Swim-up Procedure Research Articles

A hyaluronic acid-containing reagent compatible with glass-bottom dishes and capable of sustained binding of human spermatozoa.

Commercial products currently available for sperm selection utilizing hyaluronic acid (HA) binding prior to intracytoplasmic sperm injection (ICSI) are widely used but have some disadvantages. To potentially circumvent these limitations, we compared ICSI using a self-made hyaluronic acid (smHA) reagent with ICSI using SpermSlow. The binding of the reagents to spermatozoa on plastic- or glass-bottom dishes was quantitated using spermatozoa that were isolated by density-gradient centrifugation and swim-up procedures (N=10/group). Additionally, we investigated the relationship between the HA reagent used prior to ICSI and clinical outcomes after assisted reproduction with HA-ICSI (N=81). The smHA reagent exhibited extremely stable binding to human spermatozoa. The binding time of spermatozoa was significantly longer in the smHA reagent than in SpermSlow on both plastic and glass dishes (plastic: 60.0±0.0 min vs. 2.7±5.9 min, P<0.001; glass: 60.0±0.0 min vs. 2.5±1.8 min, P<0.001). There were no significant differences in the normal fertilization rate between HA-ICSI with the smHA reagent (128/160, 80.0%) and HA-ICSI with SpermSlow (171/231, 74.0%, P=0.184). The frequency of the blastocyst development from the HA-ICSI-derived zygote was significantly higher with the smHA reagent (74/101, 73.3%) than with SpermSlow (76/131, 58.0%, P=0.019). The rates of biochemical pregnancy, clinical pregnancy, fetal heart movement, live birth, and miscarriage were not significantly different between HA-ICSI with the smHA reagent and HA-ICSI with SpermSlow. The blastulation rate was higher for HA-ICSI with the smHA reagent as compared with SpermSlow. Clinical outcomes, excluding blastulation, after HA-ICSI were the same using smHA reagent and using SpermSlow. Spermatozoa binding to the smHA reagent was not attenuated over a 60-min time course. In conclusion, this reagent may shorten and simplify HA-ICSI procedures because smHA can be used with any dish material, making it easier to observe the spindle or assess intracytoplasmic morphology.

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well.

Exposure to Cumulus Cell Secretome Improves Sperm Function: New Perspectives for Sperm Selection In Vitro.

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.

Effects of Benzo[a]pyrene on Human Sperm Functions: An In Vitro Study.

Benzo(a)pyrene (BaP) is considered one of the most dangerous air pollutants for adverse health effects, including reproductive toxicity. It is found both in male and female reproductive fluids likely affecting spermatozoa after the selection process through cervical mucus, a process mimicked in vitro with the swim-up procedure. In vitro effects of BaP (1, 5, 10 µM) were evaluated both in unselected and swim-up selected spermatozoa after 3 and 24 h of incubation. BaP reduced total, progressive and hyperactivated motility and migration in a viscous medium both in swim-up selected and unselected spermatozoa. Viability was not significantly affected in swim-up selected but was reduced in unselected spermatozoa. In swim-up selected spermatozoa, increases in the percentage of spontaneous acrosome reaction and DNA fragmentation were observed after 24 h of incubation, whereas no differences between the control and BaP-treated samples were observed in caspase-3 and -7 activity, indicating no effects on apoptotic pathways. ROS species, evaluated by staining with CellROX® Orange and Dihydroethidium, did not differ in viable spermatozoa after BaP treatment. Conversely, the percentage of unviable ROS-positive spermatozoa increased. Our study suggests that BaP present in male and female genital fluids may heavily affect reproductive functions of human spermatozoa.

Ketone Body β-Hydroxy-Butyrate Sustains Progressive Motility in Capacitated Human Spermatozoa: A Possible Role in Natural Fertility.

Background Calorie restriction is recognized as a useful nutritional approach to improve the endocrine derangements and low fertility profile associated with increased body weight. This is particularly the case for dietary regimens involving ketosis, resulting in increased serum levels of ketone bodies such as β-hydroxy-butyrate (β-HB). In addition to serum, β-HB is detected in several biofluids and β-HB levels in the follicular fluid are strictly correlated with the reproductive outcome in infertile females. However, a possible direct role of ketone bodies on sperm function has not been addressed so far. Methods Semen samples were obtained from 10 normozoospermic healthy donors attending the University Andrology Unit as participants in an infertility survey programme. The effect of β-HB on cell motility in vitro was evaluated on isolated spermatozoa according to their migratory activity in a swim-up selection procedure. The effect of β-HB on spermatozoa undergone to capacitation was also assessed. Results Two hours of exposure to β-HB, 1 mM or 4 mM, proved to be ineffective in modifying the motility of freshly ejaculated spermatozoa isolated according to the migratory activity in a swim-up procedure (all p values > 0.05). Differently, sperm maintenance in 4 mM β-HB after capacitation was associated with a significantly higher percentage of sperm cells with progressive motility compared to β-HB-lacking control (respectively, 67.6 ± 3.5% vs. 55.3 ± 6.5%, p = 0.0158). Succinyl-CoA transferase inhibitor abolished the effect on motility exerted by β-HB, underpinning a major role for this enzyme. Conclusion Our results suggest a possible physiological role for β-HB that could represent an energy metabolite in support of cell motility on capacitated spermatozoa right before encountering the oocyte.

In vitro approach points to a chemotactic effect of melatonin on ram spermatozoa

Sperm orientation mechanisms, such as chemotaxis, are essential for the sperm to reach the oocyte and fertilize it. Melatonin is secreted by the cumulus cells and is also present in the follicular fluid in mammals. The presence of membrane receptors for melatonin in ram spermatozoa, and its proven involvement in the sperm functionality, may suggest a possible role in the guided movement towards the oocyte. Hence, the objective of the present work is to study the in vitro potential chemotactic action of melatonin on ram spermatozoa, analysing the influence of the season (breeding and non-breeding) and the sperm capacitation state. The first experimental approach consisted in the inclusion of melatonin in the upper layer of a swim-up selection method. During the non-breeding season, the presence of melatonin at 100 pM and 1 μM concentrations significantly increased the cell recovery rate, and induced changes in the sperm location of the MT2 melatonin receptor, compared with the standard swim-up. Moreover, the selected sperm population with 100 pM melatonin presented a higher percentage of capacitated spermatozoa. The greater recovery rate obtained with melatonin could be due to the stimulation of sperm movement in random directions, i.e., a chemokinetic effect, or due to a guided movement (chemotaxis) towards the gradient of the melatonin. To elucidate this issue, together with the study of the influence of the sperm capacitation status, we performed a second experimental approach which consisted in the use of chemotaxis chambers and an open-source software (Open-CASA) that analyses the sperm trajectories towards the hormone gradient and calculates a chemotaxis index (SL index). There was a significant difference between the SL index in the presence of 1 μM melatonin and the control without hormone. This effect was only observed in capacitated spermatozoa with cAMP-elevating agents (Cap-CK samples) obtained during the non-breeding season. These results would point to an in vitro chemotactic effect of melatonin on ram spermatozoa, although chemokinesis cannot be ruled out. Nonetheless, the inclusion of this hormone in the swim-up procedure could enhance the sperm recovery rate.

Changes in human sperm motility and DNA fragmentation index after incubation at different temperatures following density gradient centrifugation and swim-up procedures

Objective: The purpose of this study was to evaluate the sperm motility and DNA integrity at different temperatures to analyze whether the sperms are suitable on the second day for insemination of in vitro matured oocytes by intra-cytoplasmic sperm injection (ICSI) following density gradient centrifugation (DGC) and swim-up (SU) procedures. Methods: Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses. Following sperm selection by DGC and SU procedures, the liquified semen samples were divided into three groups and incubated at 4, 25, and 37°C, respectively. Following incubation for 24, 48, and 72 hours, the sperm motility and sperm DNA fragmentation index (DFI) were analyzed. Results: Following the combination of DGC and SU procedures, the sperm motility (91.8% ± 8.6% vs. 50.8% ± 13.1%) and DFI (5.1% ± 7.9% vs. 13.0% ± 11.6%) were significantly improved (P &lt;0.01) compared to those without any treatment. The sperm motility of the 3 groups significantly declined (P &lt;0.05) post-incubation compared to that of the groups prior incubation. However, sperm motility significantly increased (76.9% ± 10.4%) (P &lt;0.05) at 25°C compared to that of the other 2 groups (53.5% ± 11.0% and 47.6% ± 10.2%). Sperm DFI significantly increased (P &lt;0.05) at 37°C following incubation for 24 and 72 hours in comparison to that of the other 2 groups. However, the sperm DFI did not significantly increase when the sperm samples were incubated at 4 (5.7% ± 5.9%) and 25°C (6.8% ± 5.6%) for 24 hours compared to that before incubation (5.1% ± 7.9%). Conclusions: These results indicate that the sperm quality, in terms of motility and DFI, can be efficiently improved by DGC in combination with SU. Following which, the sperm samples can be incubated at 25°C and be used on the second day for insemination of in vitro matured oocytes by ICSI.

P-030 ACE2 receptor and its isoform short-ACE2 are expressed on human spermatozoa

Study questionDo human spermatozoa express angiotensin-converting enzyme 2 (ACE2) receptor? What would be its localization?Summary answerHuman spermatozoa express uniformly ACE2 on the sperm head and the flagellum. Moreover, the short-ACE2 isoform is concentrated on the post-acrosomal region and midpiece.What is known alreadyThe Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection is generating important concerns regarding not only the possible consequences on the respiratory system, but also on other organs, including the reproductive system. ACE2 is considered the main point of entry for the SARS-CoV-2 within the cells through the binding with the spike protein on the virus surface. Furthermore, ACE2 is expressed in human testes cells including Leydig cells, Sertoli cells and spermatogonia. However, to date, the expression and location of ACE2 in mature human spermatozoa has not been investigated yet.Study design, size, durationThis was an in vitro study for the evaluation of the expression and immune-localization of full-length ACE2 and its isoform, short-ACE2, in human spermatozoa. Thirthyfour non-immunized healthy normozoospermic volunteers were enrolled in the study. The study was conducted from May to December 2021.Participants/materials, setting, methodsSemen samples were collected by masturbation from non-immunized healthy normozoospermic voluntaries. Motile sperm suspensions were obtained by swim-up procedure. The expression of ACE2 was assessed by Western-blot analysis, while the immune-localization of ACE2 was evaluated by immune-cytochemical analysis under confocal microscopy. Flow-cytometry experiments were also performed to assess the surface protein expression on a large number of cells.Main results and the role of chanceThe Western-blot analysis of sperm extracts demonstrated two specific bands, one of approximately 120 KDa, corresponding to the glycosylated full-length ACE2, and a second one of approximately 52 KDa, the molecular weight of the protein recently termed short-ACE2. The immune-cytochemical analysis showed a uniformly localization of full-length ACE2 along both the sperm head and the flagellum, whereas the short isoform was preferentially located in the post-acrosomal region of the sperm head and the midpiece. At the flow cytometer, semen samples displayed a wide between-subject variability both in the percentage of ACE2-positive spermatozoa and the density of protein surface expression.Limitations, reasons for cautionFurther studies are needed to determine whether short-ACE2 is a cleavage product from the full-length protein or if it is originated during spermatogenesis. Moreover, the role and the interaction of ACE2 with SARS-CoV-2 in human spermatozoa should be clarified to evaluate the possible impact of the virus on sperm biology.Wider implications of the findingsSince mature spermatozoa are transcriptionally silent and SARS-CoV-2 is an RNA virus, it is unlikely that the virus could affect sperm biology by replicating itself. Nevertheless, the potential effects related to modifications of the sperm membrane or interaction with other receptors or specific proteins cannot be ruled out.Trial registration numbernot applicable

Verbascoside addition during IVM improves equine blastocyst redox status after conventional and mercury-free piezo-assisted ICSI

Oxidative stress is a major problem in in vitro handling of gametes and embryos. In this study, the effects of Verbascoside (VB), a bioactive polyphenol with known antioxidant activity, added during In Vitro Maturation (IVM) of equine oocytes on blastocyst quality were analyzed after conventional or mercury-free piezo-assisted intracytoplasmic sperm injection (ICSI). Cumulus-oocyte complexes recovered from the ovaries of slaughtered mares underwent overnight holding and IVM in the presence of 0 (control) or 100nM VB. Fresh sperm of fertile stallions was subjected to swim-up procedure and used to microinject matured oocytes by conventional (Dell'Aquila et al. Biology of Reproduction 2003;68:2065-72) or piezo-assisted ICSI (Martino et al. Reproduction Fertility and Development 2019;31:1862-73). Embryos were in vitro cultured in DMEM/F-12 with 10% FCS for 9 days under 5%O 2 , 5%CO 2 , 90%N 2 atmosphere. Blastocyst quality was assessed for bioenergetic/oxidative status by confocal laser scanning microscopy after labeling mitochondria (mt) with MitoTracker Orange CMTM Ros and reactive oxygen species (ROS) with 2’,7’-dichlorodihydrofluorescein diacetate (Marzano et al. Molecular Reproduction and Development 2019;86:1388-1404). Then, blastocysts were stained with Hoechst 33258 for total cell numberevaluation and confirmed when showing more than 64 nuclei with organization of an outer trophoblast layer. Proportions of blastocyst rates after ICSI were compared between VB-exposed group and controls by the Chi-square-test, whereas blastocyst total cell number, ROS levels and mt/ROS colocalization were compared by the Student's T-test. Values with P<0.05 were considered significantly different. VB addition during IVM did not influence oocyte maturation rate (31/55, 56% vs 38/76, 50% for VB-exposed and CTRL, respectively). After both conventional and piezo-assisted ICSI, VB had no effects on blastocyst formation rate (4/21, 19% vs 3/24, 12.5%; P>0.05 and 3/10, 30% vs 2/14, 14%; P>0.05) and total cell number (187.3±104.7 vs 143.3±63.2, P>0.05; and 247.3±109.8 vs 465±17.0, P>0.05). Instead, after both microinjection procedures, VB significantly reduced ROS levels (29.7±5.1 vs 36.1±4.2, P<0.001; and 22.8±8.9 vs 30.1±4.4, P<0.01) and increased mt/ROS colocalization (0.63±0.04 vs 0.61±0.03, P<0.001; 0.79±0.06 vs 0.74±0.12, P<0.001). These findings indicate long-term beneficial antioxidant effects of in vitro exposure to nanomolar VB concentrations during oocyte IVM on equine blastocyst quality. Acknowledgments: funded by the Italian Ministry of Health: Project GR-2011-02351396.

BoviPure® density-gradient centrifugation procedure enhances the quality of fresh and cryopreserved dog epididymal spermatozoa

BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non-selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm motilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat-cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of individual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the individual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactness.

Prospective randomized controlled study of a microfluidic chip technology for sperm selection in male infertility patients.

The purpose of this study is to evaluate the impact of a microfluidic approach for spermatozoon selection in male infertility patients undergoing intracytoplasmic sperm injection (ICSI). This research enrolled 128 individuals who had ICSI for male-factor infertility. The patients were separated into two groups according to the method used to pick the spermatozoa: group I (n=64), which used traditional swim-up procedures, and group II (n=64), which used the Fertile Chip for spermatozoon selection during ICSI therapy. Fertilization rates and embryo quality were the major outcomes. The rates of pregnancy, clinical pregnancy and live birth were used as secondary outcomes. As a result, there was no statistically significant difference between the two groups in terms of fertilization rate, total grade 1 and 2 embryos. Implantation rate was significantly higher in the Fertile Chip group than in the control group (50% vs. 31%, p=0.02). The Fertile Chip group had considerably greater pregnancy rates, clinical pregnancy rates (CPR) and live birth rates than the control group (62.5% vs. 45.3%, p=0.038; 59.4% vs. 35.9%, p=0.006 and 46.8% vs. 25%, p=0.009). Fertile Chip had no effect on fertilization rates or embryo quality in male-factor infertility couples. However, the Fertile Chip group had a statistically higher pregnancy rate, CPR and live birth rate.

P–102 GM-CSF (granulocyte-macrophage colony-stimulating factor) as a sperm medium supplement improves sperm quality in Oligoastenoteratospermia (OAT) men by activating the PI3K/Akt pathway

Abstract Study question Can GM-CSF as a sperm medium supplement improve sperm quality in OAT men? Summary answer GM-CSF can be used to improve sperm quality in OAT men by activating the PI3K/Akt pathway. What is known already OAT patients have very low sperm parameters, including morphology, count, and motility, which can adversely affect the results of assisted reproduction technologies. It seems the development of sperm media is necessary to improve the sperm parameters of these patients. GM-CSF is a natural growth factor produced by the reproductive organs, culture media supplemented with GM-CSF is widely commercially available and enhances the embryo development and implantation rate. Studies show that this growth factor in the semen of infertile men is lower than that of fertile men. However, there is no study to assess the effect of GM-CSF on sperm quality. Study design, size, duration In the present study, Semen specimens were collected from 20 OAT patients who have male infertility factors, according to WHO criteria. After the swim-up washing procedure, each of the samples is divided into two groups; experiment, and control. In the experimental group, samples are incubated with a medium containing 2 ng/ml GM-CSF for one hour, yet, in the control group, the sperms are incubated without GM-CSF for the same time. Participants/materials, setting, methods The sperm motility was examined with phase-contrast microscopy, Eosin-nigrosin staining method was used to assess sperm viability. The expression of sperm glucose transporters (GLUT 1, 3) was determined using immunofluorescent staining, the ratio of pAkt to total Akt was assessed by the Western blotting method. Image J software was used to quantify results; the data was analyzed by SPSS software. P-value&amp;lt;0.05 was considered statistically significant. Main results and the role of chance As compared to the control group, supplementation with GM-CSF improved sperm progressive motility, enhanced GLUT 1 and 3, and p-AKT/AKT expression (P &amp;lt; 0.05). There was no significant difference between the viability of the control and experimental groups. Limitations, reasons for caution This study needs further investigations, including Real-time PCR for the genes of this signaling pathway which is ongoing. Wider implications of the findings: We showed for the first time that GM-CSF can improve sperm quality by influencing motility and energy metabolism in spermatozoa which can be affected by increasing the phosphorylation of AKT. This growth factor could be an appropriate supplement in sperm media for OAT patients. Trial registration number IRCT20200519047508N1

Comparison of Results of Semen Parameters Prepared by Swim-up Technique Between the Effect of Adding Autologous Platelet-Rich Plasma and Phosphate Buffer Saline to the Human Semen

The most popular technique of making ready sperm is the swim-up procedure, in which sperm is selected on motility and ability to swim out the plasma of the semen due to the plasma contains decapacitation factors that alter sperm's ability to fertilize. This research covers 119 semen samples from men visiting the fertility clinic of the High Institute for Infertility Diagnosis and ART that included normal and abnormal sperms parameters as stated in the WHO. The semen sample was dissected into 2 portions: First grouping was affected by the equal quantity of phosphate buffer saline (250) μm contain 10% human serum albumin (HSA) while the second grouping was affected by the equal quantity of phosphate buffer saline (250) μm contain 10% human serum albumin (HSA) with added 2% platelet-rich plasma. Then samples are prepared by the swim-up method for evaluation parameters of the sperm. Swim-up PBS+PRP(HAS) media resulted in the highest sperm concentration, highest total count, highest total motility, highest progressive motility A+B, A, and B, the highest concentration of active motile sperm % and highest normal morphology sperm % after that swim up –PBS (HAS), sequentially in a highly significant manner (p &lt; 0.001). We conclude the insertion of PRP resulted in results that are technically far two methods with regarding sperm concentration, total count, total motility, progressive motility A+B, A, and B, non-progressive motility (C), immotile sperm (D), the concentration of active motile sperm % and normal morphology sperm and the values were far better in Swim-up PBS (HAS) with added 2% PRP than those produced by swim-up –PBS (HAS) alone.

Comparison of Results of Semen Parameters Prepared by Swim-up Technique Between the Effect of Adding Autologous Platelet-Rich Plasma and Phosphate Buffer Saline to the Human Semen

The most popular technique of making ready sperm is the swim-up procedure, in which sperm is selected on motility and ability to swim out the plasma of the semen due to the plasma contains decapacitation factors that alter sperm's ability to fertilize. This research covers 119 semen samples from men visiting the fertility clinic of the High Institute for Infertility Diagnosis and ART that included normal and abnormal sperms parameters as stated in the WHO. The semen sample was dissected into 2 portions: First grouping was affected by the equal quantity of phosphate buffer saline (250) μm contain 10% human serum albumin (HSA) while the second grouping was affected by the equal quantity of phosphate buffer saline (250) μm contain 10% human serum albumin (HSA) with added 2% platelet-rich plasma. Then samples are prepared by the swim-up method for evaluation parameters of the sperm. Swim-up PBS+PRP(HAS) media resulted in the highest sperm concentration, highest total count, highest total motility, highest progressive motility A+B, A, and B, the highest concentration of active motile sperm % and highest normal morphology sperm % after that swim up –PBS (HAS), sequentially in a highly significant manner (p &lt; 0.001). We conclude the insertion of PRP resulted in results that are technically far two methods with regarding sperm concentration, total count, total motility, progressive motility A+B, A, and B, non-progressive motility (C), immotile sperm (D), the concentration of active motile sperm % and normal morphology sperm and the values were far better in Swim-up PBS (HAS) with added 2% PRP than those produced by swim-up –PBS (HAS) alone

Replacement of Albumin by Preovulatory Oviductal Fluid in Swim-Up Sperm Preparation Method Modifies Boar Sperm Parameters and Improves In Vitro Penetration of Oocytes.

Simple SummaryPart of the success of assisted reproductive techniques lies in gamete manipulation before the in vitro fertilization (IVF) procedure. Current chemically defined handling media lack crucial components for embryo development that exist under in vivo conditions. Recent studies of pigs have shown that the addition of reproductive fluids during in vitro fertilization and embryo culture improves embryo quality and their epigenetic profile in vitro. Porcine oviductal fluid (pOF) has been used to supplement the handling media used for sperm selection by centrifugation. However, its effect during sperm selection by the swim-up procedure is still unknown, as is the likely beneficial effect of replacing bovine serum albumin (BSA) by pOF as a protein source in sperm preparation media. In this study, four protein combinations in the swim-up medium were tested: 1 mg/mL BSA (the regular supplementation), 1% preovulatory pOF (1% pOF), 1 mg/mL BSA plus 1% pOF; and 5 mg/mL BSA. After sperm selection, various sperm parameters were assessed, and oocytes were inseminated in vitro. Results showed that the replacement of BSA by pOF improves some sperm motion parameters and increases in vitro oocyte penetration ability, whereas the combination of BSA + pOF did not show relevant effects. High concentrations of BSA had a detrimental effect, resulting in a decrease of sperm penetration.More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure’s suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

105 Increasing fertility of interspecific hybrid males using biotechnological approaches

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

Hyaluronidase-based swim-up for semen selection in patients with human papillomavirus semen infection.

Infection by human papillomavirus (HPV) represents one of the most common sexually transmitted diseases in both men and women worldwide. Recently, the detection of HPV virions in the semen of a large percentage of sexually active men has been associated with detrimental effects on both sperm parameters and on assisted reproductive technologies (ART) treatment outcomes. Conventional semen washing procedure used in ART have proved to be ineffective in removing HPV bound to sperm, requiring the identification of more effective and specific methods. In the present study, we assessed the possible use of hyaluronidase for the detachment of HPV from sperm cell surface. Semen samples from five normozoospermic control subjects (CTRL) were incubated with HPV virus-like particles (HPV-VLP) and treated with hyaluronidase by both a modified swim-up procedure (M-SU) and single-cell approach (SCA). The treatment with hyaluronidase was associated with the complete loss of HPV-VLP signal on sperms by both M-SU and SCA. In addition, semen samples from 12 HPV-positive infertile patients were treated with hyaluronidase 80IU/mL by M-SU, resulting in the complete loss of HPV-DNA signal from sperm surface. Finally, the possible impact of hyaluronidase treatment on sperm parameters was assessed on both sperms from the five CTRL subjects and on further five oligo-astheno-terato-zoospermic (OAT) patients, both HPV negative. The treatment with hyaluronidase was equally associated with a slight reduction of sperm viability and progressive motility in both CTRL and OAT. In conclusion, the treatment with hyaluronidase removed efficiently and safely HPV virions bound to spermatozoa.

193 Effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle

Unhygienic practices at the time of parturition or AI lead to uterine infections. The uterine infections ultimately result in genetic drain by culling the elite animals. The invivo developmental competence of embryos is compromised in clinically metritic animals. The genetic potential of problematic females could be harvested by invitro embryo production (IVEP). Therefore, the objective of the present study was to evaluate the effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle. This experiment was carried out from December 2017 to April 2018. Ovaries were collected from a local abattoir (Bos indicus; 5- to 8-year-old dairy cattle, body condition score 2.75±0.25, mixed parity). These ovaries (n=982) were divided into two groups: (1) clinically metritic (n=184), and (2) healthy (n=798), based upon the presence or absence of pus in the uterine lumen. Oocytes were aspirated from follicles using an 18G needle attached to a 10-mL syringe. Cumulus-oocyte complexes (COCs) were categorized into A, B, C, and D grades based on the number of layers of cumulus cells and integrity of ooplasm. The oocytes of grades A and B were subsequently transferred in groups (10/group) in four-well plates containing 100-μL droplets. The droplets with oocytes were covered with prewarmed mineral oil and incubated for 24h at 38.5°C, 5% CO2, and 95% relative humidity. The oocytes were evaluated for IVM on the basis of cumulus expansion. Frozen semen was thawed and prepared using the sperm swim-up procedure for each group. Spermatozoa and oocytes were incubated together for a period of 18h. The presumptive zygotes were invitro cultured for 4 days in a CO2 incubator under similar culture conditions. The cleavage rate, 4-cell, and 8-cell stages were recorded on Days 2, 3, and 4 after the day of insemination, respectively. Data on oocyte recovery, oocyte quality, IVM, cleavage rate, and 4-cell and 8-cell stages were analysed by Chi-squared test using SPSS software (version 20; IBM Corp.) for Windows. Results demonstrated that recovery rate was lower (63.8% vs. 71.7%; P&amp;lt;0.05) in clinically metritic compared with healthy cattle. Similarly, oocytes of grade A and B quality were lower (41.0% vs. 51.1%; P&amp;lt;0.05), whereas those of C and D quality were higher (59.0% vs. 48.9%; P&amp;lt;0.05) in clinically metritic compared with the healthy group. Moreover, 4-cell (38.2% vs. 54.8%) and 8-cell stage embryos (11.3% vs. 29.1%), were lower (P&amp;lt;0.05) in the clinically metritic compared with the healthy group, respectively. However, maturation rate and cleavage rate did not differ (P&amp;gt;0.05) between groups. In conclusion, metritis in slaughterhouse ovaries negatively affects oocyte recovery rate, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle.

Effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine.

Background and Aim:Studies have shown that the pH of the vagina during the course of fertilization may influence the migration of X- and Y-bearing spermatozoa and thus leading to skewness in the sex of the offspring. Hence, this study was carried out to check the effect of the pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine (Bos indicus).Materials and Methods:To check the effect of pH in the enrichment of X or Y sex chromosome-bearing sperm in bovine, we used buffers of various pH ranging from 5.5 to 9.0 for swim-up procedure of sperm sample and collected upper and bottom fraction from the same buffer and checked the abundance of X- and Y-bearing spermatozoa by droplet digital polymerase chain reaction using X- and Y-chromosome-specific DNA probe.Results:The abundance of X- and Y-bearing spermatozoa was not differed significantly in either of the fraction collected.Conclusion:Thus, it appears to be unlikely that an immediate impact of pH on sperm can be a solitary impact on the sex of offspring in bovine.

What are the effects of vitamin C on sperm functional properties during direct swim-up procedure?

SummaryDirect swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P&lt;0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P&lt;0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa.