a moderate irritating effect on the skin, mucous membranes of the eyes and respiratory tract, cause allergic reactions; when ingested, they cause irritation of the gastrointestinal tract, nausea, and vomiting. The objective of our study is to assess the toxicity of detergents with antibacterial action on various experimental models and select the most dangerous of them to study its chronic effects on laboratory animals. The cytotoxicity of 34 detergents was studied on three types of cell cultures: sperm suspension, green Monkey kidney cells (Vero) and hamster epithelial cells (HTK-1). Acute toxicity of the agents was determined when administered into the stomach and abdominal cavity of white mice. Among the cell cultures, HTK-1 cells were the most sensitive. The dependence of SD cytotoxicity on the presence of an antibacterial component is better seen on the sperm suspension. In addition, the study of commercial samples showed that almost half of them do not meet the requirements of MR29 FC/4746 of December 27, 2001 (hereinafter MR29 FC/4746) [1]. In this connection, it is necessary to continue the further studies for a more in-depth studying SD toxicity. Determination of DL50 values upon administration into the abdominal cavity allows us to distribute the products by the degree of toxicity, which seems difficult when determining the DL50 values of detergents with antibacterial action intragastrically. An average correlation dependence was revealed between DL50 upon intraperitoneal administration and the established MNR on HTK-1 cells. Keywords: synthetic detergents, surfactants, cell culture, cytotoxicity, toxicity analyzer, white mice, toxicity.

High temperature induces motility of zebrafish sperm after short-term storage

Short-term storage on ice of zebrafish (Danio rerio) sperm has been successfully applied in diverse basic experimental research. The optimal and economically efficient short-term storage method to date, however, can only maintain the fertilization ability of the sperm for a few hours. In this study, we aimed to improve zebrafish sperm quality after short-term storage and to test whether this affects the nucleotide cargo of spermatozoa. Zebrafish sperm was diluted with E400 extender (130 mM KCl, 50 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM D-(+)-Glucose and 30 mM HEPES-KOH, pH 7.9, 400 mOsmol/kg) at a dilution ratio 1:20 (v/v) in tubes (500 μL) and stored for 24 h at 0–2 °C under aerobic conditions. Elevating the temperature of sperm suspension at 28 °C for 20 min significantly enhanced sperm motility during activation in both distilled water and Perchec solution with 0.25 % Pluronic F-127. Despite the observed decline in sperm motility during short-term storage, Perchec solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0, 160 mOsmol/kg) enhanced activation of the aged sperm compared to activation of distilled water. Following 24 h of sperm storage, higher fertilization, and hatching rates were observed when sperm suspension was incubated at 28 °C for 20 min and activated in Perchec solution at a ratio of approximately 6120 spermatozoa per egg, compared to activation in hatchery water. A tentative RNA-seq study identified a set of 2180 differentially expressed genes after 24 h of sperm storage, significantly enriched in the glycosaminoglycan degradation and galactose metabolism pathways. The validity was discussed due to the limited mRNA content in spermatozoa. No alterations were identified in the DNA methylation level of 24 h stored sperm and neither differentially methylated CpGs (DMCs) nor differentially methylated regions (DMRs) were detected between fresh and aged sperm. Based on these phenotypic and molecular investigations and the successful implementation of high-temperature incubation prior to sperm activation in artificial reproduction, we propose that high-temperature incubation following short-term sperm storage at low temperatures is a safe and significant approach in zebrafish and holds potential benefits for other economically important warm-water species in aquaculture.

Analysis of survival and morphometric parameters of loach embryos and prelarvae under the action of the polyethyleneglycol derivative polymeric nanocarrier

Background. Our previous studies have shown a minor embryotoxic effect of polymeric poly(VEP-co-GMA)-graft-mPEG carrier on Missgurnus fossilis L. embryos and prelarvae. At the same time, it was found that a 3-day exposure to 10-15 M polymer carrier slowed developing, which is characterized by a delayed hatching of larvae. However, in the 10-day experiment on loach larvae exposed to polymeric carrier, anomalies and defects of development have not been identified. The aim of this work was to analyze the embryos and prelarvae survival, as well as determine the morphometric indicators of the loach embryos under the action of a PEG-modified polymer carrier. Materials and Methods. Ovulation in loach females (Misgurnus fossilis L.) was stimulated by intramuscular injection of female chorionic gonadotropin (500 units), eggs were obtained 36 h after stimulation, fertilized in Petri dishes with a suspension of sperm according to A. A. Neifach. The stages of development were observed visually under a binocular microscope MBS-9 with a photo camera in a real-time mode. The experimental embryos were incubated in Goltfreter’s solution with the addition of PEG-containing carrier to a final concentration of 10 and 100 μmol/L. The morphological development of the embryo groups was evaluated according to the T. Fujimoto deve­lopment tables (2004), and the morphological parameters (diameter and area of roes, blastomers, embryos, embryo sac and blastodisk) were measured using ImageJ and Photoshop (CC 2014v15) programs. Results. Addition of the 10 μmol/L PEG-containing carrier to the medium initiated significant positive changes in the survival of loach embryos and prelarvae, in contrast to exposure to 100 μmol/L carrier. A significant increase in the area and diameter of blastomers and the embryos themselves under the influence of 100 μmol/L PEG carrier was established, which may indicate swelling of the embryos and a disruption of water-salt exchange that causes the early death of embryos. Conclusions. (1) Low embryotoxicity of the PEG-polymer was confirmed on the studied model of the loach embryos. Based on the obtained data and the original data of J. Maes et al. (2) PEG is an attractive polymeric carrier for the delivery of a variety of compounds to both embryos and prelarvae as well as other model subjects. Despite the fact that we investigated only two concentrations of the PEG-containing carrier, it is quite likely that these concentrations of the carrier are (3) biologically active in themselves and, therefore, a thorough selection of the PEG-carrier concentration for each objective of the model is needed.

Ultra-Rapid Freezing and Rapid Freezing Methods in Clinical ICSI Program: Effects on Sperm Biological Characteristics, DNA Methylation Stability, DNA Methyltransferase Activity, and Embryo Morphokinetics

The purpose of this study was to evaluate the differences in sperm function and embryo morphokinetics following sperm cryopreservation by ultra-rapid freezing or rapid freezing methods compared to fresh spermatozoa. Thirty samples of normozoospermia were divided equally into fresh, ultra-rapid freezing, and rapid freezing groups. In the rapid freezing, sperm suspension was placed horizontally on nitrogen vapor. In the ultra-rapid freezing, sperm suspension with a straw-in-straw system was directly immersed in liquid nitrogen. Sperm function was assessed in terms of motility, morphology, viability, mitochondrial membrane potential, sperm DNA fragmentation, and acrosome reaction status. Also, the effects of two cryopreservation methods were assessed on global DNA methylation and DNA methyltransferase activity. Moreover, 730 embryos in three groups were cultured using time-lapse imaging until day 6 for embryo morphokinetics. Progressive motility (38.80 ± 4.21 vs. 34.86 ± 4.19; p <0.001) and viability (64.30 ± 6.24 vs. 58.10 ± 8.69; p<0.01) in ultra-rapid freezing were significantly higher than rapid group. DNA fragmentation and acrosome reaction were significantly increased in both cryopreserved groups (p <0.001). However, DNA fragmentation (16.30 ± 1.14 vs. 14.33 ± 2.94; p<0.01) was significantly higher in the rapid than the ultra-rapid freezing group. No significant differences were noted in global DNA methylation (p>0.05) and DNA methyltransferases activity (p>0.05) in fresh compared to cryopreservation groups. The kinetic times, including tPB2, tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, and tM, showed a significant delay in cell divisions in both cryopreservation groups. Furthermore, tPNa, tPNf, and t8 occurred with a significantly higher delay in embryos fertilized by sperm from the rapid freezing compared to the ultra-rapid freezing group. In addition, blastocysts formation was similar in both cryopreservation groups. Ultra-rapid freezing preserved the sperm biological integrity and lead to better embryo morphokinetics compared to the rapid freezing method. However, both methods of sperm cryopreservation were epigenetically safe.

Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

Examining the effects of Lemongrass (Cymbopogon nardus) extract on Sperm Quality and Testosterone Level in Mice Induced with Staphylococcus aureus

There are a variety of causes for male infertility. Among them is microbial infection. Lemongrass (C. nardus) contains natural antioxidants and flavonoids that have the ability to preserve the male reproductive system. This study examined the effects of different C. nardus extract dosages on mouse sperm quality. The male Balb/C mice were separated into five groups of equal size. The negative control group was injected with 0.01ml of Na-CMC 0.5% over the course of 28 days, the positive control group was injected with 0.2ml of Staphylococcus aureus (S. aureus) on days 10, 17, and 24, and the treatment groups were injected with 0.2ml of S. aureus on days 10, 17, and 24, followed by 0.01ml of C. nardus. After the experiment, mice were slaughtered, and sperm suspension was obtained from the cauda epididymal to measure morphology, concentration, and motility. The testis was fixed in a fixative solution to analyze the spermatogenic cells, and intracardiac blood was drawn to determine the level of testosterone. Significant differences (p 0.05) were seen in the morphology, concentration, and motility of spermatozoa when C. nardus extract was administered. Given the low and middle concentrations of C. nardus extract, it can repair sperm damage caused by S. aureus infection.

Evaluation of the effect of mitoquinone on functional parameters, DNA structure, and genes expression related to the apoptotic and antioxidants of human sperm after freezing-thawing.

Sperm freezing is considered as an effective way in assisted reproductive technology (ART) programs, it has detrimental effects on sperm function, due to the production of reactive oxygen species (ROS). This study aimed to investigate the potential of Mitoquinone (MitoQ) in inhibiting the production of mitochondrial ROS during sperm freezing. A total of 20 human normozoosperm samples were collected for this study. The samples were divided into four groups, each containing different concentrations of MitoQ (0, 0.2, 2, and 20 nM), and then subjected to the freezing process. After thawing, the sperm suspensions were evaluated for parameters including motility, morphology, acrosome integrity, adenosine triphosphate (ATP) level, intracellular ROS, viability, chromatin packaging, DNA denaturation, DNA fragmentation, as well as the expression of antioxidants (GPX, SOD) and apoptotic (Bax, Bcl2) genes. The results showed that total and progressive mobility of sperms significantly increased in the 2 nM group, while significantly decreased in the 20 nM group (p ≤ 0.05). Sperm morphology did not significantly improve across all the tested concentrations (p ≥ 0.05). Intracellular ROS levels showed a significant decrease and increase in the concentrations of 2 and 20 nM, respectively (p ≤ 0.05). Furthermore, a significant increase was observed in viability, ATP, acrosome integrity, chromatin packaging, and non-denatured and non-fragmented DNA after treatment with 2 nM of MitoQ, compared with the control group (p ≤ 0.05). Regarding gene expressions, the relative expressions of oxidative stress genes were increased in the 2 nM group and decreased in the 20 nM group (p ≤ 0.05), while no significant difference was observed in the expressions of apoptotic genes compared with the control group (p ≥ 0.05). All the comparisons were made with respect to the control group. Adding the optimal concentration of MitoQ (2 nM) to the sperm freezing medium not only improves sperm functional parameters and reduces DNA damages, but also stimulates the expression of antioxidant genes, leading to even greater benefits for sperm cryopreservation.

Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

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

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

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

O-300 Human sperm selection using cumulus oophorus complexes compared with conventional sperm preparation method on sperm quality and ICSI outcomes: A pilot study

Abstract Study question We compared a sperm selection protocol using cumulus oophorus complexes with a swim up preparation, evaluating the effects on sperm parameters and ICSI outcomes Summary answer Spermatozoa selected using cumulus oophorus complexes showed higher rates of normal morphology, good motility compared with basal semen and the traditional pellet swim up protocol. What is known already It is known that the head of a mature spermatozoa has a hyaluronan receptor that allows them to bind with hyaluronan acid. Cumulus oophorus complexs (COCs) extracellular matrix is made mainly by hyaluronic acid (HA). Various studies have confirmed the importance of COCs in fertilization, demonstrating that spermatozoa that are able penetrate through a layer of COCs are more likely to have a normal morphology and be more effective in producing acrosome reactions than spermatozoa that are not able to do. Moreover, these spermatozoa have been shown to have higher chromatin integrity and zona-binding capacity, resulting in increased fertilization competence. Study design, size, duration A prospective study was conducted between July and December 2022. A total of 50 participants were recruited for the study, randomly selected between patients attending ICSI. The patients were classified as group A when the sperms were selected using pellet swim up and COC selection and as group B when it was used pellet swim up treatment alone. Patients older than 39 years, with endometriosis or repeated IVF failure or severe male factor were excluded. Participants/materials, setting, methods 3 drops of 30 mlt aligned and connected each other were prepared, covered with mineral oil. In group A, in the middle drop, was transferred COC fragments, while the central drop was empty in group B. In the left drop were added 20 mlt of sperm suspension after swim up, and from the right drops were recovered the sperms after passing the middle drop with or without COC, that were used for ICSI treatment. Main results and the role of chance The aim of the study was to verify the effectiveness of COCs, collected after ovarian stimulation, in selecting the best sperms to be used in ICSI procedure. No statistical difference was found between group A and B in the median age of female patients, FSH/LH units administred, E2 and endometrium thickness at triggering using hCG, in the sperm concentration, motility and normal morphology in the baseline seminal samples. We found a statistical difference in the traditional seminal parameters comparing the two different treatments. In particular, we found an higher rates of sperms with rapid and progressive motility and normal morphology in group A between basal semen sample and after sperm selection using COC filter (86.7 vs 31.3%; 8.2 vs 4.4 %). We found a lower sperm concentration in group A vs group B after sperm treatment (2.3 vs 4.7 M/ml), and an higher rates of rapid progressive motility and normal morphology in group A vs group B (86.7 vs 38.6 %; 8.2 vs 4.6%). We found an higher but not statistically significative pregnancy and implantation rates in group A compared with group B probably because of the limited number of patients analyzed (25 vs 19.2 %; 25 vs 20.6%) Limitations, reasons for caution The study was conducted on a small participant cohort. We analyzed the traditional sperm parameters, but it is necessary to evaluate the chromatine integrity of the sperms after COC selection to confirm that the higher sperm competence in fertilization and embryo development could be based on a better genetic pattern Wider implications of the findings Data seem to demonstrate that the use of COC allows to select higher competent sperms if compared with traditional pellet swim up treatment. The clinical and biological ICSI outcomes seem to be improved by COC treatment but it is necessary to increase the sample size in future experimental trial Trial registration number not applicable

Potential Extract of Lerak Fruit (Sapindus Rarak), Pegagan (Centella Asiatica,) Mimba Seed (Azadirachta Indica A.Juss) and Mixing of Third Extract to Motility and Viability of Spermatozoa Guinea Pigs (Cavia Porcellus)

Spermicides are substances that can kill spermatozoa. Spermicide circulating in the community contains Nonoxynol9 which, if used for a long time, can irritate the vaginal mucosa and penis. Indonesia has a variety of plants that have the potential to act as anti-fertility, including Sapindus rarak, Centella asiatica, and Azadirachta indica. Objective: This study aim to evaluate extract Sapindus rarak, Centella asiatica, and Azadirachta indica. and mixing of third etract to motility and viability of spermatozoa Guinea pigs. Methods: The sample of this research was 15 guinea pig spermatozoa, 3-4 months old male guinea pigs taken from the cauda epididymis and vas deferens. Guinea pigs were divided into 5 group treatments with each group treatment incubated for 1, 10, and 30 minutes and repeated each for 5 times. The treatments in this study is control, 200 μl of spermatozoa suspension + 50 μl of Sapindus rarak fruit pulp extract 0.9%; 200 μl of spermatozoa suspension + 50 μl of gotu kola extract 0.9%; 200 μl of spermatozoa suspension + 50 μl of 0.9% neem seed extract; 200 μl of spermatozoa suspension + 50 μl of the third mixture extract 0.9%. This study used a completely randomized design (CRD). The observations in this study were the motility of guinea pig spermatozoa categories a + b and the viability of guinea pig spermatozoa using a microscope. The data obtained were analyzed statistically using One Way ANOVA. The results of this study indicated that the administration of extract Sapindus rarak, Centella asiatica, and Azadirachta indica and a mixture of the three extracts had a significant effect (P &lt; 0.05) on the motility of guinea pig spermatozoa categories a + b and viability of guinea pig spermatozoa. Conclution: This study concludes that the mixture of the three extracts has more potential in reducing the motility of spermatozoa categories a + b and the viability of guinea pig spermatozoa compared to single extracts. The mixture of the three extracts may contribute for reduce quality of sperm.

The effects of coenzyme Q10 and curcumin supplementation in freezing medium for human sperm cryopreservation

BackgroundDespite its routine and frequent application, cryopreservation of human sperm is far from the desired efficacy, as freezing and thawing impair motility, viability, acrosomal unity, and DNA integrity. ObjectivesIn this study, the authors aimed to investigate whether adding antioxidants, coenzyme Q10, and curcumin into the freezing medium provide better efficacy in the cryopreservation of human sperm. MethodsThe semen samples from 40 healthy men aged 18–45 were collected in sterile containers by masturbation. Samples within normal reference values for sperm concentration (≥15 million/mL) and motility (progressive motile ≥ 32% and total motility ≥ 40%) were included in the study. Semen samples were equally divided into five groups and evaluated; i) pre-freezing sperm suspension, ii) frozen-thawed control (Ctrl) without any supplementation in freezing medium, iii) frozen-thawed with curcumin supplementation of 0.25 mM (Cur), iv) frozen-thawed coenzyme Q10 supplementation of 25 µM (CoQ10) and v) frozen-thawed curcumin (0.25 mM) plus coenzyme Q10 (25 µM) supplementation (CurCoQ10) into the freezing medium. Liquid nitrogen vapour freezing and rapid thawing were performed in each group (ii-v). Sperm motility, viability, acrosome integrity, and DNA fragmentation rates were compared and ultrastructural evaluations by transmission electron microscopy were undertaken between the groups. Additionally, the total antioxidant capacity/total oxidant capacity values were measured. ResultsAccording to CASA results, progressive motility was significantly higher in the CoQ10 group (9.4 ± 7.6) when compared with the Ctrl (7.1 ± 6.3), Cur (6.4 ± 4.8) and CurCoQ10 (8.1 ± 7.7) groups (p < 0.05). Flow cytometry results showed no difference in the viability and acrosome integrity values after thawing, but DNA fragmentation was significantly increased in the curcumin-added groups (p < 0.05). Acrosomal changes and sub-acrosomal defects were seen in all groups after thawing at the ultrastructural level. Mitochondrial membrane structure was preserved in CoQ10 and CurCoQ10 groups. ConclusionsOur results suggested that sperm ultrastructural morphology and motility were better preserved in the CoQ10 group during cryopreservation. In curcumin groups, DNA fragmentation and head defects were increased.

The magnetic cell separation method reveals protective effect of melatonin on human spermatozoa from peroxide-induced apoptosis

BackgroundThe selection of non-apoptotic sperm is related to successful fertilization. This study investigated the protective effects of melatonin and the role of the MACS (magnetically activated cell sorting) method to prevent oxidative damage in vitro and improve sperm quality parameters such as viability and DNA integrity.Materials and methodsSemen analysis was performed on 20 different eligible donors participating in the study. Sperm viability and concentration were checked at time of 0 (0 h). In order to conduct more studies after 24 h (24 h), the remaining sperm suspension was divided into a control group and six experimental groups. DNA fragmentation was assessed using the TUNEL assay.ResultsThe treatment of human spermatozoa with 100 µM hydrogen peroxide for 24 h induced a significant increase in phosphatidylserine externalization and significantly increases apoptotic sperm (p ≤ 0.001). TUNEL analysis of human sperm pretreated with 100 µM hydrogen peroxide for 24 h showed that the percentage of sperm with fragmented DNA was significantly reduced after sorting by MACS (P ≤ 0.001). However, pretreated human sperm with 1 μM melatonin for 24 h could effectively maintain sperm motility and progressive motility.ConclusionsPretreated human spermatozoa with 1 µM melatonin for 24 h could be effective for maintenance of sperm motility and progressive motility. Although 100 µM hydrogen peroxide-treated sperm were used, MACS was used to retain the appropriate sperm and select high-quality sperm.

A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system)

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

How I overcame problems in in vitro fertilisation of livestock animals

In my research life of 35 years, growing with IETS as a researcher of in vitro maturation and fertilisation (IVM/IVF) of porcine and cattle oocytes, I suffered from hard times related to solving problems that prevented the progress of my research and conferment of my degrees. Many researchers may have similar problems. Thus, I would like to address a few examples of how I overcame these problems related to IVF and help young researchers with similar troubles. There were four main problems to be solved in my experiments. Problem 1: Establishment of IVF using only defined medium in pigs. Problem 2: Establishment of successful in vitro culture (IVC) of IVM/IVF bovine oocytes in defined medium. Problem 3: Low rate of male pronucleus formation in IVM porcine oocytes after IVF. Problem 4: Sedimentation of Ca2+ in the sperm capacitation solution for IVF in pigs. Problem 1 was solved by a lucky accident, in which a sperm suspension that would have otherwise been discarded happened to be successfully used for IVF in pigs. Problems 2, 3 and 4 were solved by communication with scientists whose fields were different from mine, where similar problems had been solved already. Young researchers are encouraged to transcend the boundaries of their research fields and solve problems by interacting with researchers in different fields. There are many good connections or answers around us that may be effective in resolving the problems that are hindering the progress of pending research.

A novel, simplified method to prepare and preserve freeze-dried mouse sperm in plastic microtubes.

Although freeze-drying sperm can save space, reduce maintenance costs, and facilitate the transportation of genetic samples, the current method requires breakable, custom-made, and expensive glass ampoules. In the present study, we developed a simple and economical method for collecting freeze-dried (FD) sperm using commercially available plastic microtubes. Mouse epididymal sperm suspensions were placed in 1.5 ml polypropylene tubes, frozen in liquid nitrogen, and dried in an acrylic freeze-drying chamber, after which they were closed under a vacuum. The drying duration did not differ between the microtube and glass ampoule methods (control); however, the sperm recovery rate was higher using the microtube method, and the physical damage to the sperm after rehydration was also reduced. Intracytoplasmic sperm injection (ICSI) using FD sperm stored in microtubes at -30°C yielded healthy offspring without reducing the success rate, even after 9 months of storage. Air infiltration into all microtubes stored at room temperature (RT) within 2 weeks of storage caused a drastic decrease in the fertilization rate of FD sperm; underwater storage did not prevent air infiltration. RT storage of FD sperm in microtubes for 1 week resulted in healthy offspring after ICSI (5-18%), but the addition of silica gel or CaCl2 did not improve the success rate. Our novel microtube method is currently the simplest and most effective method for treating FD sperm, contributing to the development of alternative low-cost approaches for preserving and transporting genetic resources.

Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.

Elevated temperature promotes spermatozoa motility kinetics and fertilizing ability following short-term storage: An implication for artificial reproduction of common carp Cyprinus carpio in a hatchery

Short-term storage of sperm in an extender at a low temperature has been widely applied in hatchery management. In the present study, for the first time, we examined the effects of elevating temperature on spermatozoa motility kinetics and fertilization ability after short-term storage at 0–2 °C. Sperm of common carp (Cyprinus carpio L.) was diluted with an extender (110 mM NaCl, 40 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, pH 7.5, 310 mOsmol/kg) at the ratio 1:1 (v/v) in containers and stored for 6–9 days at 0–2 °C under aerobic conditions. At the end of storage, the sperm suspension was incubated at 20 °C for >10 min and spermatozoa motility, velocity [straight-line velocity (VSL) and curvilinear velocity (VCL)], viability, and fertilizing ability were recorded. Spermatozoa motility of sperm stored for 6 days was significantly increased after incubation at 20 °C for 10 and 20 min and activation in both distilled water and a saline solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0). Elevating the temperature did not affect spermatozoa viability. Spermatozoa motility, VSL and VCL, and viability were decreased at 3 days post storage (DPS) after activation in distilled water without incubation at 20 °C. However, elevating the temperature for 10–20 min at any time post sperm storage (3, 6 or 9 DPS) could promote spermatozoa motility kinetics. For sperm stored for 9 days, fertilization and hatching rates were increased when it was incubated at 20 °C for 10 and 20 min prior to fertilization at the ratio 30,000 spermatozoa per egg. Increasing the spermatozoa to egg ratio to 100,000:1, however, masked the temperature effects on fertilization and hatching rates. Finally, a large-scale experiment was performed in hatchery in which sperm stored for 8 days was incubated for 2 h at 20 °C, resulting also in an increase in spermatozoa motility and fertilizing ability. Results of the present study show that elevating the temperature following short-term storage of common carp sperm at low temperatures improved spermatozoa motility kinetics and their fertilizing ability. This method may contribute to developing artificial reproduction of economically important fish species in aquaculture.

Influence of the sodium fluoride on the development and survival of the loach embryos

Background: The study of fluoride effects at the cellular level is still essential for biophysics, medicine, and ecology as one of the most common environmental pollutants. Its impact on embryonic objects is poorly understood. Objectives: The aim of the work was: 1) to study the effect of sodium fluoride (in the minimum concentration to inhibit growth) on the morphological development of loaсh embryos; 2) evaluation of the degree of survival of embryos in the presence of sodium fluoride in the incubation medium and determination of the coefficient Ks. Materials and methods: Ovulation in loach females (Misgurnus fossilis L.) was stimulated by intramuscular injection of female chorionic gonadotropin (500 units), eggs were obtained by 36 h after stimulation, fertilized in Petri dishes with a suspension of sperm according to Neifach A. A. The stages of development were observed visually used a binocular microscope MBS-9 with a photo camera. The experimental embryos were incubated in Goltfreter's solution with the addition of sodium fluoride to a final minimum concentration to inhibit growth of 500 μmol/l. Results: Sodium fluoride inhibits the development of loach embryos and leads to developmental defects. The noticeable developmental defects caused by sodium fluoride were a reduction in the size of the larvae's head and tail, low body pigmentation, changes in the eye diameter, and embryonic touch reflex. As a result of the accumulation of fluoride in embryonic cells, on the third day of development, embryonic mortality increased to 88,9%. On 12 days under the action of sodium fluoride, the total number of larvae was about 2%. Conclusions: The ability of NaF to act as a direct teratogen was tested on the cold-blooded embryo model, the same effect was found by other investigators on the FETAX model. The possibility that sodium fluoride may cause toxic and/or neuromuscular developmental defects in human embryos also should be considered. Avoiding excessive getting of fluoride in the body by limiting the consumption of foods or beverages high in fluoride, the use of fluoride in dental care products, etc. requires detailed assessment.