Percentage Of Viable Spermatozoa Research Articles

A comparison of the biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state at 5 °C

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage.

Cooling of alpaca spermatozoa using an extender with the addition of different percentages of seminal plasma

The objective of this study was to evaluate the effect of the addition of different percentages of seminal plasma (SP) during the cooling at 5 °C of alpaca spermatozoa from vas deferens. Fifteen pools of sperm from vas deferens were evaluated and then divided into four aliquots that were diluted to a final concentration of 30 × 106 sperm/ml with either: (1) Tris with 20% egg yolk (T-EY) (control, 0% SP), (2) T-EY with 10% SP, (3) T-EY with 25% SP, and (4) T-EY with 50% SP. Samples were cooled at 5 °C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. A significant decrease in progressive and total sperm motility was observed in samples cooled with 50% SP with respect to all diluted samples, while these parameters were preserved in samples cooled with 0%, 10%, and 25% SP. The percentages of sperm viability, normal morphology, and highly condensed chromatin did not change after the cooling process and were similar between cooled samples. Although a significant decrease was observed in the percentage of spermatozoa with functional membranes and with an intact acrosome in all refrigerated samples compared to raw sperm, the greatest decrease was observed in samples cooled with 50% SP. No advantage was observed from the addition of SP to alpaca spermatozoa obtained from vas deferens and being cooled. In addition, to preserve the sperm motility of cooled samples for up to 72 h, it should be recommended to include a 10% SP in the extender.

Study the properties of ram semen preserved at 5°C for different periods after adding different levels of acai fruit extract.

This study was conducted to know the effect of adding an aqueous extract of Acai fruit (Euterpe oleracea Martius) to the semen extender of Awassi rams on semen Parameters after storage at cooling. This study was carried out in the animal field and laboratories of the Department of Animal Production-College of Agricultural Engineering University of Baghdad from November 15, 2021, to April 25, 2022. Three local Awassi rams were used in this experiment. Their ages were between 2.5 - 3 years, and they weighed Between 50-58 kg. Semen was collected from them using an artificial vagina to obtain one ejaculate from each ram per week. The samples were divided using Tris extender: 1.0, 1.5, 2.0, 2.5 mg Acai extract / 1 ml Tris in addition to the control group. The necessary tests were performed to evaluate the semen in terms of estimating individual motility, the percentage of sperm viability, the percentage of abnormal sperms, the integrity of the plasma membrane and the acrosome integrity. There was a significant increase (P<0.05) in the percentage of sperm viability during the T3 treatment periods (48 and 72 hours) compared to the control group C for the same two periods; also there were no significant differences between all groups in the two periods (0 and 24 hours) of preservation in comparison with the control treatment C. Moreover T3 recorded a significant decrease (P < 0.05) in the percentage of sperm abnormality after 24 hours of preservation when compared with the control group C in the same period, while there were no significant differences between groups T1, T2, T3 and T4 compared with the control group C in the periods 0, 48 and 72 hours. Keywords: ram semen, acai fruit, plant extracts

Impact of latent toxoplasmosis on the fertility indices of male rats

Infertility is a prevalent condition affecting approximately 70 million people worldwide and male factor contributes to about fifty percent of the issues. Studies on infectious agents as a possible cause of infertility has become prominent in the past decade. Toxoplasma gondii has emerged as a prime candidate as it has been found in the reproductive organs and semen of males of many animal species and humans. The aim of this study is to assess the effect of latent toxoplasmosis on experimental rat fertility. Ninety Toxoplasma infected rat were used as the experimental group besides, thirty control naïve ones. Both groups were observed clinically. Weekly assessment of fertility indices starting from the 7th week post infection till the 12th week were done by recording rat body weight, weight of testes, semen analysis and histo-morphometric analysis of the testes. Toxoplasma infected rats exhibited significant gradual loss of body weight and the absolute weight of the testes. The sperm characteristic parameters including percentage of motile sperm, percentage of viable sperm and sperm concentration in Toxoplasma infected rats showed highly significant decrease throughout the observation period in comparison to the control group with recording highly significant increase in the percentage of abnormal sperm forms. Pathological insults in tests of the infected rat group were denoted. Our findings demonstrated that Toxoplasma gondii is accused for affecting male rat main reproductive parameters and is implicated in the male reproductive disorders.

Counteracting the Toxic Hidroarsenicism Effects upon Reproductive-Physiological Outcomes in Male Goats: The Selenium and Vitamin-E Mitigation Approach.

This study was to evaluate whether selenium and vitamin-E counteract the toxic effects of arsenic on reproductive aspects and physiological conditions of male goats. Male goats [Criollo, n = 20, 4-5 yr-old, 72 kg live weight (LW)] were distributed in homogeneous groups (n = 5), and received during 12 weeks: (1) Sodium arsenite 2 mg/kg of LW/day (AG, LW = 69 kg); (2) Sodium selenite 6 mg + vitamin-E 420 I.U. every 14 days during the 12 weeks (SG, LW = 68 kg); (3) Both AG and SG treatments (ASG, LW = 77 kg); and (4) 1 mL of physiological solution every 14 days during the 12 weeks (CG, LW = 72 kg). The animals had access to water from a well with a concentration of 35 μg/L of arsenic. The SG had the highest percentage of sperm viability (80.6%) followed by the CG, ASG (74.7; p > 0.05), and AG (64.3; p ≤ 0.05). The ASG and SG had a lower heart rate as compared to the CG and AG (58.8, 58 vs. 65.4, 63.5; respectively, p ≤ 0.05). The CG and SG showed a lower respiratory rate than the AG and ASG (19.2, 18.7 vs. 22.1, 21.0, respectively; p ≤ 0.05). Selenium and vitamin-E were efficient in reducing the damage caused by sodium arsenite in sperm quality and maintaining heart and respiratory rates and increases in odor in male goats.

The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation.

This study investigates the effects of cryopreservation and supplementation of Azeri water buffalo's semen with proline (Lp) and fulvic acid (FA). Therefore, this study aimed to assess motility parameters, sperm viability, oxidative stress parameters, and DNA damage to detect the optimum concentrations of Lp and FA for buffalo semen cryopreservation. Thirty semen samples of three buffalo bulls were diluted in Tris-egg yolk extender and divided into 12 equal groups including control (C), Lp-10, Lp-20, Lp-40, Lp-60, Lp-80 (containing 10, 20, 40, 60, 80mM L-proline, respectively), FA-0.2, FA-0.5, FA-0.8, FA-1.1, FA-1.4 and FA-1.7 (containing 0.2%, 0.5%, 0.8%, 1.1%, 1.4% and 1.7% fulvic acid, respectively). The velocity parameters, TM and PM were improved by FA-1.7, FA-1.4, Lp-40 and Lp-60 groups compared to the C group but no significant difference was found regarding the amplitude of lateral head displacement and straightness compared to the control groups. The percentage of sperm viability and PMF were increased by FA-1.7, FA-1.4, FA-1.1, Lp-40 and Lp-60 groups compared to C group, while in terms of sperm DNA damage FA-1.7, FA-1.4, FA-1.1, Lp-10, Lp-20, Lp-40 and Lp-60 groups showed better results compared to C group. The results also showed that FA-1.7, FA-1.4, FA-1.1, Lp-20, Lp-40 and Lp-60 groups could improve TAC, SOD, GSH and decrease MDA levels. Also, FA-1.7, FA-1.4, Lp-20 and Lp-40 groups could improve GPx levels but just FA-1.7, and Lp-40 groups could improve CAT levels compared to C group. Thus, it can be concluded that L-proline and fulvic acid supplementations can improve the quality parameters of post-thawed buffalo bull semen.

Bull field fertility differences can be estimated with in vitro sperm capacitation and flow cytometry

IntroductionThis study evaluated whether post in vitro capacitation changes in sperm could be used to estimate field fertility differences between bulls.MethodsFrozen-thawed semen from five bulls (two to four ejaculates per bull) previously identified as high (48.1% and 47.7%), intermediary (45.5%) or low (40.7% and 43.1%) fertility, based on pregnancy per AI (P/AI), were evaluated for total and progressive motility, sperm plasma membrane integrity (viability), acrosome integrity (viable sperm with an intact or disrupted acrosome), reactive oxygen species (ROS; viable sperm ROS+ or ROS-), mitochondrial membrane energy potential, zinc signatures (signatures 1-to-4) and CD9 protein populations at pre-wash and post-wash (only total and progressive motility), h0 (diluted with non-capacitation media), and at h0, h0 CM, h3, h6, and h24 after dilution with capacitation media (CM) and incubation at 37ºC. Data were analyzed using the GLIMMIX procedure as repeated measures in SAS with bull, time and the interaction as fixed effects.ResultsBull by time interaction was significant (P≤0.03) for total motility, viability, viable sperm with disrupted acrosome, and zinc signature 3. There tended (P=0.06) to be a bull by time interaction for zinc signatures 1+2 combined. Time was significant (P≤0.003) in all analyses, except viable ROS- (P=0.12). There was a significant effect of bull (P≤ 0.03) for viability, viable sperm with disrupted acrosome, zinc signatures 1, 2 and 1+2, viable CD9- and dead CD9+. High and intermediary fertility bulls had greater (P≤0.04) percentages of viable sperm, zinc signature 2 and zinc signature 1+2 compared to low fertility bulls. High and intermediary fertility bulls had decreased (P≤0.05) percentage of dead CD9+ compared to low fertility bulls. Viable CD9+ differed (P=0.02) and viable sperm with an intact acrosome and viable CD9+ tended to differ (P=0.06) amongst bulls; however, association with field fertility was not observed. There was a positive correlation between P/AI and zinc signature 2 (P=0.04), and there tended to be a positive correlation between P/AI and viability (P=0.10), and zinc signature 1+2 (P=0.10).DiscussionIn summary, incubation of sperm in CM and flow cytometry analyses for viability, zinc signatures 2 and 1+2, and dead CD9+ seems promising to estimate in vivo fertility differences amongst bulls.

Dietary Supplementation with Linseed Oil Ethyl Esters Improves Sexual Behavior and Chosen Seminal Parameters in Porcine Species.

Numerous studies have shown that improvements in the sperm and semen quality of males of many species can be achieved with appropriate dietary supplements added to feed or fodder. Particularly promising seems to be the inclusion of omega polyunsaturated fatty acids in the diets of males. Among other things, it has been shown that linseed oil ethyl esters (EELO can be an excellent source of omega 3 polyunsaturated fatty acids in animal diets. These compounds are more durable and resistant to oxidation, epoxidation and resinification processes, and do not exhibit toxic properties in living organisms. At present, there is a lack of data in the literature on the enrichment of boar diets with EELO. The purpose of this study was to analyze the effects of the addition of EELO to boar diets on the properties of sperm in fresh semen. The study was conducted during the summer on semen collected from 12 boars of the line 990. Linseed oil ethyl esters were administered in each feeding at a rate of 3.0% (45 mL each) in basal diets for each boar on a daily basis for 16 weeks. Ejaculates were collected manually by the gloved-hand technique, at one-week intervals for eight-week periods, from the eighth week onwards after the start of feeding. Eight ejaculates were collected from each boar, totaling 96 samples. The addition of EELO to the diets of boars caused an increase in sperm viability (p < 0.001), semen volume (310 mL versus 216 mL, p < 0.001) and sperm concentration (331 versus 216 million per mL, p < 0.001). Furthermore, in the experimental animals, there was a decrease in the percentage of spermatozoa exhibiting DNA fragmentation. The experimental boars also showed an increase in the percentage of gametes without apoptosis and capacitation and an increase in the percentage of viable spermatozoa not showing lipid peroxidation membranes. Consequently, EELO nutritional supplementation resulted in the improved quality of the fresh semen of boars.

Semen quality characteristics of Aseel, Kadaknath and native chicken of Kerala

The present study was conducted to compare the microscopic and macroscopic parameters of semen of three Indian indigenous chicken breeds viz. Aseel, Kadaknath and native chicken of Kerala. The semen volume in native chicken of Kerala was significantly higher compared to that of Aseel and Kadaknath roosters. Significantly (p&lt;0.05) higher appearance score was observed in native chicken of Kerala and Kadaknath roosters compared to that of Aseel roosters. The pH of semen was found slightly alkaline in all the three breeds. Most of the semen samples were white in colour. Most of the semen samples were thick in Kadaknath roosters, thick and medium thick in native chicken of Kerala and thick, medium thick and watery in Aseel roosters. The major contaminants of semen observed were faeces, blood and uric acid crystals. Significantly (p&lt;0.01) higher mass activity score and initial progressive motility was measured in native chicken of Kerala compared to Aseel roosters and the values of Kadaknath roosters were comparable to both. There was no significant difference in sperm concentration and sperm abnormality among three indigenous breeds. The sperm viability percentage was significantly (p&lt;0.05) higher in native chicken of Kerala compared to Aseel and Kadaknath roosters. The characteristics of fresh semen samples collected from all the three breeds revealed that all three were fit for artificial insemination.

Severe deterioration in sperm parameters and testes of rats administered naproxen and diclofenac at pre-puberty: An experimental study.

Although naproxen and diclofenac are the most commonly used nonsteroidal drugs, their toxicity affecting male reproductivity has not been sufficiently studied, particularly when used in pre-puberty. This study aims to investigate the toxic effects of naproxen and diclofenac on sperm parameters and testes in pre-pubertal rats. In this experimental study, 15 pre-pubertal male albino rats (aged 5 wk, weighted 70-80 gr) were used. The animals were divided in to 3 groups (n = 5/each) of control (0.1 ml dimethyl sulfoxide), naproxen (50 mg/kg), and diclofenac sodium (5 mg/kg), and were orally administered with these drugs every day for 3 wk. Epididymal spermatozoa were taken to assess sperm count, viability, and morphology. After preparing tissue sections, testicular histopathological and histomorphometric analyses were performed. The body weights of rats in both naproxen and diclofenac groups significantly decreased (p 0.001 and p = 0.03, respectively) in comparison to the control group. Remarkably, the testis weight and total sperm number significantly decreased (p = 0.002, and p = 0.004 respectively) in the naproxen-administered rats only. The sperm viability percentage decreased significantly in both diclofenac and naproxen-administered groups (p 0.001 and p = 0.03 respectively). Moreover, there was a significant increase (p 0.001) in the percentage of sperm morphological anomalies in both drug-administered groups. Also, the histological and morphometric findings exhibited severe histopathological appearances in the testes and seminiferous tubules parameters in both drug-administered groups.>. Naproxen and diclofenac administrations of rats before their puberty induce considerable harm to sperm parameters and testicular histology and morphometry. These severe toxic effects can lead to potential infertility.

Green tea (Camellia sinensis) aqueous extract improved human spermatozoa functions in vitro

Background: Idiopathic causes of infertility is associated with oxidative stress. Antioxidants are known to scavenge the excessive production of reactive oxygen species (ROS). Green tea (Camellia sinensis) contains polyphenols that enhance its antioxidant potential.Aim: This study focused on the impact of aqueous green tea extract on normozoospermic human spermatozoa.Setting: Department of Medical Biosciences, University of the Western Cape (UWC), South Africa.Methods: Semen samples obtained using masturbation method following three to five days of sexual abstinence from consenting men (n = 59) at the University of the Western Cape (UWC) were liquefied and analysed. Normozoospermic samples were selected according to the World Health Organization (WHO) 5th guideline. Thereafter, semen samples (7.5 × 106 /mL) were washed in human tubular fluid (HTF; 10 min at 300 ×g) and exposed to aqueous extracts of green tea (0 μg/mL, 0.4 μg/mL, 4 μg/mL, 40 μg/mL, 405 μg/mL) for 1 h with various sperm parameters analyzed. Human tubular fluid supplemented with bovine serum albumin (HTF-BSA; 10%) served as control.Results: Sperm motility, reactive oxygen species production, across some reaction and deoxyribonucleic acid (DNA) fragmentation decreased significantly, particularly at the highest concentration (405 μg/mL; p 0.001). A substantial increase in the percentage of viable spermatozoa and those with intact mitochondrial membrane potential (MMP) were observed (p 0.001).Conclusion: Aqueous extract of green tea prolonged sperm viability and MMP while reducing sperm intracellular ROS production, capacitation and across some reaction and DNA fragmentation, and may be attributed to its antioxidant potential. However, a high concentration of the extract appears to be detrimental to the functioning of human spermatozoa.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

Influence of tropical, subtropical, and temperate climate conditions on seminal redox status and sperm quality parameters in breeding bulls

This study evaluated the effect of climate change on andrological parameters of beef bulls raised under tropical, subtropical, and temperate conditions. Bull ejaculates were collected to evaluate seminal quality parameters, sperm membrane integrity, and redox status (SOD; GPx; GSH; GRx; CARB; DCF; and SOD/GPx ratio). Bulls located in the temperate region showed a higher sperm motility rate and percentage of viable sperm (P < 0.05). When evaluating regions independently, we observed a lower GPx activity from animals in the tropical region (P < 0.05). In contrast, we found that SOD and GRx activities, GSH content, and CARB oxidative levels were higher in the tropical region, while oxidation values of DCF were lower (P < 0.05). Braford bulls showed higher CARB and DCF levels (1.23 ± 0.61 nmol/mg and 1453.60 ± 828.63 nmol/mg, respectively) compared to Hereford bulls (1.00 ± 0.43 nmol/mg and 1138.70 ± 423.24 nmol/mg, respectively) in the temperate region. However, Nellore bulls showed higher DCF levels (650.50 ± 401.53 nmol/mg) than Braford bulls (409.40 ± 286.97 nmol/mg). In addition, the SOD/GPx ratio was lower in Braford (12.44 ± 7.64 U/mg) compared to Nellore bulls in tropical conditions (87.25 ± 2.83 U/mg). A positive correlation was found in temperate conditions between DCF levels, SOD, and GRx activities (0.51, 0.58; P < 0.01, respectively), as well as in subtropical conditions between DCF levels and GRx activity (0.53; P < 0.01). A negative correlation between the temperature-humidity index and CARB content was found in subtropical and tropical regions (−0.44; P < 0.01). We concluded that Braford breeds showed lower seminal motility, DCF contents and SOD/GPx ratios compared to Nellore bulls in tropical climate conditions. Finally, in temperate environmental conditions, Braford bulls also showed lower seminal motility but higher levels of CARB and DCF contents compared to Hereford bulls. Therefore, the existence of climatic differences between the temperate and tropical regions evaluated affected Braford bulls’ seminal motility and seminal redox homeostasis.

In vitro storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of motility.

Prolonging the shelf-life of liquid-preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization-relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown. To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility. Extended semen samples (n=7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single-cell analysis of motility parameters was performed. The energy status was not affected by semen storage (p>0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: -24.2±10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2-deoxy-d-glucose prevented the re-establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r=0.44-0.70 for individual subpopulations, p<0.01). Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non-linear, and hyperactivation-like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long-term storage in vitro.

P-068 Oxidative stress and DNA fragmentation of spermatozoa in patients with cancer

Abstract Study question Does cancer increase sperm DNA fragmentation (sDF) level by increasing oxidative stress in human spermatozoa? Summary answer Cancer increases both sDF and oxidative stress, but further studies are necessary to understand whether higher levels of ROS are responsible for sperm DNA damage. What is known already In recent years, it has been emerging that not only oncological therapies but also cancer itself can induce abnormal spermatogenesis. In addition, several Authors reported that occurrence of malignancy provokes also increases of sperm DNA damage, although such finding was not confirmed by others and the possible mechanisms responsible for such damage are presently unknown Study design, size, duration This was a prospective observational study conducted from 2018 to today, conducted in 102 patients affected by cancer and in 66 control subjects. Participants/materials, setting, methods We recruited 102 patients affected by cancer and 66 male partners of infertile couples as control subjects, in the Andrology Clinic of University of Florence. Control subjects were normozoospermic with absence of leukocytospermia, semen viscosity, smoking habit and recent antibiotic therapies. In the recruited men, we evaluated standard semen parameters, sperm DNA Fragmentation with SCD (Sperm Chromatin Dispersion) Test and oxidative stress as percentage of viable spermatozoa with MitoSOX™ Red labeling on total viable spermatozoa. Main results and the role of chance We found poorer standard semen parameters (sperm motility, concentration and number) in cancer patients (both testicular and hematological ones) with respect to control group, whereas no differences were observed between the two types of cancer. Testicular, but not hematological patients, were younger than control subjects. No difference was seen in the other tested characteristics (sperm morphology, abstinence, semen volume and pH, BMI). Regarding sDF, we found higher median values [IQR] in cancer patients (total: 22.25[17.00-25.95], n = 68; hematological: 23.00[20.13-26.38], n = 28; testicular: 21.13[16.13-25.73], n = 40) vs control subjects (12.50[8.25-14.75], n = 53); p &amp;lt; 0.05, test U di Mann-Whitney. In addition, the amount of sperm oxidative stress was dramatically higher in patients with cancer (total:38.92[24.90-58.87], n = 79; hematological: 38.85[24.98-50.77], n = 34; testicular: 38.92[20.59-63.59], n = 45) vs control subjects (11.50[8.38-17.20], n = 62); p &amp;lt; 0.05, test U di Mann-Whitney. We also studied the occurrence of a correlation between levels of sDF and oxidative stress. We found a sharp correlation when both cancer patients and control subjects were analysed (Spearman coefficient = 0.62, p &amp;lt; 0.001, n = 103), but such correlation was completely lost when only cancer patients were considered (Spearman coefficient = 0.10, p &amp;gt; 0.05, n = 50). This finding suggests that mechanisms different from ROS attack to DNA could explain the increase of sDF levels in cancer patients. Limitations, reasons for caution The study did not investigate, because of scarce availability of semen samples from cancer patients, other possible mechanisms (i.e apoptosis, defects in sperm chromatin maturation, failure in DNA system repair) which could cause the observed increase of sperm DNA damage in such patients. Wider implications of the findings Cancer patients show high levels of both sDF and oxidative stress. This finding rises concern, as cancer patients cryopreserve semen for using it with Assisted Reproductive Tecnhologies and both parameters represent a threat for natural and assisted reproduction. In addition, emerging evidence suggest that oxidative stress may alter sperm epigenome. Trial registration number not applicable

Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14+/PI−), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.

21 Impact of Breed and Method of Semen Collection on Viability and Membrane Integrity of Goat Spermatozoa

Abstract Goats have potential to be genetically improved for meat and milk. Artificial insemination is an important technique used to enhance genetic gain through dissemination of selected male traits. One reason for huge variability in fertility rates in goats is the semen quality, which is affected by breed, collection method, and freezing procedures. Here we investigated effect of breeds (Saanen versus Alpine) and semen collection methods (artificial vagina; AV versus electroejaculation; EE) on the quality of goat spermatozoa. Semen collected from Saanen and Alpine bucks by either AV or EE was evaluated for its color, consistency, and volume. Besides, sperm motility, concentrations, viability, abnormalities, and membrane integrity were assessed. At least three ejaculates were collected from each breed and data were analyzed by student t-test. Results showed that colors of samples collected from the two breeds were white to yellow and the consistency ranged from watery to creamy. Irrespective to the collection method, average volume of ejaculates was higher for Alpine than Saanen (1350 vs. 880 µl for AV and 1066 vs. 987.5 µl for EE). Sperm motility and concentrations were higher (P ≤ 0.05) in Alpine than Saanen semen for both methods. Within the same breed, these parameters were higher (P ≤ 0.05) for AV than EE. Percentage of viable spermatozoa was higher for Alpine than Saanen bucks (80.0% vs. 64.5% in AV) and (52.42% vs. 41.35% in EE). However, sperm abnormalities were lower (P ≤ 0.05) in Alpine semen. Sperm membrane integrity tended to be higher in Alpine semen. In conclusion, semen collected from Alpine bucks had higher quality than those obtained from Saanen ones. Furthermore, collection of semen by AV improved sperm motility and viability than EE in both breeds. These findings suggest that both breeds and method of semen collection could influence the viability and fertility of goat sperm.

Effects of low-density lipoprotein extracted from egg yolk on preservation of chilled canine semen (4oC)

Low-density lipoprotein (LDL) derived from hen egg yolk has been considered an important component in the storage of canine semen; however, in Vietnam, there have not been many studies regarding this topic. Therefore, this study aimed to evaluate the effectiveness of LDL in the preservation of canine chilled semen in Vietnam. The assessment of semen samples was based on four criteria: storage time, motility, absolute viability, and integrity of acrosomal membrane at motility rate 0.9; 0.5 and 0.3. The semen samples collected were divided into three equal parts, each was then diluted with three different extenders and stored at 4oC. The extenders were LDL + basic extender (LDL), egg yolk + basic extender (EY), and basic extender (C). The storage time of canine semen was recorded from the beginning of monitoring until the motility decreased to 0.3. The storage time of the three extenders was in the order of EY &gt; LDL &gt; C, with 108 h, 60 h, and 48 h, respectively. The absolute viability of fertilizable sperm (Sa5) in the extender was EY (768), LDL (423) and C (280), which was significant (P &lt; 0.001). The percentage of viable sperm with intact acrosome membrane at motility time A = 0.9 in LDL was 59.31%, higher than that in EY (30.99%). In the storage period from A = 0.9 to A = 0.5, the acrosome loss percentage of both EY and LDL decreased equally (7.30% and 7.23%), but the storage time of EY (84 h) was longer than that of LDL (48 h). In conclusion, the EY gave a longer storage time, higher absolute viability and longer maintain percentage of viable sperms and intact acrosome membrane compared to the LDL. However, within the first hours of storage, the percentage of viable sperm and intact acrosome membrane in the EY was lower than that in the LDL. Therefore, to preserve canine semen for a short time (less than 48 h), the LDL extender should be used for better effectiveness.

Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm.

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Sperm chromatin condensation as an in vivo fertility biomarker in bulls: a flow cytometry approach

BackgroundGenetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations (AI) are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3 (CMA3) has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups (highly fertile and subfertile).ResultsChromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters (morphology, viability, total and progressive motility) were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve (AUC) of 0.54, 0.64 and 0.68, respectively (P > 0.05), and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73 (P = 0.05), CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78 (P = 0.02).ConclusionsChromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.