Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

In this study, we conducted a simultaneous analysis of sperm count and viability in rats by flow cytometry (FCM). Epididymal fluids were taken from the caudal epididymis of 12 to 13 week-old Sprague-Dawley rats. The fluids were weighed and mixed with Dulbecco's phosphate buffered saline (D-PBS). Propidium iodide, which can stain only dead sperm, was used to distinguish viable and dead sperm. The sperm count and viability analyzed by FCM were 1.28 x 10(6)/mg and 78.0%, respectively. These values were consistent with the corresponding values (1.39 x 10(6)/mg and 81.0%) that were directly determined microscopically in the fluids of the same sample. In addition, when the original mixture containing sperm was diluted two times and four times with D-PBS, or was diluted two times with D-PBS containing only killed sperm, the sperm count and viability determined by FCM also correlated well with the sperm count (r = 0.96, P < 0.01) and sperm motility (r = 0.99, P < 0.01) by direct microscopic observation, respectively. In conclusion, the present flow cytometric analysis would be practical for the simultaneous determination of sperm count and viability in rat epididymal fluids.

Nurture vs nature: how can we optimize sperm quality?

Assessment of sperm motility, viability and apoptosis in human spermatozoa after hydrogen peroxide exposure

Optimizing incubation conditions for the preservation of sperm motility in processed semen samples

Why the WHO recommendations for eosin-nigrosin staining techniques for human sperm vitality assessment must change.

HIGHLIGHTS Infertility has been afflicting an increasing proportion of our society. Majority of the male infertility features are attributed to oligospermia. The present corresponding article highlights the salient predispose factors. One of the dietary models that adhere to the fundamentals of a pro-fertility diet is the MD. Infertility has been afflicting an increasing proportion of our society, with an estimated more than 70 million couples of reproductive age encountering problems getting pregnant. Interestingly, half of them relate to male infertility issues in one way or the other1. Majority of the male infertility features are attributed to oligospermia (semen with low concentration of sperms), asthenozoospermia (nonmotility or decreased motility of spermatozoa), or teratozoospermia (spermatozoa with altered structure)2. The present corresponding article highlights the salient predispose factors responsible for causing male infertility problems, especially during the current era of western diets dominating the lifestyle, and the significant role of healthy dietary models with nutritional interventions such as Mediterranean diets (MDs) in enhancing fertility and ameliorating the problems related to male infertility. There has been an intricate and complex relationship between the qualitative and quantitative aspects of dietary nutrients and the quality of sperms, as evidenced through earlier studies where sperm energetic metabolism has been directly affected by not only the amount but the quality of nutrients as well. For example, a diet rich in saturated fats or a diet low in polyunsaturated fatty acids (PUFAs) having unbalanced omega-6/omega-3 PUFA negatively impact the sperm quality while supplementing the PUFA-rich diet ameliorates the above negative effects on sperm quality and preserving the male fertility3. It is suggested that a healthy diet based on plant foods or fish positively impacts sperm quality, increasing the mitochondrial enzyme activities responsible for gamete energetic metabolism and reducing oxidative damage, hence preventing male infertility. Genetic and environmental factors are known to contribute toward male infertility. Among environmental factors environmental pollution, age, smoking, emotional stress, tight clothing, exposure to high temperatures, excessive alcohol consumption, exposure to electromagnetic radiation and pesticides, and a sedentary lifestyle significantly affect male fertility1. It is certain that some factors like environmental pollution, age, or radiation cannot be evaded4–6. Nevertheless, it has been observed that antioxidants, such as resveratrol, protect the sperm from oxidative stress and may act as one of the alternative therapeutics for male infertility6,7. Moreover, recent studies pointed toward the association of diet modulating semen quality6,8,9. The 'Western diet' has emerged as the dominant dietary pattern in recent decades3. This diet is considered to be of high consumption of commercially processed foods, which are low in essential unsaturated fatty acids and dietary fiber but high in trans and saturated fats, simple carbohydrates, and animal proteins which are directly linked to augmenting oxidative stress that leads not only to metabolic disorders such as type 2 diabetes, obesity, and insulin resistance but also associated with a decline in fertility by decreasing the sperm quality10,11. Therefore, reactive oxygen species (ROS), proinflammatory cytokines, and aromatase activity, the enzyme that converts testosterone into estradiol, are all produced in greater amounts when white adipose tissue is present. However, type 2 diabetes, insulin resistance, and obesity in males increase the risk of secondary hypogonadism and reduced sex hormone-binding protein levels. Additionally, the process of fertilization and sperm motility are also negatively impacted by hyperglycemia3. The hallmarks of insulin resistance, hyperinsulinemia, and hyperglycemia, appear to be the cause of the decline in glucose uptake and metabolism in sperm12, possibly contributing to the impairment of sperm cells' glycolysis which serves as the main source of energy for spermatozoa13. This theory is supported by the findings that insulin treatment reversed the decreased sperm motility observed in diabetic male rats14. Additionally, the chronic proinflammatory condition of the testicular microenvironment and/or excurrent ductal system, caused by an increase in ROS15, which is what causes the decline in sperm quality, is significantly influenced by leptin, a hormone released from fat cells in adipose tissue15,16. Conversely, healthy dietary models are clearly connected with improved quality of sperms, implying that interventions based on nutrition may play an imperative role in the preservation of male fertility3. Male infertility prevention and/or treatment has also been shown to be quite successful with an appropriate intake of antioxidant molecules17. One of the dietary models that adhere to the fundamentals of a profertility diet is the MD. The MD is a manner of eating that is based on the classic recipes of Greece, Italy, and other Mediterranean Sea countries. Mediterranean food is popular in Italy, Greece, Turkey, Lebanon, Morocco, and a number of Middle Eastern and North African countries. As a result, no single MD exists. A significant portion of vegetables and fruit, whole grain goods, nuts, olive oil, and seafood are consumed frequently in MD; thus, this diet is labeled as rich in monounsaturated fatty acids, fiber, and antioxidants while low in saturated fatty acids. MD dietary model does bear similarities to the vegetarian diet (VD) model though VD lacks meat, meat products, seafood, etc. Some studies, though pointed out the negative impact of the VD model on semen quality18; however, it is still a matter of further investigation. The MD has been linked to numerous health benefits, mostly because of its lipid-lowering, anti-inflammatory, and antioxidant properties8. In observational studies, consuming an MD has also been linked to greater semen quality. Numerous dietary natural polyphenols present in vegetables, fruits, and edible plants have been demonstrated to affect ROS homeostasis, mitochondrial metabolism, and biogenesis19,20. These plant bioactive compounds may have a significant contribution in the enhancement of male reproductive performance by modulating mitochondrial function and reducing oxidative stress1,3. The influence of fatty acids and cholesterol on testicular glutamyl aminopeptidase and gamma glutamyl transpeptidase activities, as well as favorable effects of a diet high in virgin olive oil, a key component of the MD, on male fertility, may be mediated in part by an increase in testicular sol dipeptidyl peptidase IV activity21. Foods high in flavonoids, which are also a part of the MD, have been linked to several biological processes that can protect male fertility22. Curcumin, which is found in the powdered rhizome of Curcuma longa used in the MD (turmeric latte and Mediterranean yellow rice), is a potent antioxidant that aids in the direct elimination of ROS and the indirect activation of antioxidant enzymes. Low-carbohydrate diets have been found to reduce sperm quality and damage testicular histology; therefore, curcumin supplements may improve the impaired function of sperms and testis by reducing inflammation, oxidative stress, and apoptosis23. In men with low-quality sperm, antioxidant-rich diets may enhance sperm quality by reducing oxidative stress-induced sperm damage and increasing hormone production, spermatozoa concentration, motility, and morphology24. Men's fertility and sperm quality are also improved by increasing consumption of monounsaturated and PUFA in foods such as whole grain cereals, fish, seafood, and low-fat dairy products25. The benefits of MD can be strengthened further by increasing physical activity as part of a well-balanced healthy lifestyle. The role of simple and complex carbohydrates in diet affecting sperm function is yet to be established. However, some evidence clearly suggest the negative impact of excess simple carbohydrates on sperm quality. Moreover, the protein-deficient diet or diet lacking specific amino acids does also form a strong basis as a potential risk for male infertility. The main cause of male infertility is oxidative stress; therefore, the proper synthesis, function, and vitality of spermatozoa depend on the balance between ROS and antioxidants. Molecules of life like fats, carbohydrates, and proteins are known to affect sperm quality as they presumably act upon the oxidative stress and testosterone levels through mitochondria which are the powerhouse for energy production, mediating ROS homeostasis, and hormone biosynthesis, thus affecting male fertility, while the effect is reversed by PUFA and polyphenol-rich diet. It would be beneficial to conduct future clinical trials with large sample sizes that focus on combining several antioxidants with taking advantage of their combined mechanisms of action for promoting ameliorating male fertility. People who follow the MD or any other anti-inflammatory diet may occasionally eat significantly more or less fruit, vegetable, or dairy items than other people. Despite the use of dietary indices to identify and enhance anti-inflammatory diet adherence, there is a lack of agreement over the application of certain MD methods across various nations and areas. Because there is uncertainty surrounding the definition of anti-inflammatory diets, there is a lot of variety in how they are operationalized. In order to produce high-quality nutrition intervention studies, future research should concentrate on resolving the drawbacks. An overview of the influence of the MD in improving male fertility and ameliorating infertility problems in men is depicted in Figure 1.Figure 1: Dietary influence on male infertility. The dietary fibers enriched Mediterranean diet through gut microbiota homeostasis, bringing about the regulation of metabolism as well as pH, further resulting in an increase in seminal volume and sperm concentration leading to an overall improvement in male infertility. Moreover, unsaturated fatty acids [polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA)] could reduce oxidative stress and cholesterol, followed by lowering DNA fragmentation, again leading to improved sperm vitality, count, motility, and quality as well. Similarly, polyphenols exert antioxidative effects resulting in the lowering of proinflammatory factors and preventing sperm agglutination, hence leading to improved male infertility.There seems to be a major influence of the dietary regimen on sperm quality, especially the MD, but still, there are a variety of other lifestyle risk factors as well, such as excess alcoholism, smoking, drug addiction, obesity, psychological stresses, overuse of caffeinated drinks, and sedentary lifestyle with lack of physical exercises26. Moreover, there is a strong possibility of biomagnification of the underlying negative consequences in subsequent generations. In order to mitigate and ameliorate the negative impacts, one has to extensively work on dietary and other lifestyle modifications which could further improve the male reproductive potential. Though there is a strong need to delineate the individual risk factors involved and the sequence of events as an outcome of multiple factors involved simultaneously. However, the role of diet is certainly at center stage in the current context, as male infertility cases are constantly on the rise. Maintaining a positive dietary and healthy lifestyle, along with early counseling and appropriate clinical interventions, do hold the key to cope up with the impact of risk factors influencing sperm quality and male reproductive health. Given the relevance of the role that diet plays in male infertility, which is occurring at an exponentially increasing rate today, additional studies concentrating on the underlying molecular mechanisms of action of natural substances and nutrients are essential to formulate innovative dietary approaches to preserve and enhance male reproductive potential and treat male fertility problems. Ethical approval Not applicable. Sources of funding No funding was received. Author contribution N.S.: conceptualization, data curation, and writing – original draft preparation, reviewing, and editing. U.S., V.S., A.D., A.K.S., and K.D.: data curation and writing – original draft preparation, reviewing, and editing. T.B.E.: writing – reviewing and editing, visualization, and supervision. Conflicts of interest disclosure The authors declare that they have no financial conflict of interest with regard to the content of this report. Research registration unique identifying number (UIN) None. Guarantor Talha Bin Emran, PhD, Associate Professor, Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh. Tel: +88 030 335 6193, fax: +88 031 255 0224. https://orcid.org/0000-0003-3188-2272. Data statement The data in this correspondence article is not sensitive in nature and is accessible in the public domain. The data is therefore available and not of a confidential nature. Provenance and peer review Not commissioned, internally peer-reviewed.

The clinical content of preconception care: preconception care for men

Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique

We investigated the application of flow cytometric analysis to evaluate the rat sperm viability and number in the male reproductive toxicity studies. Flow cytometric procedure has been developed to evaluate sperm number and viability that uses fluorescent dye (propidium iodide, PI) to distinguish between viable (negative staining) and dead (positive staining) sperm. Sperm samples were collected from the caudal epididymides of SD rats (13–21 weeks old). PI staining patterns/viabilities were compared among several ranges of sperm concentrations, and several kinds of sperm viability. Viabilities determined by flow cytometry (FC) were also compared with motility by direct microscopical observation in several kinds of sperm viability. No notable changes in the PI staining patterns/viabilities were observed in the range from 1 × 105 to 6 × 106 sperm/ml. Essentially similar results were obtained from both FC and microscopical analyses for three degrees of viability sperm: live sperm (general preparation as a control), weakly viable sperm (mixed by vortex mixer for 30 seconds), and dead sperm (treated with 90°C or Triton X‐100).Viabilities of normal rat samples were 95.0 ± 4.0% in FC and 93.7 ± 4.6% in microscopical observation, indicating good correlation in both analyses. Sperm numbers with FC analysis were approximately 0.8 × 106 to 1.8 × 106 sperm per mg indicating good correlation with those by microscopy.It was concluded that the present flow cytometric procedure was objective, rapid and reliable, and that it was one of the useful methods for measuring the number and evaluating the viability of sperm collected from the caudal epididymides of rats in the male reproductive toxicity studies.

Human semen contains a large number of macromolecules, including proteins/enzymes and carbohydrates, regulating and protecting sperm cells. Proteomic analysis of human seminal fluid led to the discovery of semen amyloids derived from short peptide fragments of the proteins prostatic acid phosphatase (PAP) and semenogelin (SG) which are known to play a crucial role in enhancing HIV infection. However, the relevance of their existence in human semen and role in maintaining sperm behavior remains unclear. Distinct physiological, biochemical, and biophysical attributes might cause these amyloids to influence sperm behavior positively or negatively, affecting fertilization or other reproductive processes. We assessed the direct effect of amyloids derived from a PAP248-286 fragment, on sperm motility and viability, which are crucial parameters for assessment of sperm quality in semen. Co-incubation of human sperm with PAP248-286 amyloids at normal physiological concentrations formed in buffer led to significant reduction in sperm viability, though approximatelya 10× higher concentration was needed to show a similar effect with amyloid formed in seminal fluid. Both forms of PAP248-286 amyloid also had a significant impact on sperm motility at physiological levels, in agreement with a previous report. Our study suggests that PAP248-286 amyloids can directly influence sperm motility and viability in a concentration-dependent manner. We hypothesise that the direct toxic effect of PAP248-286 amyloid is normally mitigated by other seminal fluid ingredients, but that in pathological conditions, where PAP248-286 concentrations are elevated and it plays a role in determining sperm health and viability, with relevance for male fertility as well as sterility.

1. A relatively new instrument known as a Sperm Quality Analyzer® (SQA) offers a rapid assessment of sperm quality and quantity by providing a sperm quality index (SQI). The SQA measures a combination of the intensity of sperm activity and motile concentration by determining the number and amplitude of sperm movements per second in a capillary tube as detected through light beam interference. 2. Because the SQA has not been tested for its potential use in turkeys, the objective was to determine if the SQA could accurately respond to changes in turkey sperm concentration, viability, and motility in semen collected from turkey breeders. 3. The effect of varying concentrations of sperm on SQI values was evaluated by diluting replicate pools of semen from 4 different aged turkey breeder flocks with saline. Results from all 4 flocks showed that semen dilutions greater than 20-fold resulted in a linear decline in SQI values. 4. Additional in vitro analysis evaluated the effects of turkey sperm viability on the SQI under conditions of constant sperm concentration. Incubated, live sperm was mixed in various proportions with thawed, dead sperm to determine changes in viability. Increased proportions of dead sperm caused a decline in the SQI. 5. To assess sperm motility, turkey semen was incubated under either aerobic (motile) or anaerobic (immotile) conditions. Varied amounts of immotile and motile sperm samples were mixed. A linear increase in the SQI was observed as per cent motile sperm increased. 6. These results indicate that the SQA can respond to differences in turkey sperm concentration, viability, and motility using in vitro analyses.

Background:This study aimed to examine the effect of vitamin E injection and seasonal changes on semen characteristics (pH, ejaculation volume, mass motility, progressive motility, sperm concentration, live sperm, dead sperm, and abnormal sperm) in rams.Aim: This study aimed to evaluate the effectiveness of vitamin E injections in improving semen characteristics in rams.Methods: Twenty-one healthy rams were randomly divided into three groups (7 animals for each group) as follows: (1) control group injected with saline solution, (2) rams injected with 2 ml of vitamin E, and (3) rams injected with 4 ml of vitamin E. All doses were injected intramuscularly every 2 weeks during the cold season from December to the end of February 2022 and the hot season from June to the end of August 2023.Results: The results showed significant (p <; 0.05) differences between the treated groups in most of the semen characteristics. In summer, all of the semen characteristics were significantly increased (p <; 0.05), except for the count of dead and abnormal sperm, compared with those in winter. Rams treated with 4 ml of E vitamin caused a significant increase (p <; 0.05) in all semen characteristics, while this dose significantly decreased (p <; 0.05) the percentage of dead and abnormal sperm in comparison with the control.Conclusion:The study suggests that treating rams with 4 ml of vitamin E during the hot season could improve semen characteristics such as pH, ejaculation volume, mass motility, progressive motility, sperm concentrations, and live sperm.

In order to find an optimal hypo-osmotic swelling test (HOST) and to identify viable sperm cells from patients with asthenozoospermia for intracytoplasmic sperm injection (ICSI), we tested single motile and non-motile spermatozoa in four hypo-osmotic solutions by micromanipulation. The four solutions were: A, H2O; B, 50 mOsm NaCl; C, 150 mOsm NaCl and D, 150 mOsm sodium citrate and fructose. Eosin Y staining was then carried out for evaluation of viability of the spermatozoa after HOST. Using motile spermatozoa, no statistical difference was found in HOST-positive spermatozoa between these four solutions. There were more viable sperm cells after HOST in solutions C and D, as noted by Eosin Y staining. After non-motile spermatozoa were incubated for 1 min in the four solutions, HOST with solution C gave the best results for identification of viable sperm cells compared to the other three solutions. When motile spermatozoa were incubated in solution C or solution D for 30 min, the result of HOST with solution C (10.8% dead spermatozoa) was superior to that of solution D (49.1% dead spermatozoa). In conclusion, the HOST protocol using 150 mOsm NaCl (solution C) for 1 min yielded the best results for selection of viable spermatozoa. This procedure should be used for selection of viable spermatozoa for ICSI in patients with 100% non-motile spermatozoa.

The objective of this study was to assess the association of exposure to metal mixtures with semen quality and sperm DNA integrity of coke oven workers (n = 96). Urinary six metals (cadmium, lead, arsenic, zinc, selenium, and copper) were quantified using inductively coupled-mass spectrometry. Semen quality parameters included sperm concentration, sperm concentration, sperm motility, sperm morphology, and sperm viability. Sperm DNA fragmentation and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) adducts served as biomarkers for assessing sperm DNA integrity. Bayesian kernel machine regression with the hierarchical variable selection process was used for analyzing both individual and joint effects of the metal mixture on the outcomes of semen samples, while adjusting for age, smoking, alcohol consumption, job length, and body mass index. The metal mixture was associated with reduced sperm concentration, motility, viability, and normal morphology. It was novel that a dose-response relationship was observed between exposure of the metal mixture and semen quality. Among the metals tested, cadmium had a reverse relationship with sperm motility, viability, and normal morphology, and a non-linear relationship with sperm viability and sperm motility. The metal mixture and individual metals were not associated with sperm DNA fragmentation and 8-oxodGuo. In conclusion, exposure to metal mixtures and cadmium may exert an association with semen quality and had no association with sperm DNA breakages.