Live Sperm Research Articles

Phosphatidylserine-Positive Live Sperm as a Biomarker of Fertility Status: Evidence from Patients Undergoing Varicocele Treatment

Purpose: Phosphatidylserine exposed on live and motile sperm is necessary for sperm:egg fusion, and emerging data suggest it is a biomarker for fertilization competent sperm. Men with varicocele often display infertility, and one of the known causes is poor sperm quality. Here, we sought to determine whether the percentage of phosphatidylserine-positive live sperm (PS+ live sperm) could serve as a reliable indicator of male fertility. Materials and Methods: Semen samples from cryo-banked pregnancy-proven donors, from self-identified pregnancy-reported donors, and from men seeking infertility treatment for varicocele were analyzed for phosphatidylserine exposure on live sperm. Semen samples were also analyzed premicrosurgical and postmicrosurgical varicocele repair to determine whether varicocele repair led to a change in the percentage of PS+ live sperm. Results: Semen samples from “pregnancy-proven” donors (n = 31) had 82% ± 2% PS+ live sperm and samples from “self-identified pregnancy-reported” donors (n = 34) had 87% ± 1%. Men presenting with a varicocele (n = 80) had significantly lower PS+ live sperm with 68% ± 3%. PS+ live sperm correlated with reported pregnancies in patients with varicocele, whereas total motile sperm count did not. Varicocele repair (n = 10) led to a significant increase in PS+ live sperm, and 2 of the patients reported pregnancies after varicocele repair. Conclusions: These results suggest that the percentage of PS+ live sperm in an ejaculate correlates with male fertility status and could help in guiding treatment options for patients with varicoceles.

Efecto de la criopreservación en el potencial de membrana plasmática en espermatozoides de alpaca (<i>Vicugna pacos</i>)

Membrane potential is the electrical difference between the inside and outside of a cell, and depolarization is the reduction of the negative value of this potential. This study aimed to determine the state of the plasma membrane potential of alpaca spermatozoa after cryopreservation, using fluorescence intensity as a measure. In total, 32 epididymal sperm samples subjected to a standard cryopreservation protocol were analysed. Each sample was evaluated before and after the process. The fluorochrome DISBAC2(3) was used at 15 µm for 30 min and 12 µM propidium iodide (PI) at 37 °C for 10 min to analyse membrane depolarization and viability, respectively. Measurements were performed with a flow cytometer containing an image analyser, using a 488 nm excitation laser and the detection channels Ch03 and Ch05 for DisBAC2(3) and PI, respectively. The mean fluorescence intensity of DisBAC2(3) in fresh live spermatozoa was 3176 ± 1398, while in thawed spermatozoa it was 2356 ± 1206 (p<0.05). Results suggest that cryopreservation, on average, reduces the fluorescence intensity of DisBAC2(3), which can be interpreted as a hyperpolarization of the plasma membrane after thawing.

EFFECT OF SEASON ON METABOLITES AND SEMEN TRAITS OF BULLS

This study was conducted to investigate the effect of the winter and spring seasons on some biomarkers and semen characteristics of Holstein bulls (n = 20) during the Coronavirus pandemic period. Semen was collected for 16 weeks and evaluated weekly, along with the determination of some biomarkers. Sperm concentration, live sperm percentage, the concentrations of the most amino acids, essential, non-essential, and total amino acids, all carboxylic acids, omega 3, 6, 9, total fatty acids, and total saturated and unsaturated fatty acids, increased (P<0.05) in semen of spring compared to the winter season. Moreover, the percentage of plasma membrane integrity was higher in the winter than the spring season. The concentration of malondialdehyde in semen decreased (P<0.05) in the semen during the winter compared to the spring season. These biomarkers may have played a role in the fertility and semen quality or were affected by the season and repercussions of the Corona pandemic (feed shortage stress). In conclusion, the level of some amino and carboxylic acids can be adopted as an indicator of climate change (season) and semen quality of bulls or energy level in the diet.

Dietary supplementation with rosemary essential oil improves genital characteristics, semen parameters and testosterone concentration in Barki rams

BackgroundMale reproductive performance is an essential part of sheep production; therefore, the use of natural antioxidants to improve sperm quality and maintain reproductive performance in males is very important. Hence, oral administration of rosemary essential oil (REO) was investigated to improve the fertility rate, including the ultrasonographic testes, epididymal tail and genital glands, as well as semen parameters and testosterone concentration. Sixty animals were splitted into two groups, each with 30 rams; the rosemary group (C + REO) received 2 mg/kg/bw and the control group (C-REO). Ultrasound images and blood samples were collected at 15, 30 and 45 days of the REO treatment.ResultsThe testis and epididymal tail ultrasonographic assessments demonstrated a significant enhancement in the C + REO group compared to the C-REO group. However, the rams in the C + REO group showed significant improvements in the pampini-form plexus, seminal vesicle, Cowper's and prostate genital glands compared to the C-REO group. The data showed that the sperm cell concentration (× 109/ml) and individual motility (%) were significantly improved in the C + REO group. Furthermore, ejaculate volume (ml) in the C + REO group was significantly higher than that in the C-REO group. While the animals treated with REO did not improve live spermatozoa (%), it reduced the abnormalities of spermatozoa (%) compared to the C-REO group. Also, the C + REO group significantly increased the testosterone concentration more than the C-REO group.ConclusionIt can be concluded that supplementation with 2 mg/kg/bw REO improves genital characteristics, semen parameters and testosterone concentration in Barki rams.

Factors determining semen sample collection and semen quality parameters in African penguins Spheniscus demersus

Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes.

Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

Freezability of Semen Collected from Ganders of 12 Breeds Covered by the Polish Genetic Resources Conservation Program

Abstract A rapid decline in wild animal biodiversity, as well as in the number of local livestock breeds of lower economical values, makes species and breeds protection and creation of genetic reserves a necessity and a challenge of the 21st century. For birds, semen freezing is still the best method of gene preservation ex situ in vitro. The aim of the presented study was to assess the susceptibility to freezing process and to establish the semen gene bank of twelve goose breeds covered by the Polish genetic resources conservation program. Sexually mature ganders were randomly selected from four northern local breeds: Kartuska, Pomorska, Rypinska, Suwalska, four southern local breeds: Garbonosa, Kielecka, Lubelska, Podkarpacka, and four foreign goose breeds: Kuban, Landes, Roman and Slovakian. Each breed was represented by 12 males, twelve repetitions were carried out. Semen samples were frozen in a computer-controlled cryogenic chamber using a method developed for White Koluda goose. The basic evaluation criterion was sperm morphology in fresh and freeze-thawed semen assessed in nigrosine-eosin stained histological smears. The percentage of live in total and live normal sperm in the fresh semen differed significantly (P<0.05) between breeds. Semen of Kartuska and Suwalska ganders (northern breeds), contained the highest percent of live sperm – 94.4 and 94.1 respectively, including 52.5% and 54.2% of live normal sperm, while the lowest number of these forms was observed for Slovakian goose – 86.4% and 29.2%, respectively. The freezing process resulted in a significant (P<0.05) reduction in the total number of live and live normal sperm and an increase in dead sperm in all groups. Cryopreservation process was the most effective in case of Suwalska goose semen (28.4% of live normal cells in thawed semen) and the less profitable for Landes (7.4% of live normal cells). The analyses showed significant differences between breeds in sperm morphology in fresh and thawed semen. Moreover, the high proportion of live spermatozoa in the fresh semen did not guarantee a high proportion of spermatozoa after thawing, indicating the differences in semen susceptibility to cryopreservation process depending on goose breed.

Assessment of eruca sativa leaves extract ZnO NPs effect on the adverse effects of creatine - induced testes injury

This study was conducted to evaluate the protective role of Eruca sativa NPs on the harmful effects of creatine on the testes in male rats. This study used 48 male rats, in two experiments. The first experiment included determining the most effective nano-concentration of watercress extract, while the second experiment included evaluating the role of the nano-extract at the concentration that was chosen based on the results of the first experiment (60 mg/kg) against toxicity resulting from doses of creatine (4 g/kg) for 60 days. This study used 24 adult male rats were randomly assigned to four groups, with six animals per group, and were treated as follows:(G1) 1 ml of 0.09% physiological solution , (G2) dosed (single dose/day) with nano-extract of Eruca sativa at a concentration of (60 mg/kg) , (G3) The animals were dosed (single dose/day) with creatine monohydrate (4 g/kg), (G4 prevention group) dosed with nano-extract of Eruca sativa (60 mg/kg) followed one hour later by a dose of creatine (4 g/kg) . The results of the study indicated a non significant in Concentration of sperm in the epididymis, Percentage of live sperm ,Percentage of motile sperm , Percentage of normal sperm, LH , FSH and Testosterone in G2 and G4 compared to G1. As for the preventive group G4, it recorded a normal results near from control group. it recorded a significant decrease (P<0.05) in Concentration of sperm in the epididymis, Percentage of live sperm ,Percentage of motile sperm , Percentage of normal sperm, LH , FSH and Testosterone in G3 compared to G1 and G2 . The results of the histological examination in G3 showed that there are interstitial spaces between the seminiferous tubules and few sperm in the cavities of the tubules with reduction and disintegration of the interstitial tissue and a lack of Leydig cells and a decrease in the size of the germinal epithelial cell layer with cell degeneration necrosis of cells lining the tubules compared to G1,G2 and G4. The results of this article revealed the positive effects of Eruca sativa leaf ZnO NPs extract on the adverse effects of creatine-induced testes injury.

PSXIII-16 Kinematics and viability of spermatozoa from Criollo and Angus bulls in a low-input system

Abstract The Chihuahuan Criollo cattle is a genetic resource with special adaptive characteristics to rough, arid and semi-arid ecosystems. There is a great interest in incorporating such adaptations into beef production systems as one of the strategies for sustainable production to face the climate change challenge. Reproductive success is determinant to achieve sustainability in animal production systems. Most research efforts to improve reproductive efficiency have focused on the study of females. However, it has been shown that much of the economic losses caused by embryonic deaths are due to the bull. The aim of this study was to evaluate the quality of thawed semen from Angus and Criollo bulls kept in a low-input system. Thirteen healthy bulls (n = 7 Angus and 6 = Chihuahuan Criollo), with an average age of 4.3 ± 0.48 yr, grazing in the same rangeland and subjected to natural pasture conditions, were studied. Semen obtained by electroejaculation, were frozen and subsequently thawed for analyses. Characteristics of motility, vitality (V), morphology (M) and plasma membrane integrity (PMI) were evaluated in fresh and thawed semen, whereas movement kinetics (MK) and acrosome reaction (AR) were evaluated in thawed semen. Statistical analysis was performed using the PROC GLM of SAS Studio 2022 including the fixed effects of breed, semen condition and their interaction for variables of progressive motility (PM), V, M, PMI and the effect of breed for variables of MK and AR. As expected, PM and V were less (P < 0.05) in thawed (57.93 ± 4.1 and 53.69 ± 2.7) compared with fresh (72.64 ± 3.9 and 74.98 ± 2.6) semen, whereas no differences (P > 0.05) were found in M. However, the interaction between semen condition and breed for PMI tended to the significance (P = 0.08) with a PMI of 68.95 ± 4.6 and 58.98 ± 4.3 for Criollo and Angus, respectively, in fresh semen, whereas in thawed semen this difference was inverted (52.08 ± 4.6 and 58.40 ± 4.6). It is important to note that the semen retained quality traits with values above the minimum accepted for use in artificial insemination, despite the scarcity of resources of the system in which the animals were kept. There were no differences by breed in the main MK variables; however, AR reported as live unreacted sperm was less (P < 0.05) in Criollo (40.49 ± 4.49) compared with Angus bulls (58.48 ± 4.49). In conclusion, it is important to incorporate the assessment of membrane integrity and acrosome reaction in thawed semen evaluation to obtain a more accurate prediction of the potential fertility of samples, even if motility parameters appear to be optimal.

THE EFFECTIVENESS OF VITAMIN C AND E ADDITION IN VARIOUS DILUENTS OF BRAHMAN CATTLE SPERMATOZOA AFTER FREEZING

This research aimed to determine the quality of Brahman cattle spermatozoa after freezing in various diluent ingredients (Egg Yolk Citrate, Egg Yolk Tris, Skim Milk) which added vitamin C and vitamin E. The research was carried out using 4 treatments with 3 different diluent ingredients, each of which was Vitamins C and E are added to each diluent ingredient at a dose adjusted to the type of diluent agent. The treatment given is P0: Diluent without Vitamin C and E; P1: Diluent + Vitamin C; P2: Diluent + Vitamin E; and P3: Diluent + Vitamin C and Vitamin E. Research data were analyzed using analysis of variance and followed by Duncan's test at the 5% and/or 1% level for different variables. The results showed that the addition of vitamin C and vitamin E had a significant effect (P0.05) on motility and viability in the egg yolk tris diluent and egg yolk citrate but had no significant effect (P0.05) in the skim milk diluent. The use of different diluents had no significant effect (P0.05) on motility, viability, and abnormalities. This research concludes that Vitamin C added to the diluent tris egg yolk, egg yolk citrate, and skim milk showed higher average motility, percentage of live spermatozoa, and abnormalities of post-freezing Brahman cow spermatozoa.

Boar Sperm Performance in Polystyrene Transport Cool Box Under Various Storage Conditions and Holding Times

When transporting highly valuable boar semen samples over long distances, it is crucial to ensure that boar semen is stored ideally at approximately 15 – 17°C to minimize the impact of cold shock and oxidative damage on sperm quality. This study evaluated the performance of a commercially available semen and embryo polystyrene transport cool box, BotuFLEX®, in preserving the quality of extended boar semen under different storage conditions and holding times. Boar ejaculates were extended at 1:1 or 1:3 using a commercial mid-term extender, stored in a BotuFLEX® semen cool box, and examined 24 vs. 48 h thereafter (Experiment 1; n = 12), or 24 h after storage in air-conditioned room vs. ambient room (Experiment 2; n = 9) conditions. Comprehensive sperm motility using the Sperm Class Analyzer® CASA system revealed no significant difference in either total and progressively motile sperm, and the combined progressivity and velocity characteristics across different treatments. Almost similar results were observed on sperm vitality using the standard supravital staining technique except that percent live spermatozoa were higher at 24 h of storage using the 1:1 dilution.

Live Motile Sperm Sorting Device Improves Embryo Aneuploidy: A Retrospective Cohort Study

Background: Conventional sperm selection methods, involving centrifugation, exert a detrimental effect on sperm DNA integrity due to mechanical stress. The recent noninvasive sperm selection device, CA0, based on live sperm sorting technology, facilitates the retrieval of highly motile sperm and minimizes sperm DNA fragmentation (SDF). This study was to investigate the impact of various sperm separation methods, with and without centrifugation, on embryo ploidy status. Methods: The retrospective study comprised 82 intracytoplasmic sperm injection (ICSI) cycles involving preimplantation genetic testing for aneuploidy (PGT-A) cases, with a focus on recruiting egg donation cycles to thoroughly investigate the impact of male factors. Two populations are classified based on semen quality: normozoospermic ([Formula: see text] = 33) and non-normozoospermic ([Formula: see text] = 49). Subjects were allocated to either swim-up (SU) or CA0. Preimplantation genetic testing results were recorded. Results: When comparing male characteristics between subgroups, no significant differences were observed except for a lower normal morphology rate in the CA0 group compared to SU (SU: 3 [3–4] vs. CA0: 2 [2–2.8], [Formula: see text] < 0.0001) in the non-normozoospermic cohort. There were no differences in female factors such as age and mature oocyte count (MII) number between subgroups, indicating that this model is ideal for assessing the impact of male factors on clinical outcomes. In the normozoospermic cohort, euploidy rates were similar between SU and CA0 (SU: 71.9% vs. CA0: 64.2%). However, in the non-normozoospermic cohort, CA0 showed a significantly higher euploidy rate compared to SU (SU: 53.6% vs. CA0: 74.2%) and a lower aneuploidy rate (SU: 37.1% vs. CA0: 25.8%). Additionally, CA0 minimized the incidence of mosaic embryos, whereas a mosaicism rate of 9.3% was observed with SU. This trend highlights CA0’s distinct advantage in optimizing outcomes for non-normozoospermic cases. Conclusions: CA0 is a reliable intervention to optimize paternal genetic quality before assisted insemination, thereafter effectively reducing the incidence of embryo aneuploidy associated with male factors.

In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism

BackgroundConservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24–48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. Material and methodsProcessed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca2+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. ResultsAfter 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca2+ increased in all treatment groups compared to time 0h. Discussion and conclusionViable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.

Effect of tyrode albumin lactate pyruvate and modified Krebs Ringers broth media on in vitro capacitation of HD-K75 boar spermatozoa at different period of incubation.

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán etal., Theriogenology, 198, 2023 and 231; Oberlender etal., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo etal., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on invitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.

Body weight, scrotal parameters and semen characteristic of Kano Brown Buck Kids fed Pluerotus ostreatus solid state fermented sugarcane scrapings

The study evaluated the effect of substitution of Pleurotus ostreatus biodegraded sugarcane scrapings (BSS) for corn bran on the growth performance and reproductive potential of Kano Brown bucks. Twenty-one healthy buck kids (6 – 7 months of age, with an average initial BW of 9.44 ± 0.39 kg) were stratified into three treatments and fed: (1) a total mixed diet containing no BSS (0% BSS; control), (2) the control diet containing 15% BSS substituting 50% corn bran and (3) the control diet containing 30% BSS substituting 100% corn bran on dry matter basis (DM) for 12 weeks in a completely and fully randomized design. Intakes of DM, crude protein and organic matter, and sperm concentration varied in the order: 15% BSS &amp;gt; 0% BSS &amp;gt;30% BSS (p&amp;lt;0.05). Final BW, semen volume, initial fructose, scrotal length (SL) and scrotal circumference (SC) were greater in 15% BSS diet than 0 and 30% BSS diets. Semen pH and color, sperm progressive motility, viability and abnormalities, and live spermatozoa were not affected by diets. Whereas testosterone level was greater in 0 and 15% BSS diets, libido was lower in 30% BSS diet. Final BW was positively correlated with SC (p=0.030; r = 0.510) and SL (p = 0.048; r = 0.472). It was concluded that up to 30% biodegraded sugarcane scrapings can be used in a complete diet for bucks without negatively impacting final body weight and semen quality, though 15% BSS was more impactful and recommended.

Comparison of Nanoparticles and Single-Layer Centrifugation for Separation of Dead from Live Stallion Spermatozoa.

The goal of this study was to compare the efficacy of coated iron-core nanoparticles and single-layer centrifugation for separation of dead from live stallion spermatozoa. Our hypothesis was that nanoparticles would bind to dead sperm and allow for separation from live sperm using a magnet, resulting in a population of spermatozoa with a high percentage of total and progressive motility. Treatment Group 1 was an untreated control. Treatment Group 2 (nanoparticles, NP) utilized sperm incubated with nanoparticles followed by application of a magnet to remove dead sperm adhered to the coated nanoparticles. Treatment Group 3 (single-layer centrifugation, SLC) layered sperm above EquiPure™ followed by centrifugation. Semen samples were subsequently evaluated for sperm motility parameters, plasma membrane integrity, acrosome status, and morphology. The SLC technique yielded higher (p < 0.05) progressive motility (76 ± 9.2%) than the NP separation technique (59 ± 12.2%) or the untreated control (47.3 ± 5.1%). However, the total number of sperm recovered was higher (p < 0.05) in the NP technique (526.2 ± 96.6 × 106) than the SLC procedure (211.7 ± 70 × 106), yielding a higher total number of progressively motile sperm (317.6 ± 109 × 106) recovered using the NP technique than the SLC technique (157.8 ± 43.6 × 106). The percentage of live, acrosome intact sperm recovered was higher for SLC than NP. In summary, the SLC technique yielded a higher percentage of sperm motility, intact plasma membranes, and acrosome integrity, but yielded lower total sperm than with the nanoparticle separation technique.

Importance of L-carnitine as Biochemical Marker for Semen Quality Preservation after Cryopreservation in Stallions

Background: The aim of this work is to study the significance of carnitine fractions and NO metabolites for the assessment of spermatozoa resistance to cryopreservation, for which purpose these parameters were investigated in seminal plasma of animals divided according to the indices of spermatozoa of stallions subjected to cryopreservation. Methods: There were 18 breeding stallions with an average age of 11.28±5.46 years, all 18 were of Arabian breed. Using cluster analysis of indicators of semen quality, animals were identified into two groups: with high and low sperm resistance to cryopreservation-thawing. We assessed standard quality parameters in fresh and cryopreserved sperm, as well as carnitine and its fractions and NO metabolites in stallions’ seminal plasma, steroid hormones level and carnitine fractions in blood serum. Data were analyzed using JASP. Differences at p less than 0.05 were considered statistically significant. Result: Seminal plasma obtained from animals from the cluster with low motility after cryopreservation-thawing was characterized by a lower concentration of nitric oxide metabolites in 1,79 time (p=0.0415) and free carnitine in 1,64 time (p=0.0349). Positive moderate strength correlation were established between the level of seminal plasma free carnitine and the percentage of live spermatozoa after sperm cryopreservation-thawing.

Morphology Classification of Live Unstained Human Sperm Using Ensemble Deep Learning

Sperm morphology analysis is crucial in infertility diagnosis and treatment. However, current clinical analytical methods use either chemical stains that render cells unusable for treatment or rely on subjective manual inspection. Here, an ensemble deep‐learning model is presented for classification of live, unstained human sperm using whole‐cell morphology. This model achieves an accuracy and precision of 94% benchmarked against the consensus of three andrology scientists who classified the images independently. The model loses less than a 12% prediction performance even when image resolution is reduced by over sixfold. This ensures compatibility across varied clinical imaging setups. This model also provides a high certainty and robust classification of challenging images, which divided the experts. By providing a consistent, automated approach for classifying live, unstained cells using quantitative data, this model offers promising future opportunities for enhancing clinical sperm selection practices and reducing day‐to‐day variability in clinics.

P-042 Enhancing human sperm motility using high-frequency ultrasound

Abstract Study question Is high-frequency ultrasound effective in the treatment of low-motility sperm for use in assisted reproduction? Summary answer Our study reveals that immotile and poorly motile sperm respond to ultrasound treatment and show up to 266% boost to motility parameters upon ultrasound exposure What is known already Sperm motility is central to both natural and assisted reproduction. This crucial importance has led to the utilization of chemical drugs such as pentoxifylline, theophylline, and other phosphodiesterase inhibitors to improve motility. However, these drugs are embryotoxic with a potentially harmful effect on sperm longevity. In a recent study, we showed that sperm cells at the population level showed a 32% boost to motility after 15 seconds of exposure to ultrasound. However, to translate these findings into clinical applications, it is crucial to identify which subset of sperm within a population is most affected by the treatment. Study design, size, duration We conducted single-cell study aimed at comprehensively assessing how different sperm motility grades are affected by ultrasound exposure. We utilized a droplet acoustofluidic system using which sperm cells were isolated in droplets and their responses to ultrasound were monitored. A total of 239 individual sperm from three biologically independent human samples were included in this study and parameters such as sperm motility, viability, DNA integrity, and mitochondrial membrane potential (MMP) upon ultrasound exposure were analysed. Participants/materials, setting, methods Fresh human semen samples were collected from healthy donors with the approval of the Monash University Human Research Ethics Committee. Three human donors contributed to a total of sixteen independent experiments performed in this work. The manual tracking plugin in ImageJ was used to measure sperm motility. The LIVE/DEAD™ Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability and JC-1 (Invitrogen, USA) was used to monitor the MMP. Main results and the role of chance We classified the sperm cells based on their pre-exposure straight-line velocity (VSL) into three categories of grade C (non-progressive, VSL &amp;lt; 5 µm s-1), grade B (slow progressive, 5 ≤ VSL&amp;lt; 25 µm s-1), and grade A (rapid progressive, 25 µm s-1 ≤ VSL) cells. Our single-cell analysis results show that 20 seconds of ultrasound exposure boosts the motility of grade C sperm up to 266% and as a result, 90% of total sperm are graded as progressively motile after exposure. Through ANOVA tests, we showed that non-progressive sperm cells show a boost to all of the motility parameters more than the other grades. In addition, ultrasound exposure renders 34% of live immotile sperm motile with 24% of the cells graded as progressive and the remaining 10% showed a twitching motility after exposure. We also showed that ultrasound exposure induces flagellum deformation to 16% of total live immotile sperm cells, providing opportunities to detect live sperm in testicular samples using this approach. Finally, we explored the non-invasiveness of the ultrasound platform and showed that there is no detrimental effect on sperm DNA integrity and viability post exposure. Limitations, reasons for caution A larger sample size could provide a higher statistical confidence to our study. Treatment of patient samples (testicular or asthenozoospermic) and evaluation of embryo safety must be included in future studies to evaluate the effectiveness of this method for clinical use. Wider implications of the findings Our acoustic mechanotherapy method could enhance sperm motility to potentially benefit assisted reproduction outcomes. It also enables new opportunities for detecting live sperm in testicular samples. Trial registration number not applicable

P-043 Preliminary clinical outcome using the Q300™ device in a reproductive laboratory environment

Abstract Study question How does the Q300™ Device impact clinical outcomes in a reproductive laboratory environment compared to established standards? Summary answer The device showed favorable clinical outcomes and was user-friendly. More research is needed to identify the patient subgroups that can benefit the most from system What is known already Embryologists struggle with accurately assessing sperm morphology without staining, prompting interest in emerging technologies like AI. These methods evaluate motility parameters, offering non-invasive sperm selection. A previous study comparing Q300 to manual staining methods showed high agreement and repeatability, indicating Q300’s accuracy in measuring live sperm cells. It aids in selecting cells per WHO2021 guidelines without staining, potentially revolutionizing sperm assessment. Study design, size, duration The study is an open-label, non-randomized study. Semen samples intended for ICSI were imaged by the Q300™ device before micro-injection to aid in selecting morphologically compliant sperm cells. The data collected was compared to the Key Performance Indicators (KPI) for ART laboratories according to the Vienna Consensus. Up to 5 sites will be enrolled in the study, with up to 100 couples in total from all sites. Study duration: 18 months Participants/materials, setting, methods The Q300™ device is an optical system using quantitative phase microscopy and holographic imaging to analyze human semen morphology. The device aids embryologists in selecting live and motile sperm cells for ICSI by providing automatic, objective analysis. Couples meeting inclusion criteria, including female age ≤40 and availability of motile sperm, were recruited for ICSI procedures. There were no restrictions on previous IVF/ICSI cycles. Endpoints: fertilization, day-3 embryo development, good blastocyst development rates, and implantation rates. Main results and the role of chance 14 couples were recruited from Barzilay Medical Center’s IVF unit in Ashkelon, Israel. The average fertilization rate is 79.1% (Vienna consensus competency is ≥ 65%), the day 3 embryo development rate is 55% (Vienna consensus competency is ≥ 45%), and the good blastocyst development rate is 42.5% (Vienna consensus competency is ≥ 30%). The implantation rate (day 3+day5) is 36.8% (Vienna consensus competency is ≥ 25% for day 3 embryo transfers and ≥ 35% for blastocyst transfers). The intended users performed the procedures with no reported malfunctions. 270 sperm imaging procedures were performed using the Q300 device to select 109 morphologically suitable cells for ICSI. The utilization of the product resulted in approximately a 40% yield of correct selections by the embryologist. Consequently, out of every 10 cells deemed compliant by the embryologist, only 4 were confirmed by the device, with roughly 3 falling within a margin of 10% around the WHO measurement range. Moreover, only about 1 of these selections fell within the boundaries defined by the WHO standards. This observation offers insights into the characteristics of cells routinely chosen for injection into oocytes. Limitations, reasons for caution The study recruited 14 couples from a single site; it is important to validate and statistically power the outcome measures and compare them with clinical control groups in the future. In the next generation of the product, incorporating an objective selection based on sperm motility can further improve its functionality. Wider implications of the findings The findings offer insights into optimizing sperm selection processes, potentially improving fertilization, embryo development, and implantation rates in couples undergoing ICSI. Moreover, the study highlights the importance of continued research and refinement of technological innovations in ART to better serve patient needs and improve treatment outcomes. Trial registration number NCT06232720