Capacitating Conditions Research Articles

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity.

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

Succinate dehydrogenase participation in porcine gamete function

Context Porcine gametes require energy for the physiological processes that allow fertilisation. Succinate dehydrogenase (SDH) plays a pivotal role in both, the tricarboxylic acid (TCA) cycle and the respiratory chain. Aims The aim of this work was to study the participation of SDH in the in vitro oocyte maturation, sperm capacitation and acrosome reaction in porcine species. Methods Cumulus–oocyte complexes (COCs) from abattoir-derived porcine ovaries were collected by aspiration and were incubated in maturation media, with the addition of increasing concentrations (0, 1, 5 and 10 mM) of malonate (a specific inhibitor of SDH). Nuclear maturation and cytoplasmatic maturation were analysed. Semen samples were incubated for 2 h in capacitating medium with 40 mM sodium bicarbonate, as sperm capacitation inducer, and the addition of increasing concentrations of malonate (0, 1, 5 and 10 mM). Sperm capacitation state and true acrosomal reaction were evaluated. SDH activity was determined in sperm and oocyte extracts by the spectrophotometric method. Key results The addition of 10 mM of malonate decreased both nuclear and cytoplasmic maturation rates (P &lt; 0.05) without affecting COC viability (assessed using fluorescein diacetate). A lower level of capacitation (induced by bicarbonate) and acrosome reaction (induced by follicular fluid) was observed with the addition of 5 mM of malonate (P &lt; 0.05) without affecting motility and viability of sperm at this concentration. The activity of SDH was 0.35 ± 0.1 × 10−5 and 2.37 ± 0.9 × 10−5 U/COC for immature and in vitro matured COC extracts (P &lt; 0.05) respectively, and 0.44 ± 0.16 U/1010 sperm for boar sperm extracts. Conclusions In conclusion, because it has been proposed that aerobic and anaerobic metabolic pathways of cells are changed depending on the oxygen availability and the composition of metabolic substrates in their environment, our results suggest that energy obtained through the mitochondrial respiration (TCA cycle and oxidative phosphorylation) is necessary to support oocyte maturation, sperm capacitation and acrosome reaction in the porcine species. Implications The study of enzymatic activity in gametes is essential for understanding the mechanisms that control the energy production required to achieve successful fertilisation. This knowledge has significant implications for the development of assisted reproductive technologies.

Acquisition of fertilization competence in guinea pig spermatozoa under different capacitation protocols

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs.

Analysis of Synchronous Generator Self-Excitation under Capacitive Load Condition in Variable-Frequency Aviation Power System

As power electronic converters become more widely used in aviation power systems, the associated capacitive loads in the harmonic filter circuits increase accordingly. The risk of self-excitation of aeronautical synchronous generators due to capacitive loads is thus increased. Compared with the self-excitation of a generator in a conventional fixed-frequency power system, this process is more complicated in a variable-frequency aviation power supply (360–800 Hz), as both the varied frequency and the loading conditions contribute to the self-excitation. To quantify this effect, in our study, a series-parallel model of simplified RLC loads under a variable-frequency power supply was built. The criterion of generator self-excitation, given in terms of the generator impedance and the load impedance, was then derived. To facilitate the load configuration design in the case of an aviation power system, a comprehensive analysis of the influences of the varied load power and system frequency on the load impedance was conducted. A graphical approach was proposed to determine self-excitation by comparing the series load reactance and resistor with three critical impedances corresponding to three self-excitation criteria, which is more intuitive and enables one to visualize the tendency of self-excitation with varied frequencies and loading conditions more effectively. Finally, the influence of variable frequency on the self-excitation of the aeronautical synchronous generator was verified by the simulation results.

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.

Analog Compatible Logic Functions Using EXCCIIs and an Extended Application for Sigmoid Activation Function

This paper introduces the use of an extra X current conveyor (EXCCII) for realizing logic operations, suited for mixed signal design, without necessitating separate digital blocks. The NOR and OR logic functions are thus shown to be realized using the current-mode analog building block operable at a low voltage. The use of [Formula: see text][Formula: see text]V supply makes the new circuits operable at low voltage. The EXCCII bias current used in the design is 25[Formula: see text][Formula: see text]A. The input currents for defining logic 0 and logic 1 are, respectively, taken as 0[Formula: see text]mA and 0.5[Formula: see text]mA. The proposed circuits’ delay is found to be 10[Formula: see text]ns for low-to-high transition, while 2[Formula: see text]ns for high-to-low transition for NOR gate under capacitive loading conditions. The use of the current-mode approach makes the new proposed circuit apt for current-mode signal processing. The new proposed logic function circuits are compatible with analog circuits, thus providing an easy merger for mixed signal systems design, paving the way to a modular design approach. As an extended application in artificial neural networks, the proposed circuit is shown to generate a sigmoid activation function with convincing results. Several results are included by varying the reference current, and the circuit’s robustness aspect is also tested in the presence of fluctuation in the bias current of EXCCII. The novel approach of utilizing an analog building block for realizing digital functions is, therefore, verified with promising future applications.

Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

Post-Thaw Characteristics of the Simmental Sperm Function in Different Ages of Bulls

This study aims to determine the effect of the age difference of Simmental bulls on motion characteristics, capacitation status, and DNA fragmentation of post-thawing sperms. The frozen semen used was collected from twelve bulls, which were divided into four groups of age, which include a group of two, four, ≥ 10 years old with high semen rejection (≥ 10 HR), and ≥ 10 years old with low semen rejection (≥ 10 LR). Computer Assisted Sperm Analysis (CASA) was used to determine sperm motion characteristics, capacitation status by chlortetracycline (CTC) staining, plasma membrane integrity, and viability using eosin-nigrosine staining. In contrast, the DNA fragmentation index was determined using the Sperm-Bos-halomax kit. The results showed that the four year old group had a higher total and progressive motility percentage than the others (p<0.05). In all groups, there was no significant difference among sperm kinematics such as VAP, VSL, VCL, STR, and ALH. However, the LIN, WOB, and BCF of the ≥ 10 HR year old groups were significantly lower (p<0.05) than those of the other groups. However, un-capacitated sperm was higher (p<0.05) in the two years and four years old groups compared to the ≥ 10 years old, while the four years old group had lower capacitated and acrosome-reacted (p<0.05) than the other groups. Furthermore, the sperm membrane integrity, viability, and DNA fragmentation index of the ≥ 10 years old groups were significantly higher (p<0.05) than those of the other groups. The research concludes that aging in the Simmental bull affects motion characteristics, capacitation status, and DNA fragmentation of post-thawing sperm. However, the semen rejection rate in the older bull did not directly affect the post-thawing sperm quality.

Chemotaxis of capacitated ram spermatozoa to ewe follicular fluid

Sperm orientation mechanisms, as chemotaxis, are essential for the sperm to reach the oocyte and then fertilize it. The follicular fluid is the biochemical environment where the oocyte develops until its ovulation, and its composition changes throughout the estrous cycle. The follicular fluid is released together with the oocyte at the moment of ovulation, and it has been proposed as a chemoattractant for spermatozoa. This chemotactic ability has been demonstrated in some mammals, but to date, no studies have been done in the ovine species. Also, it has been reported that only capacitated and hyperactivated spermatozoa can respond to chemotactic stimuli. Therefore, the aim of this work was to study the chemotactic response of ovine spermatozoa to ewe follicular fluid, obtained at various stages of the estrous cycle, and to evaluate the influence of the capacitation status in the chemotaxis mechanism. In order to perform the experiments, ovine follicular fluids were obtained in the early and late follicular and luteal phases, and two concentrations, 2 and 10%, were tested. Semen samples were collected from nine Rasa Aragonesa rams, and spermatozoa were selected by the swim-up/dextran method. In vitro capacitation was induced by incubation without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail). After sperm quality evaluation (motility, viability, and capacitation state), the chemotactic assays were carried out with the aim of the commercial device (IBIDI® µ-Slide Chemotaxis chamber, Ibidi GmbH). Swim- up, cap-TALP or cap-Cocktail samples were loaded in the chambers in the presence of follicular fluid. Individual trajectories were analyzed by the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (Version 8.0.1). Follicular fluid at 2 and 10% concentrations, obtained in luteal and early follicular phases, induced sperm chemoattraction. This chemotactic response was only observed in capacitated spermatozoa, which supports previous evidence in other species. These results reveal, for the first time, the chemotactic effect of ewe follicular fluid in ram spermatozoa. Grants: AGL-2017- 83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).

Analysis of the chemotactic response of ram spermatozoa to steroid hormones

Some substances in follicular fluid may be acting as sperm attractants, and steroid hormones progesterone (P4) and estradiol (E2), both secreted by the cumulus cells, are possible candidates. We have previously observed a chemotactic effect of the ewe follicular fluid in ram spermatozoa (data not published). The presence of progesterone and estrogen receptors in ram spermatozoa suggests the involvement of these hormones in the sperm functionality, including guided movement towards the oocyte. P4 acts as a chemoattractant in human and rabbit sperm, but there is no information on its effect, or E2 effect, on ram spermatozoa. The objective of this work was to study the putative chemotactic action of P4 and E2 on ram spermatozoa, analyzing the influence of the sperm capacitation state. Swim-up selected spermatozoa from nine Rasa Aragonesa rams were incubated without (cap-TALP) or with cAMP-elevating agents (cap-Cocktail) to induce in vitro capacitation. After evaluating the sperm quality parameters in non- capacitated and incubated samples (motility, viability, capacitation state), IBIDI® µ-Slide Chemotaxis chamber (Ibidi GmbH) was loaded with swim-up, cap-TALP or cap-Cocktail sperm samples according to the manufacturer’s instructions. P4 or E2 were added to the chambers at 100 pM or 1 µM concentration each. Medium with 0.01% dimethyl sulfoxide (DMSO) was used as control treatment. After 5 min of equilibration, 200 fps videos were recorded for 3 sec with a video camera mounted on a microscope. Sperm trajectories and chemotaxis were analyzed with the OpenCASA software (Alquezar-Baeta et al., PLoS Comput Biol, 15(1), 2019), which calculates a chemotaxis index. Differences were analysed by χ² test, except for the chemotaxis index (ANOVA) using GraphPad PRISM® (versión 8.0.1). A chemotactic response was only observed in capacitated spermatozoa, as we previously described in presence of follicular fluid. P4 at both concentrations had a chemotactic effect on capacitated ram spermatozoa. However, E2 had no effect on any of the studied samples. These results reveal, for the first time, the chemotactic effect of P4 in ram spermatozoa. Grants: AGL-2017-83799-R and DGA A07-17R. S.M.-J. has a predoctoral contract from the Diputación General de Aragón (DGA).

Effect of hypothermal storage on motility, viability, acrosome integrity and intracellular zinc of boar sperm

Boar semen is commonly stored at 17°C to preserve sperm quality to be used for insemination within 3 d. However, the first stages of decline of sperm quality occurs as sublethal damage, but eventually result in sperm death and loss of fertility potential. Alternatively, hypothermal (5°C) storage is useful to control bacterial growth but limiting due to the high sensitivity of boar sperm to chilling injuries. Therefore, we aimed to evaluate the effect of hypothermal storage on sperm quality and intracellular Zinc2+ as indicative of capacitation status. Semen from commercial boars (n=15) was collected, extended in Androstar®Plus (Minitube) at 3 ×109 sperm/75 ml and shipped (17°C) overnight. Paired-samples were stored at 17 and 5°C. Motility (IVOS II, Hamilton Thorne) and flow cytometric assessment of viability, acrosome integrity [100 nM SYBR®14, 10 µm propidium iodide (PI), 5.6 µg/ml PNA Alexa- FluorTM647; Molecular Probes] and intracellular Zinc2+ [8 µg/mL Hoecsht-33342, 1 µM FluoZin™-3-AM (FluoZin), 10 µM PI] were performed at Days -1, -4 and -7 after collection. Results were analyzed by 2-factor mixed model for repeated measure and Tukey-adjusted pairwise comparisons and expressed in percentages (mean±SEM). Total motility was higher (P<0.05) at Day -1 (85.9±3.1) compared to Day -4 (17°C: 78.9±3.1; 5°C: 73.7±3.1) or Day -7 (17°C: 71.8±3.1; 5°C: 73.5±3.1), with no differences between and within temperatures. Progressive motility remained similar (P>0.05) during 17°C-storage (Day -1: 47.5±4.1; Day - 4: 48.1±4.1; Day 7: 53.8±4.1), but it was reduced (P<0.05) at Day -4 (36.9±4.1) compared to Day -7 (47.4±4.1) at 5°C and to samples held at 17°C. For live sperm with intact acrosome (SYBR+/PI−/PNA−), percentages of 17°C (Day -4: 85.3±2.1; Day -7: 82.8±2.1) or 5°C-storage (Day -4: 78.9±2.1; Day -7: 81.3±2.1) did not differ compared to Day -1 (84.3±2). However, live sperm with intact acrosome at Day -4 was higher (P<0.05) at 17°C than at 5°C. Samples stored at 17°C had reduced (P<0.05) intracellular Zinc2+ and plasma membrane integrity (FluoZin+/PI–) at Day -4 and Day -7 (62.2±4.3; 67.4±4.3) than Day -1 (77.6±4.3), but they did not differ from samples kept at 5°C (Day -4: 61.5±4.3; Day -7:67.9±4.3). We concluded that storing boar sperm either at 5 or 17°C reduced motility and plasma membrane integrity, but live sperm subpopulation maintain high intracellular Zinc2+, indicative of non-capacitated status, further than 4 d of storage.

Effects of Cell Concentration During Cryopreservation On the Post-thaw Quality of Santa Inês Ram Sperm

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe's cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G – 400, G – 800, G – 1200, and G – 1600 x 10 6 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 ºC, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 ºC/20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G – 400 treatment resulted in samples with the highest linearity and progressive motion (P &lt; 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P &lt; 0.05). Overall, use of the G – 400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G – 800 and G – 1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single rtificial insemination protocol is desirable.

Evaluation of Cryoprotective, Cryocapacitation Inhibitory and Fertility Enhancing Potential of RU-486, Sericin and Taurine in Cattle and Buffalo Semen

Background: Artificial insemination with frozen semen is of great value for livestock breeding and improvement. However, the procedure of cryopreservation leads to varying degree of oxidative and cryodamage to sperms deteriorating its quality, capacitation status and fertility. Hence, a variety of protocols, cryoprotectants and additives have been tried to protect sperms from such damages. This study was aimed to evaluate the antioxidant and cryocapacitation inhibitory potential of Mifepristone, Sericin and Taurine in routine extender on cryopreserved bull semen including in vivo fertility. Methods: Semen ejaculates of 3 Gir and 3 Murrah bulls with greater than 75% initial motility were split-diluted @ 100 million spermatozoa ml-1 using TFYG extender without and with Mifepristone (10 µg/ml), Sericin (5 mg/ml) and Taurine (4 mg/ml) and frozen in LN2 using a programmable biofreezer. The freshly diluted as well as frozen-thawed samples were assessed for sperm motility, viability, plasma membrane integrity, CTC (chlortetracycline) fluorescence assay and seminal plasma oxidative markers. The straws frozen as above were used to inseminate 100-375 cows and/or buffaloes with each treatment involving well trained field AI technicians. Conception rates were determined based on confirmation of pregnancy per-rectum 45-60 days after first AI in non-return cases. Result: The mean percentages, on dilution and at post-thaw stage in particular, of motile, live, HOS reactive and non-capacitated sperm were significantly (p less than 0.01) higher with reduced lipid peroxidation (MDA, SOD and GPx) whereas capacitated and acrosome reacted sperm were lower in Mifepristone supplemented extender than in control extender and the values for Sericin and Taurine fortified extender were intermediary in both the species. The first AI conception rates obtained for Gir and Murrah bull semen cryopreserved in control extender and extender supplemented with Mifepristone, Sericin and Taurine were 44.48, 51.11, 47.22 and 48.72% in cattle and 44.44, 55.40, 51.57 and 52.05% in buffaloes, respectively, suggesting that all additives used and Mefipristone in particular can be advantageously incorporated in the semen extender for cryopreservation of both cattle and buffalo semen.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences.

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

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

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

CRYOCAPACITATION AND ITS ASSOCIATION WITH OXIDATIVE FEATURES IN CRYOPRESERVED BOVINE SPERMATOZOA

The goal of our research was to evaluate the impact of cryopreservation on the antioxidant activity, lipid peroxidation and protein carbonylation as well as its connection with the progress of cryocapacitation in bovine spermatozoa. As biological material we used semen obtained from 20 sexually mature Holstein bulls. Each ejaculate was divided into three equal aliquots as follows: the first part or control (CTRL) was incubated in physiological saline solution while the second part (CAP) was incubated in a capacitation medium at specific conditions. The third part of each sample was cryopreserved (CRYO) and stored in liquid nitrogen at -196°C for further analysis. The motility of spermatozoa was assessed with CASA (computer assisted sperm analysis), while the capacitation status was evaluated by the chlortetracycline (CTC) fluorescent staining. Total antioxidant capacity (TAC) and the level of lipid peroxidation (LPO) was measured with a combined spectro-fluoro-luminometer. The presence of protein oxidation was detected by the traditional DNPH (2,4-dinitrophenylhydrazine) method and evaluated spectrophotometrically. Based on our data, the motility was significantly decreased (P&lt;0.0001; P&lt;0.01) in the CRYO group against CAP and CTRL. There was a significant increase (P&lt;0.01; P&lt;0.05) of acrosome-reacted spermatozoa (AR-pattern) in the CRYO group when compared to CTRL and CAP, while TAC was statistically decreased (P&lt;0.05) between the CRYO and CAP experimental group. In the case of protein oxidation and LPO, both parameters were significantly higher (P&lt;0.0001) in the CRYO group when compared to CAP or CTRL. In summary, cryopreservation induces capacitation-like changes and promotes oxidative damage in frozen-thawed bovine spermatozoa.

Reproductive toxicity of ritonavir in male: Insight into mouse sperm capacitation

Since COVID-19 began in 2019, therapeutic agents are being developed for its treatment. Among the numerous potential therapeutic agents, ritonavir (RTV), an anti-viral agent, has recently been identified as an important element of the COVID-19 treatment. Moreover, RTV has also been applied in the drug repurposing of cancer cells. However, previous studies have shown that RTV has toxic effects on various cell types. In addition, RTV regulates AKT phosphorylation within cancer cells, and AKT is known to control sperm functions (motility, capacitation, and so on). Although deleterious effects of RTV have been reported, it is not known whether RTV has male reproduction toxicity. Therefore, in this study, we aimed to investigate the effects of RTV on sperm function and male fertility. In the present study, sperm collected from the cauda epididymis of mice were incubated with various concentrations of RTV (0, 0.1, 1, 10, and 100 μM). The expression levels of AKT, phospho-AKT (Thr308 and Ser473), and phospho-tyrosine proteins, sperm motility, motion kinematics, capacitation status, and cell viability were assessed after capacitation. The results revealed that AKT phosphorylation at Thr308 and Ser473 was significantly increased, and the levels of tyrosine-phosphorylated proteins (at approximately 25 and 100 kDa) were significantly increased in a dose-dependent manner. In addition, RTV adversely affected sperm motility, motion kinematics, and cell viability. Taken together, RTV may have negative effects on sperm function through an abnormal increase in tyrosine phosphorylation and phospho-AKT levels. Therefore, individuals taking or prescribing RTV should be aware of its reproductive toxicity.

MEN WITH LOW CAP-SCORE™ HAVE SIMILAR FERTILIZATION RATES WITH ICSI, SIMILAR EUPLOIDY RATES, AND SIMILAR FET LIVE BIRTHS COMPARED TO MEN WITH NORMAL SCORES

Men are often assumed fertile if they have enough morphologically normal, motile sperm. This is despite the fact that it is well known that most male fertility problems are a result of poor sperm function/fertilizing ability that are not detected by traditional semen analysis. Lack of such a diagnostic assessment of fertilizing ability has led to most male infertility cases being classified as “idiopathic,” or unexplained. One quantifiable measure of sperm function is capacitation status. Use of the percentage of sperm in an ejaculate that capacitate (the Cap-Score Test, Androvia LifeSciences, Mountainside, NJ), has been validated and used commercially to assess capacitation status of a semen sample.

Second-order associative memory circuit hardware implemented by the evolution from battery-like capacitance to resistive switching memory

SummaryMemristor-based Pavlov associative memory circuit presented today only realizes the simple condition reflex process. The secondary condition reflex endows the simple condition reflex process with more bionic, but it is only demonstrated in design and involves the large number of redundant circuits. A FeOx-based memristor exhibits an evolution process from battery-like capacitance (BLC) state to resistive switching (RS) memory as the I-V sweeping increase. The BLC is triggered by the active metal ion and hydroxide ion originated from water molecule splitting at different interfaces, while the RS memory behavior is dominated by the diffusion and migration of ion in the FeOx switching function layer. The evolution processes share the nearly same biophysical mechanism with the second-order conditioning. It enables a hardware-implemented second-order associative memory circuit to be feasible and simple. This work provides a novel path to realize the associative memory circuit with the second-order conditioning at hardware level.

GRP78 plays a key role in sperm function via the PI3K/PDK1/AKT pathway

Glucose-regulated protein 78 (GRP78), which is commonly found in the endoplasmic reticulum (ER), is involved in stabilizing ER proteins and inducing the unfolded protein response. Furthermore, GRP78 is expressed on the surface of most common cancer cells, such as cells of breast, lung, liver, and prostate cancers, and plays a role in apoptosis and cell proliferation via the PI3K/PDK1/AKT signaling pathway. Therefore, various trials have been performed for evaluating cancer treatment by inhibiting GRP78. Moreover, GRP78 is expressed on the surface of spermatozoa; however, its role in spermatozoa physiology remains unclear. Therefore, this study was designed to investigate the effects of GRP78 on sperm function during capacitation and elucidate the underlying mechanisms. Boar spermatozoa were exposed to various concentrations of HA15, a GRP78 antagonist, and sperm kinematic parameters, capacitation status, cell viability, levels of PI3K/PDK1/AKT-pathway related proteins, and tyrosine phosphorylation were evaluated. GRP78 inhibition significantly decreased sperm motility, kinematic parameters, capacitated and acrosome-reacted spermatozoa counts, and cell viability. Moreover, GRP78 expression was significantly decreased in HA15-treated spermatozoa compared to that in the control group, and levels of PI3K/PDK1/AKT-pathway related proteins changed significantly. Furthermore, tyrosine phosphorylation was significantly altered in the HA15-treated group. The results of this study suggest that GRP78 inhibition in cancer therapy may negatively affect sperm function. These results lay a strong foundation for future studies aiming to identify the molecular mechanisms related to GRP78 in spermatozoa.