Live Sperm Research Articles

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

The effect of ferulic acid on 2-methoxyethanol-induced spermatotoxicity, hematotoxicity and hepatotoxicity in rats

AbstractFerulic acid (FA), also known as hydroxycinnamic acid, is a sweet-smelling and natural antioxidant present in the cell walls of plants and bran of cereals, making it a common dietary component. FA possesses diverse medicinal properties, including anti-inflammatory, anti-arrhythmic, antioxidant, anti-bacterial, anti-thrombotic, anti-cancer, anti-diabetic, anti-cardiovascular, neuroprotective, anti-apoptotic, antifibrotic, anti-platelet, anti-aging, anti-melanogenesis, angiogenesis promoting, and skin damage reducing effects. Judging by these outstanding credentials of FA, this study investigated the hepatoprotective, spermato-protective, and hemato-protective effects of FA against 2MEETH-induced spermatotoxicity, hematotoxicity, and hepatotoxicity in rats. After oral administration of 2MEETH for 30 days, the number of abnormal and dead spermatozoa, as well as serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and activity of alcohol dehydrogenase (ADH), were significantly increased. In contrast, the number of normal and live spermatozoa, sperm motility and concentration, serum total cholesterol level, red blood cell (RBC), packed cell volume (PCV), and white blood cell (WBC) counts were significantly decreased compared to the control group. FA treatments did not have any effect on all the sperm and hematological parameters checked, but significantly lowered the serum levels of AST, ALT, and ADH activity compared to rats administered with only 2MEETH. Based on the results, it is concluded that FA may not possess spermato- and hemato-protective effects against 2MEETH-induced spermatotoxicity and hematotoxicity but may possess a hepatoprotective effect in rats.

Mitigation of endogenous oxidative stress and improving growth, hemato-biochemical parameters, and reproductive performance of Zaraibi goat bucks by dietary supplementation with Chlorella vulgaris or/and vitamin C

This study was conducted to explore the effects of dietary inclusion of Chlorella vulgaris (CV) or/and vitamin C (VC) on growth, hemato-biochemical parameters, oxidative and antioxidant status, reproductive hormones, and semen quality variables, and scrotal-testicular dimensions of Zaraibi goat bucks. Twenty sexually mature bucks (41.49 ± 0.91 kg BW) were randomly divided into 4 groups (5 bucks/group). The control group was fed the control diet, while the other three groups received a diet supplemented with VC (2 g/animal /day), CV (5 g/animal/day), and CV plus VC (the same levels), respectively, for 8 weeks (treatment period), and then semen was collected for 8 weeks. Results showed that dietary supplementation with CV-VC combination significantly increased the final body weight, weight gain, packed cell volume, hemoglobin, red blood cells, white blood cells, and lymphocytes; elevated serum total protein, globulin, testosterone, estradiol, superoxide dismutase, glutathione peroxidase with a significant reduction in Malondialdehyde in serum and seminal plasma. Also, the CV-VC combination significantly improved the ejaculate volume, total sperm output, sperm concentration, and live sperm, and reduced reaction time and sperm abnormality of bucks. Either CV or VC given separately or in combination, at the chosen levels, had no detrimental effects on animal physiological responses with normal hepatic and renal functions. Therefore, the CV-VC combination could be safely utilized as a dietary supplement in buck’s diets to improve antioxidant defenses, scavenge free radicals, and potentiate buck’s reproductive activities under normal conditions.

The synergistic impact of Spirulina and selenium nanoparticles mitigates the adverse effects of heat stress on the physiology of rabbits bucks.

Heat stress has a detrimental effect on animal fertility, particularly testicular functions, including reduced sperm output and quality, which causes an economic loss in the production of rabbits. The present trial investigated the efficacy of dietary Spirulina (SP) (Arthrospira platensis), selenium nanoparticles (SeNPs), and their combination (SP-SeNPs) on semen quality, haemato-biochemical, oxidative stress, immunity, and sperm quality of heat-stressed (HS) rabbit bucks. Sixty mature bucks (APRI line) were distributed into 6 groups of ten replicates under controlled conditions. Bucks in the 1st group (control-NC) were kept under normal conditions (11-22°C; 40-45% RH% = relative humidity), while the 2nd group (control-HS) was kept under heat stress conditions (32±0.50°C; 60-66% RH %). The control groups were fed a commercial pelleted diet and the other four heat-stressed groups were fed a commercial pelleted diet with 1 g SP, 25 mg SeNPs, 1 g SP+25 mg SeNPs, and 1 g SP+50 mg SeNPs per kg diet, respectively. The dietary inclusion of SP, SeNPs, and their combinations significantly increased hemoglobin, platelets, total serum protein, high-density lipoproteins, glutathione, glutathione peroxidase, superoxide dismutase, and seminal plasma testosterone while decreased triglycerides, total cholesterol, urea, creatinine, and malondialdehyde compared with the control-HS. Red blood cells, packed cell volume, serum albumin, and testosterone significantly increased, while SeNPs, SP+SeNPs25, and SP+SeNPs50 significantly decreased low-density lipoproteins, aspartate, and alanine amino transferees. Total antioxidant capacity substantially increased in serum and seminal plasma, while seminal plasma malondialdehyde decreased in 25 or 50 mg of SeNPs+SP/kg groups. All supplements significantly improved libido, sperm livability, concentration, intact acrosome, membrane integrity, total output in fresh semen, and sperm quality in cryopreserved semen. SP-SeNPs50 had higher synergistic effect than SP-SeNPs25 on most different variables studied. In conclusion, the dietary inclusion of SP plus SeNPs50 has a synergistic effect and is considered a suitable dietary supplement for improving reproductive efficiency, health, oxidative stress, and immunity of bucks in the breeding strategy under hot climates.

Estimation of genetic parameters for semen traits in Egyptian buffalo bulls

This study was conducted to characterize semen traits (ejaculate volume (VOL), mass motility (MM), sperm livability (LS), percentage of abnormal sperms (AS), and sperm concentration (CONC)) of Egyptian buffalo bulls and evaluate the importance of some nongenetic factors (year (YC) and season (SC) of semen collection and age of bull genetically and environmentally at collection (ABC)) affecting the investigated traits. A total of 7761 normal semen ejaculates were collected from 26 bulls from 2009 to 2019. Single-trait and bivariate repeatability animal models using Bayesian methods were used to estimate variance components, heritability, repeatability, and genetic correlations among the investigated semen traits. YC and ABC exerted significant effects on most semen traits, whereas SC exerted no significant effect on all the investigated semen traits. Heritability estimates were 0.08, 0.52, 0.51, 0.04, and 0.49 for VOL, MM, LS, AS, and CONC, respectively. Repeatability estimates were 0.14, 0.82, 0.79, 0.06, and 0.78 for VOL, MM, LS, AS, and CONC, respectively. The genetic correlations between MM and each of LS and CONC were highly significant (0.99 ± 0.01 and 0.95 ± 0.14, respectively), and that between LS and CONC was also highly significant (0.92 ± 0.20). The high heritability estimates for MM, LS, and CONC combined with the favorable high significant genetic correlations between these traits indicated that direct selection for MM may be an effective method to enhance semen quality in Egyptian buffalo bulls and consequently improve fertility.

Effect of ascorbic acid on metabolic status, lipid peroxidation, antioxidant activity and quality of frozen Indian red jungle fowl (Gallus gallus murghi) semen.

The Indian red jungle fowl population is decreasing in its natural habitat. Its conservation through semen cryopreservation with sufficient live sperm recovery rate is requisite where ascorbic acid could play significant role to mitigate cryo-incited injuries. The objective was to elucidate the effect of ascorbic acid on freezability of Indian red jungle fowl sperm. Pooled semen was aliquoted and diluted (1:5) with red fowl extender having ascorbic acid: 0.0 (control), 1.0, 2.0 and 4.0 mM. Diluted samples were cryopreserved and semen quality was assessed at post-dilution, cooling, equilibration and freeze-thawing stages. Sperm metabolic status, antioxidant potential and lipid peroxidation were studied at post-dilution and freeze-thawing. Sperm motility did not differ (p > .05) in experimental extenders and control at post-dilution and cooling; however, it was recorded higher (p < .05) with ascorbic acid at 2.0 mM compared with other levels at post-equilibration and post-thawing stage. Sperm viability, plasma membrane and acrosome intactness were recorded higher (p < .05) with 2.0 mM ascorbic acid compared with other concentrations of ascorbic acid at all stages of cryopreservation. Sperm metabolic status and antioxidant potential were recorded higher (p < .05), while lipid peroxidation was recorded lowest (p < .05) with 2.0 mM ascorbic acid compared with 1.0, 4.0 mM and control. In conclusion, ascorbic acid at 2.0 mM in red fowl extender improves quality, metabolic status and antioxidant potential of frozen Indian red jungle fowl semen through ameliorating lipid peroxidation.

Automated Orientation Control of Motile Deformable Cells

Automated manipulation of deformable objects is challenging due to the object&#x2019;s deformation behavior. Different from still deformable objects such as wires and cloth, biological organisms such as sperm and worms are both deformable and motile, requiring the control of both deformation and motion. This paper reports automated orientation control of live sperm, as an example of motile deformable cells. Robotic manipulation of human sperm was performed by using a glass micropipette, which is a standard clinical tool, to rotate individual motile sperm. Sperm rotation must be performed before immobilization, as required in clinical cell surgery for infertility treatment. To control tail deformation during sperm rotation, a path planner was designed based on kinematic analysis and manipulation point update. To deal with the intrinsic motion of a motile sperm, a motorized stage was controlled to compensate for sperm swimming motion, and an observer was designed to decouple sperm orientation from its wiggling motion. A sliding mode controller was designed to cope with stiffness variances along the sperm tail and among different sperm. Deep neural networks were developed for robust sperm tail detection, and Kalman filter was used to predict tail motion. Experimental results demonstrated that automated sperm manipulation achieved an orientation error of 0.8<inline-formula> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and operation time of 6.8 s, both significantly less than those of manual operation. The designed observer was effective to reduce sperm orientation error by reducing the disturbance from sperm wiggling motion. The developed sliding mode controller outperformed the PID controller in operation time, reducing the time of oocyte exposure to the ambient environment. <i>Note to Practitioners</i>&#x2014;This work tackled the challenge of rotating a fast-swimming and deformable sperm in clinical cell surgeries. Automated manipulation of deformable objects has wide applications in industrial and service settings such as manipulating wires and folding cloth. However, the intrinsic motion of a motile sperm and the lack of a rotational degree of freedom in standard micromanipulators pose difficulties to automated sperm manipulation. In this paper, we propose automation techniques for sperm orientation control. For sperm tail detection, deep learning was used to handle the variances of shape and length among different sperm. A path planning strategy and a controller were designed to achieve automated rotation of motile sperm, with its deformation and motion both controlled. The developed methods can be generalized to the manipulation of other deformable objects such as wires, cables and cloth. These objects exhibit significant variance of mechanical properties, and calibration is often time-consuming. The designed controller can be used to manipulate deformable objects with robustness to varied mechanical parameters. Path planning was designed by updating the manipulation point based on the object&#x2019;s deformation behavior, and is suitable in manipulation where constraints are imposed such as the object&#x2019;s strain.

Semen quality of progeny-tested breeding bulls maintained at the National Artificial Insemination Centre, Arusha, Tanzania

The success or failure of artificial insemination (AI) depends on the quality of semen used. This study was conducted to investigate the effect of breed, age, collection period, and ejaculate number on semen quality collected from 15 breeding bulls available at the centre with different ages (4 Friesian, 4 Ayrshire, 3 Jersey, 2 Simmental and 2 Boran breeds) maintained at National Artificial Insemination Center (NAIC), Arusha, Tanzania. A total of 600 ejaculates were collected for four weeks per month consecutively from January to May 2022, weekly per bull and twice a day; morning (6.00 - 9.00 AM) and evening (3.00 - 6.00 PM) sessions). The ejaculates were first examined visually for volume, color, foreign bodies, and consistency whereas sperm concentration (SPC), progressive motility (PM) and total motility (TM) were assessed using Computer Assisted Semen Analysis System (CASA). Viability/vitality (SPVI) and morphology (SPNR) of sperms were analyzed microscopically after staining with Eosin-Nigrosin. Data were subjected to SAS program and results showed that ejaculate volume, progressive motility, total motility, sperm viability, and morphology were significantly (P&lt;0.05) affected by age and collection period. Simmental, Friesian, and Ayrshire bulls produced more voluminous semen than Jersey and Boran bulls. Jersey bulls had higher (P&lt;0.05) sperm concentrations and live spermatozoa as compared to other bulls. Boran and Jersey's bulls exhibited higher PM and TM than the other bulls. The period of semen collection (morning versus evening) negatively affected semen volume, total motility, and semen viability in which morning harvests performed better than evening collections. Middle aged bulls (3-5 years) produced semen of higher quality compared to other groups (&lt; 3 years and &gt; 5 years old bulls). In conclusion, age, breed, period of semen production and ejaculate number revealed significant effect on bovine semen characteristics.

Semen quality parameters of the Tanzanian Horasi chicken ecotype

Predicting potential fertility and eventual egg hatchability in chickens can be done by evaluating the quality of the semen. This study's objective was to describe the semen characteristics of three different age groups of Horasi chicken ecotype cocks (24-32, 28-36, and 32-40 weeks). From 20 Horasi cocks; 6 cocks (24-32 weeks), 6 cocks (28-36 weeks), and 8 cocks (32-40 weeks) over the course of four weeks (5-29th Dec 2022), 80 semen samples in total were taken. Sperm was harvested from cocks using the abdominal massage technique. The amount of semen, pH, sperm motility, sperm concentration, live spermatozoa percentage, and spermatozoa with normal morphology percentage were all measured. Between the three age groups, the semen volume, pH, sperm motility, sperm concentration, percentage of live spermatozoa, and percentage of spermatozoa with normal morphology ranged from 0.52 ± 0.03mL to 0.68 ± 0.03mL, 7.24 ± 0.01 to 7.31 ± 0.01, 75.16 ± 0.91% to 76.67 ± 1.07%, 4.31 ± 2.27 to 4.56 ± 3.34 × 109 sperm cells/mL, 90.00 ± 0.55% to 91.04 ± 0.85% and 87.78 ± 0.51 to 89.71 ± 0.68% respectively. Only differences in ejaculate volume between the three age groups of Horasi chicken ecotype cocks were significant at p&lt;0.05. The semen quality parameters had extremely low to moderate Pearson correlation coefficients, with values ranging from - 0.22 to 0.38 between sperm concentration and sperm motility, and between the percentage of morphologically normal sperm cells and the percentage of spermatozoa that are alive respectively. The Horasi chicken ecotype's semen parameters are within the normal range, notwithstanding the possibility that individual cocks may differ from one another in some semen characteristics as a result of environmental influences. Using artificial insemination, breeding programs can therefore exploit the Horasi chicken ecotype to increase the productivity of indigenous chickens.

O-019 Decreased Sperm Motility Associated With Bacterial Vaginosis Modeled in a Human Cervix Chip

Abstract Study question What is the underlying cause of infertility associated with bacterial vaginosis (BV)? Summary answer We provide the first evidence to suggest that BV-associated infertility could be the result of sperm dysfunction induced by exposure to poor cervical microbiome. What is known already Infertility is a global health concern, impacting 186 million individuals worldwide. In 25% of infertile couples the underlying cause remains unexplained. The complex interactions between the host mucosal epithelium, microbiome, and other microenvironmental factors play a significant role in health and disease of the reproductive tract. BV affects 30% of reproductive-aged women and is linked to overgrowth of pathogenic Gardnerella vaginalis bacteria. BV has a strong association with infertility, but it is not clear whether the resulting changes in the vaginal microbiome play a causal role, and as such BV screening is not currently included in infertility evaluation or treatment Study design, size, duration Female reproductive tract protective factors such as cervical mucus helps sperm retain their ability to fertilize for days compared to hours in suspension. We previously leveraged human organ-on-a-chip microfluidic culture technology to develop a physiological in vitro model of human cervix (Cervix Chip) that is lined with primary cervical epithelium interfaced with stromal fibroblasts. The epithelium forms a functional tissue barrier and produces mucus with compositional, biophysical, and hormone-responsive properties similar to the living cervix. Participants/materials, setting, methods We used 2D culture of sperm and bacteria as a screening and validated the effect of the identified bacteria on physiological in vitro Cervix Chip. A BV consortium containing E2 and E4 Gardnerella vaginalis bacteria was co-cultured with the Cervix Chip for 48 hours before adding human sperm samples. The motility of fluorescently labeled sperm was non-invasively evaluated in live chips using time-lapse imaging and quantitatively analyzed to obtain the percentage of motile sperm. Main results and the role of chance We initially analyzed the effects of bacterial co-cultur L. crispatus (optimal) or G. vaginalis (suboptimal) bacteria on sperm motility using a conventional 2D culture system. We observed a significant drop in sperm motility within 2 hours when exposed to G. vaginalis compared to L. crispatus or the control condition (no bacteria). We have established sperm staining and live tracking on Cervix Chip. Motile sperm was tracked on Cervix Chip for more than 7 days compared to 12 hours in suspension. We then tested this effect on-chip and observed that co-culture of live sperm with dysbiotic Cervix Chip (infected with G. vaginalis) resulted in a significant reduction in the sperm motility within 24 hours after exposure compared to the sperm exposed to the chips without bacteria. The dysbiotic Cervix Chip also exhibited a disease phenotype with elevated levels of proinflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, and TNF-α in the effluents of the chip's epithelial channel as well as significantly reduced thickness of the cervical mucus layer that correlated with reduced sperm motility. This finding is consistent with the observation that successful fertility is often associated with the quality of cervical mucus, which can be compromised in the BV dysbiotic condition. Limitations, reasons for caution Primary cervical epithelium cells used in the study were sourced from a single donor. Frozen sperm samples prepared for intrauterine insemination were used in the study Wider implications of the findings Our study suggests that infertility related to BV may be caused by sperm dysfunction, and BV screening should be considered in patients with unexplained infertility. We propose a model that can be used to identify dysbiotic conditions in the female reproductive tract that are unfavorable to sperm motility. Trial registration number N/A

Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa.

Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5mm), pyruvate (10mm), and lactate (10mm) for 0.5 to 4h. Response to calcium ionophore A23187 (5μm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Incubation with lactate alone for 2h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.

Assess the quality of Murrah Buffalo (Bubalus bubalis) Bull Semen regarding seasonal and geographical variability in Nepal

We carried out this study to assess the quality of semen regarding the seasonal and geographical variability of Murrah buffalo breeding bull in Nepal. We aimed to access the quality of seme n of murrah buffalo bull considering the seasonal and geographical variability to help standardize quality semen producing protocol and concern. A total of eight Murrah buffalo bulls were used. Semen collection was done at once week and two ejaculations were considered at an interval of 30 minutes during the Summer (June to August), Autumn (September to November), Winter (December to February), and Spring (March to May) from 2017 to 2020 AD. Immediately after collection, initial motility, sperm concentration, pre-filling motility, post-thaw motility, live sperm, and abnormality of sperms were evaluated. Findings revealed that the season had affect (p&lt;0.005) to the semen volume, individual motility, and post- thawing motility except for semen volume and sperm abnormality(p&gt;0.05). However, initial motility, sperm concentration, and pre filling motility did not vary in months (p&gt;0.05). It is thus well reflected that several traits related to quality and quantity of semen Murrah buffalo bulls may vary depending on the season of collection, location and age (p&lt;0.05) with the semen traits of initial motility, sperm concentration, and pre-filling motility. Accordingly the semen collected during the autumn and spring season is revealed the percentage of average post thaw motility above the 52% that considered the success of artificial insemination is good for buffaloes.

Effect of addition of honey and skim milk and cooled cauda epididymal spermatozoa of Awassi ram

The current study intends to assess the effectiveness of combining honey and skim milk in an extender on individual motility, livability, and abnormalities of cauda epididymal Awassi ram spermatozoa after diluting and cooling. Nine pairs of testicles Awassi rams were collected after the slaughter at the abattoir. Honey and skim milk combined were prepared. The cauda epididymal spermatozoa were divided into four equal parts and diluted in a Tris-based extender. (Control, basic diluents), HSM1 (basic diluents containing 0.5 ml honey and 9.5ml skim milk), HSM2 (basic diluents containing 1 ml honey and 9ml skim milk), HSM3 (basic diluents containing 1.5 ml honey and 8.5ml skim milk) and cooled 4ºC for evaluation of the percentages of sperm individual motility, live and abnormalities spermatozoa (including head, midpiece, and tail) at 0, 24, 48, and 72h. Results showed that individual motility spermatozoa preserved (P&lt;0.05) in HSM1 and HSM2 groups at 24h and 48h. Livability spermatozoa were increased (P&lt;0.05) in the HSM1 group at 48h and 72h. Groups HSM1, HSM2, and HSM3 decreased total abnormalities (P&lt;0.05) at 48 h and 72h while decreasing tail abnormalities (P&lt;0.05) at 24h and 48h than control. The HSM2 group was lower (P&lt;0.05) in head abnormality of spermatozoa at 48 h, whereas the HSM1 group was at 72 h. In conclusion, the nourishing and protective effects of lower honey concentrations in extender favorably impact cauda epididymal spermatozoa of Awassi rams. Keywords: cauda epididymal spermatozoa, honey, skim milk, motility, livability, and abnormalities.

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting.

A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Influence of sperm viability on uterine leukocyte chemotaxis

The immune system can differentiate live from dead microorganisms, generating a response as necessary; there has been limited study of equine sperm. We aimed to compare the uterine cellular immune response to artificial insemination (AI) with live versus dead equine sperm. Twenty mares in estrus (follicles ≥ 35 mm, uterine edema), without intrauterine fluid or polymorphonuclear leukocytes (PMNs) were selected from a population of horses sent to an abattoir during the breeding season. Their average age was 10.8 ± 0.9 years (range 6 - 20), body condition score > 3 (scale 1 - 5), and they were clinically healthy. Mares were randomly allocated into two groups. Frozen semen, a pool from two stallions, was inseminated into the uterine body: either LIVE (n = 10), 3.0 mL of 900 × 106 sperm (≥ 35% progressive motility), or DEAD (n = 10), 3.0 mL of 900 × 106 sperm killed by snap freezing in liquid nitrogen. Four hours post AI, mares were slaughtered; ex vivo uteri were subjected to a low-volume lavage (LVL) with 50 mL of PBS and three endometrial tissue samples were collected (both uterine horns and uterine body). The fluid recovered was analyzed in a Neubauer chamber. Endometrial samples were processed for histologicalevaluation using bright-field microscopy. For each mare, the three uterine samples were pooled for analysis. The number of PMNs in the stratum compactum (SC) and spongiosum (SS) was counted in 5 fields per uterine sample at 400x magnification, totaling 15 fields/stratum/mare. Groups were considered independent factors. Leukocyte concentrations in the LVL and PMN presence in both strata were considered dependent factors. Leukocyte concentrations in the LVL were analyzed with a Mann-Whitney test (median ± IQR) and the number of PMNs per field of each stratum using a t-test (mean ± SEM). The significance level was defined as p ≤ 0.05. Mares in the DEAD group presented a lower concentration of leukocytes in the LVL than in the LIVE group (33.2 × 106/mL ± 62.6 versus 63.2 × 106/mL ± 56.5; p = 0.05). Correspondingly, the mean PMN number per field in the SC (50.5 ± 5.2/field versus 69.0 ± 5.3/field; p = 0.02) and SS (32.8 ± 6.2/field versus 54.4 ± 5.7/field; p = 0.02) of mares in the DEAD group were lower than mares in the LIVE group. In conclusion, dead sperm elicited a less intense inflammatory response 4 hours after AI compared to live sperm, suggesting that as with bacteria, the immune system may differentiate sperm according to viability.

Evaluation of equine semen frozen in extenders free of animal proteins

Various countries are reticent regarding importation of semen that contains egg-yolk, limiting its commercialization. Egg-yolk composition is complex, varying with the diet of the chicken and egg-yolk granules are similar in size to sperm, interfering with analysis. Therefore, development of a chemically defined cryopreservation extender that maintains seminal parameters is relevant. Twelve ejaculates from 4 stallions of proven fertility (n= 4; r=3) were diluted in one of 5 extenders: 1) EDTA-glucose with 11% lactose, Equex, 20% egg-yolk and 5% dimethylformamide (EY); 2) casein salt and cyclodextrin cholesterol extender (CE); 3) CE with 3% dimethylformamide (CE-3); 4) OptiXcell (OP); 5) BioXcell (BIO). Cryopreservation was carried out in a Cryogen, Neovet automatic freezing machine: -0.5°C/minute to 5°C, 180 minutes equilibration, then -5°C/minute to -20°C and -20°C/minute to -100°C, then plunging in N2. Thawing was carried out at 37°C for 1 minute and sperm kinematic parameters (CASA, AndroVision, Minitube), viability and acrosome status (FITC-PNA-PI stain), membrane lipoperoxidation (BODIPY stain) and DNA fragmentation (SCD) were evaluated. Data were analyzed using ANOVA or Friedman test and results are reported as mean ± SD. Higher (P<0.05) percentages of total and progressively motile sperm were observed in samplescryopreserved in EY (29.25 ± 11 and 21.17 ± 10), CE-3 (30.4 ± 15 and 22 ± 12) and OP (23.1 ± 14 and 15.5 ± 10) compared to those in CE (4.75 ± 3 and 1.58 ± 2) and BIO (8 ± 4 and 3.25 ± 3). Straight-line, curvilinear and average-path velocity parameters, amplitude of lateral head and beat-cross frequency were significantly higher in samples frozen with EY, CE-3 and OP compared to CE and BIO media. Straight-line and average-path velocities were also significantly higher in OP compared to EY medium. Live acrosome-intact sperm were significantly higher in EY (45 ± 14) compared to the rest of the extenders; with CE-3 (25 ± 10) being significantly higher than CE (8 ± 5), OP (9 ± 5) and BIO (13 ± 5). Live, non-peroxidized sperm were significantly higher in EY (47 ± 15) compared to the other extenders; with CE-3 (29 ± 15) being significantly higher than CE (14±10), OP (21 ± 15) and BIO (23 ± 14). No significant differences were observed in the percentages of sperm with intact DNA in any of the extenders: EY (95 ± 4); CE (95 ± 2); CE-3 (93 ± 8); OP (94 ± 7) and BIO (94 ± 5). To conclude, the casein salt and cyclodextrin cholesterol extender with 3% dimethylformamide, without egg-yolk could be a suitable alternative for extenders with animal proteins. Further studies are ongoing to evaluate in vivo fertility of ejaculates cryopreserved in this extender.

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 106 mL−1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 μM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 μM (CCCP), uncoupling agent; antimycin A 1 μg/mL (ANTI), complex III inhibitor; oligomycin 5 μM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O2•– production and H2O2 intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

Protective Effect of Acetyl L-Carnitine on Rat Spermatogenesis Defects Induced by Lead Acetate

In this study the influences of the ameliorative effects of Acetyl-L-carnitine, when exposed to lead acetate on functional aspects in spermatogenesis process in the testes of albino male rats, studied, 24 male albino rats weight 178-250 g were randomly distributed into four groups, 6 rats in each, the control group received distilled water orally, Treatment 1 lead acetate group received 10mgkg body weight orally, Treatment 2 acetyl L-carnitine(ALCAR) group received 100mgkg body weight orally, and Treatment 3 lead acetate and acetyl L-carnitine group received 10mgkg body weight and 100mgkg body weight orally respectively. After the end of the experiment the weight of sexual organs were taken, in addition, the total sperm count number, and percentage of dead, live, with abnormal spermatozoa also have been calculated. Results showed that the total sperm count in the lead acetate group was decreased compared with the control and ALCAR groups. The lead acetate with ALCAR showed an increase in total sperm count and live sperm percentage in comparison with control and ALCAR groups. Dead and abnormal sperm percentage increased in the lead acetate group and in lead with ALCAR groups in compared with control and ALCAR grou