Nickel-induced oxidative stress causes cell death in testicles: implications for male infertility.

Aligarh region is well known for its lock industry. This lock industry utilises nickel for electroplating. There have been informal reports of infertility in men and women living near the lock industry. We analysed field water samples to investigate this link, and the results showed considerable nickel contamination. To further validate our results, we exposed male rats to relevant nickel levels in drinking water. This experimental exposure resulted in abnormal sperm morphology, decline in sperm count, significant change in activities of antioxidant enzymes, pronounced oxidative stress in the rat spermatocytes and decrease in serum testosterone level, as well as damage in the hypothalamus and pituitary (in all cases, the changes were most significant at the highest concentration used i.e 2.5 mg/l). The breeding experiments showed decline in live birth rate, while pups did not survive post birth in cages where males were given 2 and 2.5 mg/l concentrations of nickel in drinking water prior to mating. Our data strongly indicate a link between industrial nickel exposure and male infertility.

Influence of Storage Time on the DNA Integrity and Viability of Spermatozoa of the Spider Crab Maja brachydactyla.

Natural populations of the spider crab Maja brachydactyla constitute a fishery resource of great economic importance in many countries. As in the rest of eubrachyurans, the females of this species have ventral-type seminal receptacles where they store sperm from copulations. Sperm can be stored in these structures for months and even years before egg fertilisation, with the consequent degradation of the sperm cells during the time. In this work, we analyse the viability and the possible genetic damage in sperm accumulated in the seminal receptacles of M. brachydactyla females as a function of the storage time (from 0 to 14 months) using the comet assay technique. On one hand, we developed an algorithm for comet image analysis that improves the comet segmentation compared with the free software Open comet v1.3.1 (97% vs. 76% of detection). In addition, our software allows the manual modification of the contours wrongly delimited via the automatic tool. On the other hand, our data show a sharp decline in sperm viability and DNA integrity in the first four months of storage, which could lead to a decrease in the fecundity rate and/or viability of the embryos or larvae from the second and third clutches of the annual cycle if the repair capacity in these gametic cells is low.

Characterization of sperm and implications for male fertility in the last of the Rhynchocephalians.

Managing a species of conservation concern can be best achieved when there is information on the reproductive physiology of both sexes available; however, many species lack this critical, baseline information. One such species, the tuatara (Sphenodon punctatus), is the last surviving member of one of the four reptile orders (Rhynchocephalia) and is the only reptile known to lack a male intromittent organ. Culturally and evolutionarily significant, the conservation of this species is a global priority for the maintenance of biodiversity. In light of this, we characterized the morphology, viability and swim speed of mature tuatara sperm for the first time. We found that tuatara sperm are filiform and bear the remarkably conserved three-part sperm structure seen across the animal kingdom. Tuatara sperm are long (mean total length 166 µm), with an approximate head:midpiece:tail ratio of 15:1:17. While tuatara sperm are capable of high levels of within-mating viability (94.53%), the mean viability across all samples was 58.80%. Finally, tuatara sperm had a mean curvilinear velocity swim speed (µ × s - 1) of 82.28. At the population level, there were no differences in viability or mean swim speed between sperm collected from a male's first mating of a season and repeat matings; however, the maximum sperm swim speed increased in observed repeated matings relative to first matings. Interestingly, faster sperm samples had shorter midpieces, but had greater viability and longer head and tail sections. This work expands our understanding of male reproductive characteristics and their variation to a new order, provides wild references for the assessment of captive individuals, lays the groundwork for potential assisted reproductive techniques and highlights variation in male reproductive potential as an important factor for consideration in future conservation programs for this unique species.

Changes of the Protein CoAlation Pattern in Response to Oxidative Stress and Capacitation in Human Spermatozoa.

The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H2O2), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H2O2 and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation.

Lactate-induced spontaneous acrosomal exocytosis as a method to study acrosome function in stallion sperm.

Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 µM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.

Spermatophore development in drones indicates the metabolite support for sperm storage in honey bees (Apis cerana).

Developing effective long-term sperm storage strategies to maintain activity requires an understanding of the underlying spermatophore developmental phase in drones. Here we compared the developmental processes and metabolites about seminal vesicles of drones from different parentages (0-24 d)in honeybee colonies, including mated queens, virgin queens, and worker bees. The results showed a similar developmental trend of seminal vesicles in thethree groups of drones on the whole, although there were significant differences in developmental levels, as well as in other indicators. Correlation analysis showed significant positive correlations between seminal vesicle width and sperm viability. The metabolomics of the seminal vesicles in drones from mated queens showed differences of the metabolites in each stage. Particularly, squalene identified among them was validated a protective effect on sperm vitality in vitro experiments. Together the results of these assays support that there were significant differences in the developmental levels of seminal vesicles among the three groups of drones in honeybees, wherein a significant correlation between sperm viability and the developmental levels of seminal vesicles were dissected. The metabolomics analysis and semen storage experiments in vitro display signatures of squalene that may act as an effective protective agent in maintaining sperm viability. Collectively, our findings indicate that spermatophore development in drones provides metabolite support, which contributes to research on the differences of sperm viability among drones in the future.

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

A novel, patented method for semen collection in dromedary camel (Camelus dromedarius).

In the current article, a developed, patented method denoted the 'Camel Semen Collection Kit-CSCK', was designed to solve the problem of semen collection in dromedary camels. CSCK is composed of three main parts: (1) Semen collection sac: made from supersensitive flexible low-density polyethylene- (LDPE); (2) Metal stainless steel applicator: designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of a female camel through air insufflation; (3) Fixation sticker: a cushion sheet sticker is used to secure the outer portion of the collection sac to the female's perineal area. Semen was collected twice a week from eight dromedary bulls by using electroejaculation (EJ), artificial vagina (AV) and CSCK. Successful semen collections were 81.3%, 84.4% and 43.8% using EJ, CSCK and AV techniques respectively. Semen obtained by EJ technique showed lower semen volume, gross activity, sperm concentration, total sperm motility and percentage of live sperm cells compared to the other two techniques. Semen collected by CSCK showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared to AV technique. The advantages and disadvantages of the three techniques were compared and discussed. In conclusion, CSCK represents a practical and easy method to reliably collect high-quality semen from any untrained male dromedary camel and may facilitate the widespread application of assisted reproductive technologies (ARTs) on a large scale in this species.

Sperm Quality Parameters and Oxidative Stress: Exploring Correlation in Fluoride-Intoxicated Rats.

Background: Assessment of male fertility needs evaluation of sperm quality parameters, namely sperm count, viability, motility and morphology. Aims: The present study aimed to analyse and correlate oxidative stress with sperm quality parameters. Settings and Design: The male Wistar albino rats, weighing between 100 and 150 g, were employed in the present study under the Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines with ethical clearance from the Institutional Ethical Committee. These rats were categorised into four groups with six rats in each as control and test animals. Materials and Methods: Young male Wistar albino rats, weighing between 100 and 150 g, were divided into four groups of six rats each. The first group of rats served as control (n = 6) and was maintained under normal laboratory condition and was provided with clean drinking water, whereas rats in the second (n = 6), third (n = 6) and fourth (n = 6) groups were orally intubated with sodium fluoride of 100 ppm, 200 ppm and 300 ppm, respectively, for 40 days. Statistical Analysis Used: After the treatment period of 40 days, animals were sacrificed and alterations in sperm quality parameters were analysed by complete randomised design SAS 9.4 and Statistical Package for the Social Sciences (SPSS) IBM 17 and judged significant if P < 0.05. Results: In the experiment, a negative correlation emerged between sperm motility, viability, count versus malondialdehyde (MDA) levels, whereas the level of MDA has a positive correlation with sperm abnormalities. Sperm motility, viability and count were positively correlated with activities of superoxide dismutase and catalase, whereas decreased activities of antioxidants were related to increased sperm morphological abnormalities. Conclusion: These results suggest that MDA causes a decline in sperm motility, count and viability and an increase in morphological abnormalities via oxidative damage of membrane lipids.

Analysis of reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin.

Cypermethrin contamination was a potential threat to soil organisms. In the present work, reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin was investigated. It was found that earthworms could absorb and accumulate residual cypermethrin in soil, and also earthworm activities helped accelerate the degradation of cypermethrin in soil. The accumulation of cypermethrin in earthworms induced sperm damage, and cypermethrin not only caused the imbalance of calcium homeostasis in earthworm sperm cells by inhibiting earthworm sperm Ca2+-ATP and Ca2+-Mg2+-ATP enzyme activities but also caused barriers in acrosome reaction. It also affected sperm energy supply of earthworms by inhibiting the activity of Na+-K+-ATPase and Mg2+-ATPase of earthworm sperm. Meanwhile, the inhibition of acrosome enzyme activity of earthworm sperm by cypermethrin led to hinder fertilization and reduced cocoon production of earthworms, and the damage of cypermethrin to sperm of earthworm was a significant cause of its reproductive toxicity. The results of the evaluation of IBR index showed that reproductive toxicity of cypermethrin to earthworms reduced with the increasing time. The decreased reproductive toxicity of cypermethrin to earthworms at the later stage of exposure (42-56 d) might be due to a combination of reduced absorption of cypermethrin in soil by earthworms, decreased accumulation of cypermethrin in the body, and improved sperm capacitation.

Green Tea Extract in the Extender Improved the Post-Thawed Semen Quality and Decreased Amino Acid Mutation of Kacang Buck Sperm.

This study was the first to combine the addition of antioxidants to a skim milk-egg yolk (SM-EY) extender and different equilibration periods to obtain higher quality post-thawed Kacang buck semen. This study aimed to determine the effects of green tea extract (GTE) on the quality of frozen Kacang goat sperm equilibrated for one and two hours. The pool of Kacang buck ejaculate was equally divided into four portions and was diluted in an SM-EY extender that contained four doses of 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 groups, respectively. The aliquots were treated for an equilibration period of 1-2 h before further processing as frozen semen. Post-thawed semen quality was evaluated for sperm quality. The Sanger method was used for DNA sequencing, and the amino acid sequence was read using MEGA v.7.0. The post-thawed semen of the T2 group that was equilibrated for one hour had the highest semen quality. Pre-freezing motility had the highest determination coefficient compared to post-thawed sperm motility. This study is the first to report amino acid mutation due to freeze-thawing. The frequency of amino acid mutations revealed that T2 was the least mutated amino acid. Glycine, valine, leucine, serine, and asparagine strongly correlated to post-thawed sperm motility. It can be concluded that a combination of 0.1 mg GTE/100 mL extender as an antioxidant and one-hour equilibration period resulted in the best post-thawed Kacang buck semen quality.

How does sperm apoptosis affect the outcome of intrauterine insemination and intracytoplasmic sperm injection?

Up to 20% of male infertility is caused by abnormal DNA organization of the sperm and anomalies of the sperm apoptosis. The aim of this study was to investigate the sperm DNA apoptosis and viability in patients undergoing intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI). In the second part of the analysis, sperm DNA apoptosis and viability were investigated in patients with oligozoospermia and normospermia respectively. A total of 45 IUI and 38 ICSI patients were included in this study. Annexin V analysis was performed to investigate the sperm viability, and TUNEL assay was used to evaluate the sperm DNA apoptosis. Further investigations using 12 oligozoospermia and 11 control samples for sperm viability and sperm DNA apoptosis at different incubation periods and temperatures were performed. The results of this study showed a negative correlation between the sperm DNA apoptosis in IUI patients, but no relationship was observed for the ICSI patients. The second part of this study showed that incubation of semen samples at 37°C for 3 h has detrimental effects on the sperm DNA integrity. In conclusion, the incubation of semen at high temperatures affects the sperm quality. The results of this study showed that these tests can be beneficial for the infertile couples to achieve pregnancy.

Reproductive effects of sulfoxaflor in male Sprague Dawley rats.

The study objective was to evaluate the potential reproductive toxicity of sulfoxaflor (SFX) insecticide in male Sprague Dawley rats. To attain these objectives, forty male Sprague Dawley rats of 10-12 weeks old were randomly divided into four equal groups; the 1st group was used as a control group; the other three groups were exposed to 25, 100, and 500 mg/kg body weight SFX by oral gavage for 4 weeks. Relative testicular weight, testosterone, FSH, LH, MDA, and GPx levels, sperm viability, sperm morphology, sperm DNA damage, and histopathological changes in testes, epididymis, and seminal vesical of these rats were investigated after 4 weeks. The results showed that SFX exposure resulted in a significant increase in FSH, LH, MDA, and GPx levels as well as the percentage of dead and abnormal sperms and DNA damage in rat sperms. Histopathological examination of testes established testicular degeneration with coagulative necrosis as well as the proliferation of interstitial connective tissue infiltrated with inflammatory cells with congestion of intertubular blood vessels in epididymis and degeneration of lining epithelium of seminal vesicles.

The reversible effects of gossypol toxicity on male pigeons' reproductive performance.

Background and Aim: Gossypol, a cotton seed derivative, is well known for its reversible antifertility in male reproduction across species. Its antifertility and reversibility effects on male reproductive function vary among species in dose-and time-dependent manners. In this study, the antifertility potential of gossypol in pigeons was evaluated for the first time to determine whether it might be used as a dietary supplement for pigeon population control. Materials and Methods: Male pigeons were assigned into three experimental groups: The gossypol-treated group (n = 12), the sham control group (n = 6), and the negative control group (n = 6). There were two experimental periods: A gossypol-feeding period of 28 days and a gossypol-free period of 28 days. During the gossypol-feeding period, birds in the gossypol-treated group were fed 4 mg of gossypol extract per day. Birds in the sham control group were fed 0.5 mL of mixed ethanol and sunflower oil, while those in the negative control group were fed 0.5 mL of phosphate buffer saline. After the gossypol-feeding phase was completed, all remaining pigeons in all groups continued to receive their regular diet for an additional 28 days (gossypol-free phase). The body weight and semen quality of the birds in the experimental groups were compared to evaluate gossypol's antifertility effect. Results: In the gossypol-treated group as compared to the control groups, the percentages of sperm motility and viability were significantly lower at 21 days, and the percentage of normal sperm morphology was significantly lower at 28 days during the gossypol-feeding period. After gossypol withdrawal, these antifertility effects were resumed and reached a comparable semen quality to the control groups within 14 days. Conclusion: Gossypol supplementation (4 mg/day for 28 days) could lower male pigeons' reproductive performance in terms of sperm motility, viability, and sperm morphology. Such infertility was, however, reversible within 14 days after gossypol withdrawal without any side effects on the pigeons, suggesting its application as a safe contraceptive feeding for male pigeons.

Redox Regulation to Modulate Phosphorylation Events in Human Spermatozoa.

Significance: Spermatozoa are complex and compartmentalized cells that undergo capacitation, a series of biochemical and morphological changes to acquire the ability to fertilize oocytes. Reactive oxygen species (ROS) have a prominent dual role in capacitation. At physiological levels, ROS regulate numerous cellular processes, including increases of cyclic adenosine monophosphate, calcium, and activation of phosphorylation events needed for capacitation. On the contrary, at high concentrations that do not impair sperm viability, ROS can cause loss of motility and inhibition of capacitation. Higher ROS concentrations promote oxidation of lipids, proteins, and DNA leading to cell death, and these damages have been associated with male infertility. Critical Issues: When incubated under specific conditions, spermatozoa can produce low and controlled amounts of ROS that are not harmful but instead regulate numerous cellular processes, including the phosphorylation of tyrosine, serine, and threonine residues in critical proteins needed for sperm capacitation. Here, we outline the complex redox signaling in human spermatozoa needed to achieve fertility and the role of ROS as physiological mediators that trigger phosphorylation cascades. Moreover, we illustrate the importance of various phosphoproteins in spermatozoa capacitation, viability, and hyperactive motility. Future Directions: Further studies to elucidate the different phosphorylation players during sperm capacitation and acrosome reaction (the regulated exocytotic event that releases proteolytic enzymes allowing the spermatozoon to penetrate the zona pellucida and fertilize the oocyte) are essential to understand how the spermatozoon acquires the fertilizing ability to fertilize the oocyte. This knowledge will serve to develop novel diagnostic tools and therapy for male infertility. Antioxid. Redox Signal. 37, 437-450.

In vitro spermicidal effect of Thevetia Peruviana leaves on human spermatozoa.

An in vitro spermicidal effect of aqua-methanolic (2:3) extract of Thevetia peruviana leaves on human spermatozoa was evaluated in a dose-dependent manner (20, 40, 80 and 160 mg/ml) at a 1:1 ratio. Sperm motility, viability, hypo-osmotic swelling (HOS) and acrosomal status and function tests were performed immediately (20 s), and after 5 and 10 min of exposure of the spermatozoa to the extract of Thevetia peruviana leaves at different dose concentrations. Nuclear chromatin decondensation (NCD) test, DNA fragmentation test and sperm revival test were also evaluated. The sperm motility was affected immediately at a dose of 20 mg/ml and reduced gradually at doses of 40 and 80 mg/ml of Thevetia peruviana extract. Complete immobilisation of spermatozoa was observed at 160 mg /ml dose of this extract treatment within 5 min. 50% immobilisation of spermatozoa (EC50) was observed at 28 mg/ml dose of Thevetia peruviana extract within 20 s. The sperm viability decreased significantly at a higher concentration of extract, and all spermatozoa were found to be non-viable after 10 min when treated with 160 mg/ml dose of Thevetia peruviana extract. HOS and NCD of spermatozoa also reduced gradually at a higher concentration of extract administration. The percentage of DNA damage in spermatozoa was four times greater than in the control group. The findings indicate that the hydro-methanolic extract of Thevetia peruviana leaves possesses appreciably potent spermicidal activity through an in vitro model, which may explore an effective vaginal contraceptive.

Bull sperm selection by attachment to hyaluronic acid semi-interpenetrated hydrogels.

We report the development of a hydrogel-based approach to select bull spermatozoa, a crucial step for successful assisted reproductive techniques (ARTs). Hyaluronic acid (HA) semi-interpenetrated N-isopropylacrylamide (PNIPAM) co-20% N-Tris (hydroxymethyl) methyl acrylamide (HMA) hydrogels were synthetized on glass surfaces and cultured in presence of frozen-thawed bull spermatozoa. A fraction of motile bull spermatozoa population strongly attached to hydrogels and was partially released by treatment with hyaluronidase. Fifty-nine (59 ± 7.24) per cent of sperm cells attached to PNIPAM-HMA-HA hydrogels and 31.16 ± 4.81% of them were released upon treatment with medium containing hyaluronidase. This attached-released sperm fraction has acceptable characteristics of progressive motility (50.0 ± 5.0%), vigour (4), high viability (58.7 ± 11.7%) and low percentage of acrosome reacted spermatozoa (23.36 ± 4.1%). Our findings indicate that PNIPAM-HMA-HA hydrogels are non-toxic and allow the selection of high-quality sperm cells for ART.

Duration-dependent effects of high dose of phthalate exposure on semen quality in adult male rats.

OBJECTIVE: To determine the length of exposure to high doses of phthalate that might affect sperm quality in adult male Wistar rats. METHODS: Forty-two (42) adult male Wistar rats (weighing 150-200 g) were randomly assigned into six groups (n=7): Group A received 0.5 mL of distilled water - placebo - and served as controls; groups B, C, D, E and F received Phthalate (750 mg/kgbw) for 1, 3, 5, 7 and 9 weeks, respectively. The data obtained from the study was expressed as Mean ± SEM with a p-value <0.05 considered significant. The data was analyzed with one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test using GraphPad Prism, version 8. RESULTS: The results showed a statistically significant (p<0.05) decrease in testicular weight in the rats exposed to 750 mg/kg of phthalate for 3, 5, 7 and 9 weeks when compared with the controls. Sperm count, motility and viability were also significantly (p<0.05) reduced, while sperm cells with abnormal morphology had increased counts in the groups exposed for 3, 5, 7 and 9 weeks when compared with controls. Serum zinc and magnesium were also significantly reduced (p<0.05) in the subjects treated for 1, 3, 5, 7 and 9 weeks when compared with controls. CONCLUSIONS: The dosage of phthalate adopted in this study was deleterious to testicular function when rats were exposed to it for as short a period as three weeks.

Effects of Three Semen Extenders, Breeding Season Month and Freezing-Thawing Cycle on Spermatozoa Preservation of Portuguese Merino Sheep.

This study aimed to evaluate and compare the effect of three semen extenders (S-EXT) on 22 spermatozoa (SPZ) parameters (subjective and computer-assisted sperm analysis evaluations), before and after semen cryopreservation throughout different months of the breeding season in the Portuguese Merino breed. According to the multivariable model, the SPZ viability (alive %), kinetics subjective individual motility, total motility, total progressive motility and its subpopulations, and beat cross frequency) were higher in the egg yolk-based S-EXT improved by Estação Zootécnica National (Portugal) than in Ovixcell® or Andromed® extenders. All the differences were only observed in thawed semen, except for total motility and total progressive motility, in which Ovixcell® also showed the poorest results on fresh semen. An interaction effect between S-EXT and semen processing was observed on 72.3% (17/22) of the evaluated parameters, evidencing a variable cryoprotective action between S-EXT. The SPZ viability was poorer in the onset of the breeding season (end of April/early May) than in the previous middle breeding season (November/early December), suggesting the influence of a short anoestrous season on ejaculate quality, even though the volume and SPZ concentration of the ejaculates remained stable throughout the experiment. Additionally, S-EXT x semen processing x month interaction effect on 59.1% (13/22) of the evaluated parameters evidenced the importance of SPZ time collection in a natural environment to cryopreserve ram's semen. We concluded that, overall, the egg yolk-based S-EXT provided a greater value to the cryopreservation of Merino rams´ semen. Nevertheless, the causes of the interaction effect between S-EXT, semen processing and/or month on several SPZ parameters should be addressed, including SPZ molecular research in new studies, in order to improve egg yolk-based as well as in egg yolk-free-based S-EXT.

Analysis of correlated responses in key ejaculatory traits to artificial selection on a diversifying secondary sexual trait.

Positive genetic covariance between male sexual display traits and fertilizing capacity can arise through different mechanisms and has important implications for sexual trait evolution. Evidence for such genetic covariance is rare, and when it has been found, specific physiological traits underlying variation in fertilization success linked to trait expression have not been identified. A previous study of correlated responses to bidirectional artificial selection on the male sex comb, a secondary sexual trait, in Drosophila bipectinata Duda documented a positive genetic correlation between sexual trait size and competitive fertilization success, and found that transcript levels of multiple seminal fluid proteins (SFPs) were significantly increased in the large sex comb (high) genetic lines. These results suggest that changes in SFP activity may be a causal factor underlying the increased fertilizing capacity of high line males. Here, we tested for correlated responses to this selection in a suite of additional reproductive traits, measured in the context of variation in male age and exposure to rivals. Whereas several traits including sperm length, number and viability, and accessory gland size, increased with age, only sperm viability was influenced by selection treatment, but in complex fashion. Sperm viability of high line males surpassed that of their smaller-combed counterparts when they had been housed with rivals and were 5-6 days old or older. Interestingly, this interaction effect was evident for sperm sampled from the female seminal receptacle, but not from the male seminal vesicles (where sperm have yet to be combined with accessory gland products), consistent with the differential SFP activity between the lines previously found. Our results suggest that differences in sperm quality (as viability) may be a contributing factor to the positive genetic correlation between sexual trait size and competitive fertilization capacity in D. bipectinata.