Activation Of Sperm Motility Research Articles

Bicarbonate, calcium ions, hydrogen peroxide and trypsin modulate activation of Anopheles gambiae sperm motility and protein tyrosine phosphorylation.

With the increasing concern of potential loss of transgenic mosquitoes which are candidates as new tools for mosquito-borne disease control, methods for cryopreservation are actively under investigation. Methods to cryopreserve Anopheles gambiae sperm have recently been developed, but there are no artificial insemination or in vitro fertilization tools available. As a step to achieve this, we sought to identify a suitable medium for in vitro incubation of An. gambiae sperm and to tease out critical components that are involved in the sperm motility activation process. Using two cell viability assays, we identified the Biggers-Whitten-Whittingham (BWW) medium as suitable for in vitro incubation of An. gambiae sperm isolated from testes. We then modified the medium for motility assays by testing different HCO3- and Ca2+ concentrations. Our results show that there is an HCO3- and Ca2+ concentration-dependent activation of An. gambiae sperm motility. We further demonstrated that H2O2 can be produced by the testes in vitro and that the addition of 5.3 μM of H2O2 to the medium improves sperm motility and increases protein tyrosine phosphorylation in An. gambiae. Finally, we show a dose-dependent activation of sperm motility by the addition of trypsin to the medium and more than a 2-fold increase in sperm motility when modified BWW (mBWW) medium is supplemented with H2O2 and trypsin. Our in vitro results suggest that protein tyrosine phosphorylation, intracellular ionic influx, intrinsic production of H2O2 and trypsin-like proteases play a vital role in signal transduction that leads to the activation of An. gambiae sperm motility.

Novel combination with maca improves sperm parameters in vitro of asthenozoospermic men.

Infertility is an inability to conceive after a reasonable period of time (12 months) without the use of contraception or due to a person's incapacity to procreate, whether independently or with a spouse. Problems with the production and maturation of sperm are the most common causes of male infertility additionally; the motility is the major functional character that determines the fertilizing ability of spermatozoa. Therefore the goal of this study is to get better certain sperm function parameters in vitro of asthenozoospermic patient. The World Health Organization (WHO) and many studies considered the infertility as a disease and so many couples complaining from unsuccessful assisted reproductive technologies (ART) procedures to overcome their problem. The goal of this study is to improve certain sperm function feature in vitro of asthenozoospermic semen patients by using combination of motility inducing namely; Maca, L-carnitine and Pentoxifylline that enhance the medium to improve certain sperm characters that might be utilized for ART centers. Semen aliquots were collected from ninety patients with asthenozoospermia who participated in present study, the volume of semen samples with normal ejaculate when was ranged between 1.4-6ml and can be measured by using a measure pipette or conical graduated tube; Inclusion criteria was Asthenozoospermia, oligozoospermia and teratozoospermia men, Infertile idiopathic men also, fertile normozoospermic men. While Exclusion criteria was Azoospermic men, Alcoholic, Patients under treatment with antibiotics and men with Varicocele.The samples split into two equal groups at random. Using Ham's F12 medium, one portion served as the control group, and the other was the treatment group, which was mixing by combining the following ingredients, Maca powder extracts (Lepidium meyenii) (M) 1 mg/ml, 0.5 mg/ml of L-Carnitine (LC), and 10 mg/ml of Pentoxifylline (PTX). The data were analyzed using Statistical Package for Social Sciences (SPSS) version 23.0. The descriptive statistics including frequency, range, mean and standard error, Data from treated and control groups were expressed as mean ± SEM and to compare value between experimental and control groups using Students t-test. Layering approach is used to investigate sperm parameters before and after in vitro activation. The information showed a very large (p< 0.001) increase in active sperm motility grade A, percentage of progressive motility with significant increase in morphologically normal sperm (MNS) with decreased in DNA fragmentation index after activation by layering technique with novel combination medium compared to Hams F12 medium and before activation. The present work stated that novel combination medium (LC, maca and PXT) have potential effects to improve sperm characters in male infertility factors and suggested to be used for sperm preparation and activation in ART programs.

Treatment of human sperm with GYY4137 increases sperm motility and resistance to oxidative stress.

Hydrogen sulfide (H2S) has been shown to play a significant role in oxidative stress across various tissues and cells; however, its role in sperm function remains poorly understood. This study aimed to investigate the protective effect of GYY4137, a slow-releasing H2S compound, on sperm damage induced by H2O2. We assessed the effects of GYY4137 on motility, viability, lipid peroxidation and caspase-3 activity in human spermatozoa in vitro following oxidative damage mediated by H2O2. Spermatozoa from 25 healthy men were selected using a density gradient centrifugation method and cultured in the presence or absence of 10 μM H2O2, followed by incubation with varying concentrations of GYY4137 (0.625-2.5 μM). After 24 h of incubation, sperm motility, viability, lipid peroxidation, and caspase-3 activity were evaluated. The results indicated that H2O2 adversely affected sperm parameters, reducing motility and viability, while increasing oxidative stress, as evidenced by elevated lipid peroxidation and caspase-3 activity. GYY4137 provided dose-dependent protection against H2O2-induced oxidative stress (OS). We concluded that supplementation with GYY4137 may offer antioxidant protection during in vitro sperm preparation for assisted reproductive technology.

Aquaporin-3a Dysfunction Impairs Osmoadaptation in Post-Activated Marine Fish Spermatozoa.

Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility

Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2 µg/ml) in "TALP-Sperm” and “TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.

Seasonal seminal quality variations and vitrification prospects in black flounder Paralichthys orbignyanus

Long-term preservation of gametes has been identified as a tool to improve broodstock management and increase the number of juveniles produced by artificial fertilization. Paralichthys orbignyanus is an important commercial and recreational species distributed in marine and estuarine waters from Rio de Janeiro (Brazil) to the San Matías Gulf (Argentina). This work focused on studying the seminal quality of tank-reared P. orbignyanus, demonstrating that males are fluent year-round, with the highest yields at the early reproductive season. Fresh sperm exhibited good forward swimming, and samples could be refrigerated up to 48 h while retaining their motility after activation. The optimal conditions for P. orbignyanus sperm motility activation were established as 950 mOsmol/Kg and pH values between 7 and 7.9. Additionally, a well-defined protocol for semen vitrification was developed to assess the cryotolerance of this species’ sperm. We successfully produced high-quality sperm samples, using two vitrification formulations containing trehalose and both z-1000 and x-1000 polymers, that can be used in a near-future in vitro embryo production program.

Fertility after photodynamic inactivation of bacteria in extended boar semen.

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.

Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl− (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region.Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage.cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls.Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

Conservation of Protein Kinase A Substrates in the Cnidarian Coral Spermatozoa Among Animals and Their Molecular Evolution.

The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning.

Artificial activation of sperm motility in vitro.

The sperm activation method is a modern methodological approach that is used more and more often in practice. The number of studies focused on methods of artificial activation of human sperm motility are constantly increasing. Standard sperm selection methods can fail in some cases, among other things, because very young sperm are isolated that have not yet completed their development. In these cases, artificial stimulation of their movement can have a positive effect and greatly facilitate and faster the process of selecting suitable sperm. Methylxanthines are most often used as activating agents. However, opinions on the safety of using these substances on sperm are not uniform. The aim of the thesis is to present current knowledge about artificial activation of sperm motility for in vitro fertilization and subsequent embryonic development. Research of relevant literature in Web of Science, Scopus, PubMed/Medline databases. The literature analysis shows that this method is safe and effective in the selection of immotile spermatozoa. Scientific studies have been conducted to verify the safety and reliability of this method. The conclusion of these studies is the positive impact of this method of selection, especially in cases of sperm obtained from testicular tissue after method testicular sperm extraction. In these cases, the method of artificial sperm activation facilitated and accelerated the selection of sperm before intracytoplasmic sperm injection. Undamaged spermatozoa, which are immobile due to incomplete maturation, were activated.

Alpha-lipoic acid improves cryopreservation of rooster semen by reducing oxidative stress

Inhibiting oxidative stress is key for ensuring sperm motility during semen cryopreservation. The aim of this study was to investigate the effect of adding alpha-lipoic acid (ALA) as an extender in rooster semen cryopreservation. Different concentrations of ALA were added to the frozen diluent of rooster semen; subsequently, computer-aided semen analysis was used to determine membrane functional integrity, acrosome integrity, antioxidant capacity (based on T-AOC, GSH-Px, SOD, CAT, and MDA contents), and mitochondrial integrity. The frozen sperm ultrastructure was observed using transmission electron microscopy. The results showed that the addition of different concentrations of ALA partially to greatly improved the quality of frozen sperm; in particular, 8 μg/mL ALA significantly improved multiple parameters of sperm quality, including sperm motility and antioxidant enzyme activity, after freeze-thaw. The results of this study provide empirical and theoretical support for effective rooster semen cryopreservation and can inform the development of new protective agents in the field of livestock reproduction.

The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.

Кінематичні показники та дихальна активність деконсервованих сперміїв баранів за додавання наноцитрату Mn, Zn та Cu до середовища для кріоконсервування

The aim of the work was to find out the effect of adding nanocitrate of Mn, Zn and Cu to the medium for cryopreservation of ram sperm on kinematic indicators and respiratory activity of thawed sperm. The experiment was conducted on six clinically healthy breeder rams of the Texel breed aged 2–4 years. After receiving the ejaculates of the rams, they were evaluated for the volume, concentration and motility of the sperm and were divided into control and experimental groups. Control sperm samples were diluted with lactose-yolk-tris-citrate-glycerol medium (LYTCGM). Nanocitrates of trace elements were added to the medium in experimental samples of ram sperm in the following doses: Zn and Mn — 2.5, 5.0 and 7.5 μg/l, Cu — 1.25, 2.5 and 3.75 μg/l. Diluted sperm was packaged in straws, equilibrated for 2.5 hours and frozen. After thawing of sperm, motility, morphological damage of sperm, kinematic parameters of sperm motility (CASA), oxidation and reduction activity of sperm were determined. A dose-dependent effect of Mn, Zn, and Cu nanocitrates upon their addition to LYTCGM was established. The addition of Mn and Zn nanocitrate at a dose of 5.0 μg/l to LYTCGM significantly (P&lt;0.05–0.01) increases the activity of thawed ram sperm, while the addition of Cu nanocitrate in increasing doses significantly reduces the motility of sperm in thawed ram sperm. Addition of Mn and Zn nanocitrate in an optimal dose of 5.0 μg/l to LYTCGM significantly (P&lt;0.05–0.01) reduces the number of spermatozoa degenerated and with damaged acrosomes, and with the addition of Cu nanocitrate in increasing doses, morphological disorders of germ cell significantly increase cells The addition of Mn and Zn nanocitrate at a dose of 5.0 μg/l to LYTCGM significantly (P&lt;0.01–0.001) increases the kinematic parameters of thawed ram sperm, and the addition of Cu nanocitrate in increasing doses significantly reduces the indicators of germ cell motility. The addition of Mn and Zn nanocitrates to the medium for cryopreservation of ram sperm increases the oxidation and inhibits the reduction activity of thawed sperm. Addition of Cu nanocitrate to LYTCGM in increasing doses reduces the oxidation and increases the reduction activity of thawed ram sperm.

Manipulated sperm motility via soot nanoparticles-induced biochemical alterations in human seminal plasma

Obtaining spermatozoa with progressive motility, via postejaculatory activation with pharmacological agents such as theophylline and pentoxifylline, is crucial for the success rate of assisted reproduction in couples with severe male factor infertility. Regrettably, the possibility of premature acrosome reactions and impared oocyte function questions the practical applicability of phosphodiesterase inhibitors. The rapid development of nanotechnologies promotes the use of hydrophobic rapeseed oil soot as a non-cytotoxic biomaterial for sperm motility activation, but the scarcity of knowledge regarding the interactions of soot with components from the seminal plasma hinders the eventual commercialization of this cutting-edge approach. Aiming to eliminate this shortcoming, the current study shows for the first time how the soot nanomaterials alter the biochemistry of human seminal plasma. Upon 270 min incubation with soot nanoparticles, the activity of AST, ALT, CK, LDH and GGT enzymes in the seminal plasma of ten patients changes inversely to the registered sperm motility (i.e., lower enzyme activity, higher sperm motility and vice versa). This phenomenon is primarily related to termination of the enzymes-substrate binding or extraction of enzymes from the gametes via chemical bonding with the soot. These novel mechanisms depend on the physicochemical features of used carbon nanomaterials, revealing opportunities for predictable tuning of the sperm reproductive potential.

Characterization of a sperm motility signalling pathway in a gonochoric coral suggests conservation across cnidarian sexual systems.

Most stony corals liberate their gametes into the water column via broadcast spawning, where fertilization hinges upon the activation of directional sperm motility. Sperm from gonochoric and hermaphroditic corals display distinct morphological and molecular phenotypes, yet it is unknown whether the signalling pathways controlling sperm motility are also distinct between these sexual systems. Here, we addressed this knowledge gap using the gonochoric, broadcast spawning coral Astrangia poculata. We found that cytosolic alkalinization of sperm activates the pH-sensing enzyme soluble adenylyl cyclase (sAC), which is required for motility. Additionally, we demonstrate for the first time in any cnidarian that sAC activity leads to protein kinase A (PKA) activation, and that PKA activity contributes to sperm motility activation. Ultrastructures of A. poculata sperm displayed morphological homology with other gonochoric cnidarians, and sAC exhibited broad structural and functional conservation across this phylum. These results indicate a conserved role for pH-dependent sAC-cAMP-PKA signalling in sperm motility across coral sexual systems, and suggest that the role of this pathway in sperm motility may be ancestral in metazoans. Finally, the dynamics of this pH-sensitive pathway may play a critical role in determining the sensitivity of marine invertebrate reproduction to anthropogenic ocean acidification.

Serum lipid profile levels and semen quality: new insights and clinical perspectives for male infertility and men's health.

Several clinical scenarios regulate the final ejaculated semen, which is pivotal to reproductive success. Sperm motility and plasma membrane fusogenic activity primarily rely on the peculiar sperm lipid composition, influenced by the patient's metabolism, genetics, nutritional, environmental status, and concomitant clinical entities such as varicocele. This study aimed to determine the relationship between serum lipid profile and testicular function (semen quality and testosterone levels). This retrospective study uses medical charts of 278 infertile men who attended andrological care between 2000 and 2019. Seminal analysis data, lipid profile, and total serum testosterone were collected. A multiple linear regression analysis was performed to evaluate the influence of the lipid parameters on the seminal variables. Statistical analyses were carried out with p ≤ 0.05 considered statistically significant. Seminal creatine kinase activity (p = 0.024) is negatively related to HDL (p = 0.032) and triglycerides (p = 0.037), while total testosterone (p < 0.0001) and seminal volume (p = 0.046) appeared both to be negatively related to triglycerides (p = 0.030 and p = 0.033, respectively). Medical advice commonly advocated to prevent endothelial dysfunction and cardiovascular disease and improve HDL-cholesterol and triglyceride levels in dyslipidemic patients should also be given to infertile men. Physicians should give patients a thorough assessment, including the blood lipid profile, hormonal status, and routine seminal examinations. We propose a more comprehensive men´s health check-up for the infertile male population, not limited to a simple evaluation of basic sperm parameters.

P-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P &amp;lt; 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable

Role of Panchakarma in Male Infertility w.s.r to High Altitude Induced Asthenozoospermia: A Case Report

Background of the Study: Infertility is a diseased condition of the reproductive system which can be defined as failure to achieve a clinical pregnancy after 12 months or more of regular unprotected intercourse. It is a major health issue affecting 8%-12% of couples worldwide. Male factor contributes to 20-30%, in India the contribution rate is 23%. Asthenozoospermia is a common cause of male infertility defined as reduced sperm motility, lower than 40%. Present study is looking at the impact of chronic exposure of high altitude (environmental hypoxia) on male infertility. Hypoxia acts directly on testicular seminiferous tubules resulting in impaired quantity and quality of sperm cells. Ayurveda explains male oriented infertility under the wide classification of Ashtshukradosha and asthenozoospermia which can be correlated with Ksheena Shukra. In case of Ksheenshukrta there is predominance of vitiated Vata and Pitta. Ayurvedic literature clearly mentioned Virechan Karma in Ksheena Shukra. Aim &amp; Objectives: The main aim of this study was to evaluate the effect of high-altitude exposure on seminal parameters along with its successful management with Virechana Karma. Result: The current case study of a 29 years old male visiting the OPD section with 20% active sperm motility treated with Virechana Karma after Snehana and Swedana which resulted in the positive effect and the female partner conceived the very next month after Virechana Karma. Conclusion: The case study established the positive impact of Virechan karma in male infertility due to asthenozoospermia.

Bacterial development in semen stored at ambient temperature in INRA96®

Sperm motility and mitochondrial activity can be maintained at room temperature for 24h in INRA96®. However bacterial development remains possible. Furthermore, very few studies evaluated the sensitivity of bacteria to the antibiotics included in INRA96. In experiment 1, semen from 8 stallions (28 ejaculates) was diluted to 20 × 106 sperm/mL in INRA96. The total amount of bacteria, dead or alive, was evaluated using Easykit6® (IMV-Technologies) immediately after dilution, then after 24h and 48h. Bacterial concentration remained stable during 48h cooled storage in Neopor® box (Minitube): mean±sem 9.8±2 × 105, 8.5±2 × 105, 8.4±12 × 105 bacteria/mL at 0h, 24h, 48h, respectively. Bacterial concentration significantly increased after 48h at ambient temperature between 13 and 26°C: 9.7±2 × 105a, 10±2 × 105ab, 28±6 × 105b bacteria/mL at 0h, 24h, 48h, and was significantly higher at ambient temperature than in cooled samples both after 24h and 48h. However, when data were analysed individually, the significant increase occured only for 3 of the 8 stallions. In experiment 2, semen from 7 stallions with 3 ejaculates each was evaluated in duplicate after 0h, 24h and 48h at ambient temperature. Easykit6® showed a significant increase in bacterial concentration for 2 stallions. Conventional bacteriology demonstrated an increase in CFU concentration for 3 stallions including the 2 mentioned above, and a decrease for another stallion. For example, stallion S showed 8±8a, 592±298ab, 8567±4493bCFU/mL at 0h, 24h, 48h, respectively, whereas stallion L showed 1259±504b, 92±53a,117±72aCFU/mL. Sixteen bacterialspecies (38 strains) and one yeast were identified. All the strains of Enterococcus fecalis, Listeria grayi, Streptococcus zooepidemicus, and some strains of Aerococcus viridans, Aeromonas salmonicida, Pasteurella multocida, Pseudomonas sepacia, Staphylococcus hominis, Staphylococcus xylosus, were resistant to both antibiotics of INRA96: 0.028g/L penicillinG and 0.077g/L gentamicin. Ten of the 20 strains identified at 0h and resistant to those antibiotics were not observed anymore at 24h. Eighteen of the 20 strains identified at 48h were resistant to those antibiotics. Eight of them have not been detected at 0h. Candida famata and the 17 bacterial strains identified at 0h and sensitive to those antibiotics were not observed anymore at 24h.The antibiotics and amphotericin B present in INRA96 seem enough to prevent microbial development at ambient temperature, provided that micro-organisms are sensitive to those molecules. Competition occurs between strains: those sensitive to the antibiotics of the extender cannot develop during storage, whereas others, even under the level of detection in raw semen, grow during storage because they are resistant. Bacterial concentration was not significantly correlated with sperm quality (motility, morphological abnormalities). Such bacterial level may not affect spermatozoa. Whether it could induce endometritis in inseminated mares needs investigation. Financial support: IFCE scientific council (Spermicrobes).