Forward Progressive Motility Research Articles

Sperm quality and sexual function after the first COVID-19 infection during the omicron surge: an observational study in southwest China.

As the Omicron variant became the dominant coronavirus disease 2019 (COVID-19) strain and the threat to human health decreased, the impact of COVID-19 on male reproductive health and semen quality may have changed. In this prospective observational study, we aimed to investigate the changes in semen quality and sexual function before and after the Omicron variant infection by self-controlled study and to explore the influence of Omicron variant infection on male reproductive health. We recruited 373 participants who provided semen samples before their first COVID-19 infection. During the subsequent follow-up, only 154 participants provided the second semen samples, 11 of whom were not infected with COVID-19. Sperm quality was assessed approximately 45 and 90 days after COVID-19, as well as before infection. Semen parameters, including total sperm count, total forward sperm, progressive motility, and sperm concentration, significantly declined 45 days after COVID-19 infection. At 90 days after infection, the total sperm count, total forward sperm, and sperm concentration gradually increased to pre-infection levels. Participants who experienced fever showed worse semen quality in terms of total forward sperm, sperm concentration, and sperm progressive rate. However, this phenomenon was not observed in the other group infected with COVID-19 without fever or not infected. In addition, 49 (34.3%) of 143 persons confirmed that the COVID-19 infection resulted in changes in sexual function, with an increase in premature ejaculation diagnostic tool (PEDT) scores. The results of our self-controlled study indicate that COVID-19 is associated with impaired semen quality early after disease onset. After 1-2 spermatogenesis cycles, the semen quality gradually recovers from the infection. These findings are beneficial for better understanding the COVID-19-associated sequelae, which are fundamental for semen collection during assisted reproduction.

P-083 Short ejaculatory abstinence time of 2 hours results in higher pregnancy outcomes compared to the recommended abstinence period of 2-7 days

Abstract Study question What is the impact of a short abstinence (2h) compared to the recommended (2-7 days) ejaculatory abstinence time on embryo utilization rate and pregnancy outcome? Summary answer A short abstinence of 2 hours results in similar embryo utilization rates, but in reduced sperm DNA fragmentation and higher pregnancy rates. What is known already Semen parameters exhibit variability among and within individuals, and ejaculatory abstinence time (EA) is a known factor contributing to intra-individual variation. Despite the challenge of providing a specific recommendation for the optimal timeframe, recent studies suggested that a shorter abstinence is likely to decrease DNA fragmentation levels in semen and improve pregnancy and live birth rates following assisted reproductive technology. At present, ESHRE recommends a more limited range than the WHO manual, advising an ejaculatory abstinence of 3 to 4 days. Study design, size, duration This prospective proof-of-concept sibling oocyte study (n = 100 cycles), conducted between January 2018 and June 2023, required two sperm samples per patient. The initial sperm sample was produced on the day of oocyte retrieval following the requested abstinence period of 2-7 days. The second sperm sample was collected 2 hours later. Sibling oocytes were randomly allocated to injection with the first or second sample. Sibling oocytes of each patient were cultured under the same conditions. Participants/materials, setting, methods Inclusion criteria were ≥ 6 mature oocytes (MII), fresh ejaculates with concentration >1x106/ml, ICSI with subsequent blastocyst culture. Endpoints were pregnancy outcome, fertilization, blastocyst development, utilization rate, sperm concentration/motility and DNA fragmentation index (DFI) measured by TUNEL. Statistical analysis was done taking into account female age and clustering. A total of 104 couples consented to provide two samples on the day of oocyte retrieval and were enrolled. Main results and the role of chance Average abstinence periods of 98:48h±20:01 vs 02:05h±00:18 were observed. Sperm concentration (x106/ml), progressive forward motility and DFI, were 46.53±39.6 vs 37.0±37.1 (p < 0.001), 52.14%±20.1 vs 49.79%±20.4 (p = 0.152) and 6.76%±7.0 vs 5.37%±7.2 (p = 0.024), respectively. Of the 1282 collected oocytes, 634 were injected with the first sperm sample and 648 with the ‘2h EA sample’. Fertilization rates per MII (79.3% vs 80.7%, p = 0.154) and good-quality day 3 embryos (76.7% vs 77.6%, p = 0.276) were similar between both groups. From the 93 patients with blastocyst culture (with average female age of 33.9±5.0 years; average male age of 36.4±6.6 years), 1173 oocytes were stratified for injection: 580 (first sample) and 593 (second sample), respectively. Fertilization rates (81.4% vs 82.3%, p = 0.074) and good-quality day 5 blastocysts (46.2% vs 49.5%, p = 0.534) did not differ between the groups. Utilization rate per inseminated (30.5% vs 35.8%, p = 0.126) or fertilized (37.5% vs 43.4%, p = 0.153) oocyte was similar between the groups. Single fresh and frozen embryo transfers were compared between embryos generated after the requested (2-7 days; n = 91) and short (2h; n = 94) abstinence. Pregnancy rate was significantly higher after short abstinence: 49.5% (45/91) vs 62.8% (59/94); p = 0.01. Ongoing pregnancy rate was 31.9% (29/91) and 40.4% (38/94); p = 0.10. Limitations, reasons for caution This is a prospective proof-of-concept study, not powered to detect differences in utilization and pregnancy outcomes. Not all patient are capable of producing two sperm samples within 2 hours from each other. Wider implications of the findings The results indicate a significant, yet not relevant difference in DNA fragmentation index (DFI) but a relevant difference in pregnancy rate between the two groups. If increased live birth rates are obtained after short abstinence, recommendations concerning EA might need adaptation. Trial registration number NCT03323892

Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation.

The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9-GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.

Correlation analysis of age and MTHFR C677T polymorphism with sperm motility and sperm DNA integrity.

Several factors, including age and polymorphisms in genes such as MTHFR, affect sperm quality. However, the relationship between MTHFR C677T polymorphism and infertility remains unclear. Hence, the purpose of this study was to investigate the effects of age and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on the forward progressive sperm motility and sperm DNA integrity. For this purpose, a total of 845 men were recruited from the hospital's infertility clinic for semen analysis, sperm DNA integrity testing, and MTHFR C677T genotyping. The participants were grouped by age (<30 years, 30-35 years, >35 years) to determine the relationship between age and the progressive motility of sperms (PR%) and sperm DNA integrity. The effect of MTHFR C677T polymorphism on sperm motility and sperm DNA integrity was also analyzed in all the age groups. Results showed that PR% and DNA fragmentation index (DFI) differed significantly among the groups (P<0.001). The correlation analysis showed that age was positively correlated with DFI and negatively correlated with PR%. DFI correlated negatively with PR% (P <0.01). When the age groups were classified based on the C677T genotype, we observed that PR% and DFI did not differ significantly between different genotypes within the same age range (P>0.05). The conclusion was that age exerted a negative effect on sperm motility and sperm DNA integrity. Age correlated positively with DFI and negatively with PR%. MTHFR C677T polymorphism did not affect forward motile sperm count and sperm DNA integrity. Our observations will be useful for fertility guidance and MTHFR genotype interpretation in the clinic for couples of childbearing age.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

Influence of trans-ferulic acid on the quality of ram semen upon cold preservation.

Due to lower antioxidant capacity and higher amounts of polyunsaturated fatty acids, ram spermatozoa are very susceptible during cooling process. The objective was to examine the effect of the trans-ferulic acid (t-FA) on the ram semen during liquid preservation. Semen samples were collected from the Qezel rams, pooled, and extended with the Tris-based diluent. Pooled samples enriched with different amounts of the t-FA (0, 2.5, 5, 10, and 25mM) and preserved at 4°C for 72h. Spermatozoa's kinematics, membrane functionality, and viability were assessed by CASA system, hypoosmotic swelling test, and eosin-nigrosin staining, respectively. Moreover, biochemical parameters were measured at 0, 24, 48, and 72h. Results showed that 5 and 10mM t-FA improved forward progressive motility (FPM) and curvilinear velocity compared to the other groups at 72h (p < 0.05). Samples treated with 25mM t-FA showed the lowest total motility, FPM, and viability at 24, 48, and 72h of storage (p < 0.05). Higher total antioxidant activity levels were observed in the 10mM t-FA-treated group compared to the negative control at 72h (p < 0.05). Treatment with 25mM t-FA increased malondialdehyde amounts and decreased superoxide dismutase activity compared to other groups at the final time assessment (p < 0.05). Nitrate-nitrite and lipid hydroperoxides values were not affected by treatment. The current study indicates the positive and negative influences of different concentrations of t-FA on the ram semen upon cold storage.

The effect of varying concentrations of chicken plasma egg yolk and glycerol on the viability of canine sperm following short and long-term preservation.

Plasma egg yolk (PEY), due to simple preparation and easier access, could be a suitable alternative to raw egg yolk for preserving canine semen. The present study investigated suitable concentrations of PEY and glycerol for preservation of canine semen. Semen was collected by digital manipulation (seven replicates from four dogs). Following initial raw semen evaluation, the semen was diluted in a tris-based extender supplemented with varying concentrations of chicken PEY (0, 20, and 40% v/v) and glycerol (3%; v/v). After cooling the specimen to 4°C within 1 h, the specimens were diluted with an equal volume of freezing extender consisting of similar concentrations of chicken PEY and 0 and 7% glycerol to reach the final glycerol concentration of 1.5 and 5% for short-term storage of canine semen. Samples with different concentrations of PEY and 5% glycerol were frozen. The sperm viability parameters including total motility, progressive forward motility, plasma membrane integrity, and live percentage of sperm were assessed following short and long-term storage. Sperm viability parameters of semen extended in an extender supplemented with 20 or 40% chicken PEY with either 1.5 or 5% glycerol remained superior until 72 h after semen collection compared to the specimen that did not receive any PEY (P<0.05). Post-thaw sperm viability was also greater in samples extended in extender supplemented with either 20 or 40% PEY compared to 0% PEY. Tris-based extender supplemented with either 20% chicken PEY could be suitable for short and long-term preservation of canine semen.

Effects of wild, semi‐captive, and captive management on male Chiricahua leopard frog sperm quality with implications for conservation breeding programs

AbstractThe Chiricahua leopard frog (Lithobates chiricahuensis) is a threatened species endemic to the southwestern United States and Northern Mexico. Captive breeding programs were established to support reintroduction efforts, yet reproductive output has been lower than needed for recovery of the species. This study aimed to evaluate the effects of captivity on amphibian reproduction by (1) determining if captive, semi‐captive, and wild male L. chiricahuensis produce sperm at similar rates and concentration in response to hormone treatment; and (2) evaluating the quality of sperm obtained over time from these populations. Males from captive, semi‐captive, and wild locations were administered a combination of human chorionic gonadotropin and gonadotropin‐releasing hormone to stimulate sperm production and release. A high percentage of males in the captive (60%), semi‐captive (100%), and wild (95.3%) populations produced sperm following treatment. Sperm quality (forward progressive motility and total sperm motility) did not differ between groups. However, sperm quantity (sperm/ml) differed (p &lt; .05) between populations, with semi‐captive and wild males producing higher concentrations of sperm than captive males. These results suggest that Chiricahua leopard frog sperm quantity, but not quality, may become negatively impacted by long‐term captivity in indoor, controlled settings.

Enterotypes in asthenospermia patients with obesity

The essence of enterotypes is stratifying the entire human gut microbiome, which modulates the association between diet and disease risk. A study was designed at the Center of Reproductive Medicine, Shengjing Hospital of China Medical University and Jinghua Hospital of Shenyang. Prevotella and Bacteroides were analyzed in 407 samples of stool, including 178 men with enterotype B (61 normal, 117 overweight/obese) and 229 men with enterotype P (74 normal, 155 overweight/obese). The ratio between Prevotella and Bacteroides abundance, P/B, was used as a simplified way to distinguish the predominant enterotype. In enterotype P group (P/B ≥ 0.01), obesity was a risk factor for a reduced rate of forward progressive sperm motility (odds ratio [OR] 3.350; 95% confidence interval [CI] 1.881–5.966; P < 0.001), and a reduced rate of total sperm motility (OR 4.298; 95% CI 2.365–7.809; P < 0.001). Obesity was also an independent risk factor (OR 3.131; 95% CI 1.749–5.607; P < 0.001) after adjusting follicle-stimulating hormone. In enterotype P, body mass index, as a diagnostic indicator of a reduced rate of forward progressive sperm motility and a decreased rate of decreased total sperm motility, had AUC values of 0.627 (P = 0.001) and 0.675 (P < 0.0001), respectively, which were significantly higher than the predicted values in all patients. However, in enterotype B group (P < 0.01), obesity was not a risk factor for asthenospermia, where no significant difference between obesity and sperm quality parameters was observed. This study is tried to introduce enterotypes as a population-based individualized classification index to investigate the correlation between BMI and asthenospermia. In our study, overweight/obese men with enterotype P were found to have poorer sperm quality. however, sperm quality was not associated with overweight/obese in men with enterotype B. Thereof, BMI is a risk factor for asthenospermia only in men with enterotype P, but not in men with enterotype B.

P-117 A novel method to select spermatozoa with the best morphokinetic characteristics and superior genomic integrity

Abstract Study question Can an automated device based on membrane electrophoresis be used to select spermatozoa with superior morphokinetic characteristics and lower sperm chromatin fragmentation (SCF)? Summary answer Albeit at an expense of concentration, a membrane-electrophoretic device was able to isolate a proportion of spermatozoa with the highest motility, morphology, and genomic integrity. What is known already Conventional sperm selection based on density gradient centrifugation (DGC) can enhance the proportion of progressively motile spermatozoa; however, DGC methods are limited in their ability to select spermatozoa with higher genomic integrity. Membrane-based microfluidic technologies (MFSS) have already been used clinically to select for spermatozoa with a superior chromatin status; however, these methods are based on the intrinsic characteristics of motile spermatozoa capable of selecting themselves. By incorporating an extrinsic electrophoretic drive, membrane electrophoresis would overcome a limitation of progressive motility in the semen sample and be able to isolate viable gametes with better morphology and reduced SCF. Study design, size, duration From August 2020 to December 2021, semen samples from 68 men were evaluated by standard semen analysis and simultaneously processed by DGC or a novel membrane-electrophoretic sperm sorter (EPSS) to select for progressively motile spermatozoa. Concentration, motility, progressive motility, morphology, and SCF were measured and compared between raw, DGC-, and EPSS-processed specimens. Participants/materials, setting, methods Fresh ejaculates were evaluated by standard semen analysis according to WHO 2021 criteria. Following complete liquefaction, specimens were divided into two equal aliquots for DGC and EPSS sorting. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay under fluorescent microscopy; at least 500 spermatozoa were evaluated for each specimen with a normal threshold of ≤ 15%. Main results and the role of chance A total of 68 men with an average age of 38.1±6 years had the following average semen parameters: volume of 3.1±1 mL, concentration of 58.2±33 x106/mL, 43.3±9% motility, 40.5±9% forward progression motility, and a normal morphology of 2.9±0.8%. When comparing the final parameters between the EPSS and DGC methods, despite a reduced sperm concentration (7.3±9 x106/mL vs 45.0±36 x106/mL, P&amp;lt;0.0001, respectively), EPSS was highly selective for motile spermatozoa, yielding significantly higher motility (93.1±16% vs. 86.8±15% P&amp;lt;0.0001) and forward progressive motility (92.3±17% vs 85.2±15%, P&amp;lt;0.0001). Despite an overall lower sperm recovery rate (21.4±25% vs 75.4±17%, P&amp;lt;0.0001), normal morphology improved to 3.4±0.8% (P&amp;lt;0.0001) after EPSS but remained unchanged in the DGC-processed sample. Although both EPSS and DGC improved SCF from 12.2±9% in the raw specimen to 4.8±6% and 6.6±7% (P &amp;lt; 0.0001), respectively, EPSS outperformed DGC (P &amp;lt; 0.05). Moreover, the DGC method took up to 60 min to process, whereas the EPSS technique took a total of 6 min. Limitations, reasons for caution The selection of spermatozoa by EPSS is a promising technique to isolate progressively motile spermatozoa with enhanced morphology and superior chromatin integrity, albeit at a lower concentration. This is a preliminary study; the benefit and safety of this method must be further demonstrated by insemination or IVF treatment. Wider implications of the findings A membrane electrophoresis device may be a viable alternative method to MFSS to identify spermatozoa with superior morphokinetic characteristics and intact chromatin. Moreover, incorporating an automated device can reduce gamete processing time while minimizing labor costs and inter-operator errors. Trial registration number N/A

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm

For amphibian species that display external fertilization in an aquatic environment, hypoosmotic shock to sperm cells can quickly result in damage to cellular structure and function. This study sought to determine how fertilization media osmolality, temperature, and time impact the stability of the mitochondrial vesicle's association with the sperm head and thus motility and quality of forward progression. The presence of the mitochondrial vesicle and its relationship with sperm motility and quality of forward progression were analyzed in sperm samples from the Fowler's toad (Anaxyrus fowleri) (n = 10) when held for six hours under two temperatures and four osmolalities. Results indicated that the presence of the mitochondrial vesicle is needed for sperm motility over time (p < 0.001), where higher osmolalities (p < 0.001) and lower temperatures (p < 0.001) correlated with maintaining the vesicle attachment to the spermatozoa. The higher osmolality of spermic urine was the most important factor for maintaining higher quality of forward progressive motility (p < 0.01) of spermatozoa. Sperm samples held at 4 °C and 40 mOsm/kg had the longest half-life for motility (540 min) and quality of forward progression (276 min), whereas sperm held at 22 °C and 2.5 mOsm/kg had the shortest half-life for motility (7 min) and quality of forward progression (18 min). Special attention should be given to the osmolality and temperature of fertilization solutions, or breeding tank water, when developing cold storage protocols for toad sperm or reproducing animals to ensure the retention of the mitochondrial vesicle for maximum fertilization capability.

Development of assisted reproductive technologies for the conservation of Atelopus sp. (spumarius complex)

Amphibians are in peril, given the ongoing sixth mass extinction of wildlife. Thus, Conservation Breeding Programs (CBPs) are attempting to breed some species under laboratory conditions. The incorporation of assisted reproduction technologies (ARTs), such as hormonal stimulation, sperm collection and cryopreservation, and in vitro fertilization is contributing to successful CBPs. The objective of this study was to apply ARTs in sexually mature individuals of an undescribed species of Atelopus (spumarius complex) (harlequin frog). Our procedure involves hormonal induction of gametogenesis in this species. We were able to induce gamete release through administration of human chorionic gonadotropin (hCG) in males, and in females this has been achieved through the sequential administration of hCG (priming doses), and combinations of hCG with gonadotropin releasing hormone analogue, GnRHa (ovulary dose). We standardized sperm cryopreservation by performing toxicity tests of cryoprotectants, fast/slow freezing and thawing, as well as supplementation of non-penetrating cryoprotectants (sugars). Next, we performed in vitro fertilization, evaluated the fertilization capacity of the cryopreserved sperm, and describe external features of fresh and cryopreserved sperm. We found that 10 IU/g hCG induced the release of the highest sperm concentrations between 3 and 5 h post-injection, while 2.5 IU/g hCG induced the release of eggs in most treated females. Under cryopreservation conditions, the highest recovery of forward progressive motility or FPM was 26.3 ± 3.5%, which was obtained in cryosuspensions prepared with the 5% DMF and 2.5% sucrose. Cryopreserved sperm showed narrower mitochondrial vesicles after thawing, while in frozen samples without cryodiluent showed 31% of spermatozoa lost their tails. In most cases, our attempts of in vitro fertilization were successful. However, only ∼10% of embryos were viable. Overall, our study demonstrates that the development of ARTs in individuals of Atelopus sp. (spumarius complex) bred in laboratory can be successful, which result in viable offspring through in vitro fertilization. Our study provides a baseline for assisted breeding protocols applicable to other harlequin frogs of the genus Atelopus.

Protective effect of vitamin E on sperm parameters, chromatin quality, and DNA fragmentation in mice treated with different doses of ethanol: An experimental study.

BackgroundExcessive consumption of alcohol induces an increase in oxidative stress production and can lead to detrimental effects on the male reproductive system.ObjectiveTo evaluate the possible protective effects of coadministration of vitamin (vit) E on the detrimental changes in the sperm quality of mice administered ethanol.Materials and MethodsFifty-four BALB/c mice were categorized into nine groups (n = 6/each). The control group received a basal diet while the eight experimental groups received ethanol 10%; ethanol 20%; vit. E 100 mg; vit. E 200 mg; ethanol 10% + vit. E 100 mg; ethanol 10% + vit. E 200 mg; ethanol 20% + vit. E 100 mg; ethanol 20% + vit. E 200 mg. After 35 days, the sperm parameters and sperm chromatin were assessed.ResultsThe results demonstrated a significant reduction in the motility rate, normal morphology rate, viability rate, increase in abnormal DNA structure and packaging (TB staining), and DNA damage (TUNEL) in ethanol consumer groups. In addition, the findings showed a significant increase in the aforementioned parameters in ethanol- and vit. E-consumer groups compared to the ethanol-only consumer groups. The ethanol group received 20% of the most damage among the groups. The group receiving vit. E 100 mg and those receiving ethanol 10% + vit. E 200 mg gained the highest benefit among the groups.Conclusion Sperm forward progressive motility, normal morphology rate, and viability decreased in the ethanol groups. Also, the rates of spermatozoa with abnormal DNA structure and DNA fragmentation increased in the ethanol groups. Our findings revealed that the coadministration of vit. E and ethanol can protect destructive changes in DNA structure and damage.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species’ reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Royal jelly may improve sperm characteristics during preservation of rooster semen: Gene expression of antioxidant enzymes.

Sustainable production and the increasing number of embryonated hatching eggs are critical aspects of the poultry production industry. The present paper aims to appraise the effectiveness of royal jelly (RJ) on the semen characteristics of Native Mazandaran roosters in both liquid and frozen storage conditions. Semen collected from 10 sexually mature roosters and following dilution was supplemented with RJ at 0.0 (control), 5 (RJ5), 10 (RJ10), 20 (RJ20) and 40 (RJ 40) mg/ml. After cooling and freezing-thawing, the percentage of forward progressive motility, viability, abnormality, hypo-osmotic swelling test (HOST) and the mRNA abundance of antioxidant enzymes of spermatozoa were measured. Our results revealed that the addition of 5mg/ml RJ to the semen extender significantly increased (p<.05) the percentages of forward progressive motility, viability and HOST during liquid and frozen storage. The abnormality of spermatozoa in the RJ5 group was significantly lower compared to the other groups. During liquid storage, a significant decrease in forward progressive motility was found after 48hr in comparison with 24hr at 4°C. High levels of RJ (from 10 to 40mg/ml) were severely decreased the characteristics of rooster spermatozoa in comparison with RJ5 and the control group. The inclusion of RJ at 5mg/ml to the semen extender enhanced the mRNA transcript of antioxidant enzymes of spermatozoa during liquid preservation. The mRNA abundance of antioxidant enzymes did not influence by cryostorage. Overall, these data suggest that supplementation of RJ at 5mg/ml to the extender improved semen characteristics and redox status of rooster spermatozoa.

The association of seminal oxidation reduction potential with sperm parameters in patients with unexplained and male factor ınfertility.

ABSTRACTPurpose:Understanding the effects of high oxidation reduction potential (ORP) levels on sperm parameters will help to identify patients with unexplained and male factor infertility who may have seminal oxidative stress and determine if ORP testing is needed. This study aimed to evaluate the association between seminal ORP and conventional sperm parameters.Materials and Methods:A total of 58 patients who provided a semen sample for simultaneous evaluation of sperm parameters and ORP between January and September 2019 were enrolled in this retrospective study. To identify normal and high ORP levels, a static ORP (sORP) cut-off value of 1.36mV/106sperm/mL was used. Sperm parameters were compared between infertile men with normal sORP (control group, n=23) and high sORP values (study group, n=35).Results:Men with sORP values >1.36mV/106sperm/mL had significantly lower total sperm count (TSC) (p <0.001), sperm concentration (p <0.001) and total motile sperm count (TMSC) (p <0.001). In addition, progressive motility (p=0.04) and fast forward progressive motility (p <0.001) were significantly lower in the study group. A negative correlation was found between sORP and TSC (r=-0.820, p <0.001), sperm concentration (r=-0.822, p <0.001), TMSC (r=-0.808, p <0.001) and progressive motility (r=-0.378, p=0.004). Non-progressive motility positively correlated with sORP (r=0.344, p=0.010).Conclusions:This study has shown that TSC, sperm concentration, progressive motility and TMSC are associated with seminal oxidative stress, indicated by a sORP cut-off of 1.36mV/106sperm/mL. Presence of oligozoospermia, reduced progressive motilty or low TMSC in sperm analysis should raise the suspicion of oxidative stress and warrants seminal ROS testing.

Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa.

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.

Spermiation response to exogenous hormone therapy in hibernated and non-hibernated boreal toads (Anaxyrus boreas boreas).

Conservation programs for threatened high- elevation amphibian species rely on hibernation to trigger appropriate male reproductive behaviours and gametogenesis. Although common practice and anecdotal observations have supported the practice of hibernation, there is limited empirical evidence documenting the effects on reproduction in these species. In this study, the effect of hibernation on sperm quantity and quality was evaluated for the alpine species Anaxyrus boreas boreas . Hibernated (n =19) and non-hibernated (n =21) male toads were administered 10IUg-1 body weight (BW) human chorionic gonadotropin (hCG) and spermic urine was collected over 24h. Hibernation had no effect on the number of males undergoing spermatogenesis, but hibernated males produced sperm in higher concentrations. Sperm quality was measured in terms of total motility, forward progressive motility and quality of forward progression. Although there was no difference in the total sperm motility of samples from hibernated and non-hibernated toads, the percentage of sperm exhibiting forward progressive motility and the quality of forward progression was significantly greater from hibernated toads. These results support our hypothesis that hibernation impacts both sperm quantity and quality in male boreal toads. This study will better inform captive breeding management decisions for threatened alpine species, in imminent danger of extinction.

Oral administration of royal jelly may improve the preservation of rooster spermatozoa.

The aim of the present study was to investigate the effect of dietary supplementation of royal jelly (RJ) on liquid and frozen storage of rooster spermatozoa. Twenty-five 30-week-old of Mazandaran native breeder roosters were randomly divided into five treatments (n=5 roosters/group). Experimental treatments are designed to include a control group and various levels (0.0 (RJ0), 100 (RJ100), 200 (RJ200), 300 (RJ300) mgkg-1 BW-1 ) of royal jelly (RJ) that were fed to the roosters using force-feed method. The percentage of forward progressive motility, abnormal spermatozoa, membrane integrity and viability of spermatozoa evaluated after 24 and 48hr of cooling (at 4°C) and after the freeze-thawing process. Also, mitochondrial activity and DNA fragmentation in fresh (24hr) and post-thawed spermatozoa were assessed. The result of this study showed that the spermatozoa forward progressive motility, abnormality, membrane integrity, and viability were improved by the RJ100 group compared to the other groups after 24 and 48hr storage period at 4°C. The percentage of membrane integrity and forward progressive motility after freeze-thawing in the RJ100 group was significantly higher than the other groups, and the percentage of abnormal spermatozoa was lower. A significant decrease in semen quality parameters was seen after 24 and 48hr of refrigeration, but there was no observed change between 2 and 24hr in the RJ100. The viability percentage of spermatozoa in both RJ100 and RJ200 groups was not different. Moreover, after freeze-thawing, DNA integrity and mitochondrial activity in the RJ100 group were significantly higher than the other groups. According to our results, feeding of RJ at 100mgkg-1 BW-1 to the roosters was improved spermatozoa characteristics during liquid and cryopreservation conditions.