Intact Plasma Membrane Research Articles

Estimation of sperm concentration limits to produce intrauterine insemination doses in swine.

This study evaluated the effect of sperm concentration of boar semen doses, for intrauterine artificial insemination (IUAI), on semen quality and established concentration limits for their production. Twenty ejaculates from four crossbred mature PIC® boars were collected to produce 50 mL semen doses in a split sample, reaching the following sperm concentrations: ~20, 30, 60, and 100 × 106 cells/mL. Doses were produced using Androstar® Plus, stored at 17°C, and evaluated until 120 h of storage. There was a linear decrease in sperm motility as the sperm concentration increased (p linear < .01). The concentration which no longer affected the total and progressive motility was 59 and 55 × 106 cells/mL, respectively (corresponding to 71% and 62%, respectively). The pH linearly decreased as the sperm concentration increased (p < .01); yet, at 72 and 120 h, the parameter dramatically reduced in boar semen doses with 60 and 100 × 106 cells/mL. The percentage of cells with intact plasma and acrosomal membranes or with high mitochondrial membrane potential was not influenced by the sperm concentration (p ≥ .15). In conclusion, sperm motility was negatively affected in highly (60 and 100 × 106 cells/mL) concentrated doses. To achieve suitable sperm motility, boar semen doses may not surpass the sperm concentration of 55 × 106 cells/mL. The effect of low-concentrated boar semen doses on sperm quality still needs to be better evaluated, mainly considering the influence of extender type and thermo-resistance conditions.

Effect of Force Field Resolution on Membrane Mechanical Response and Mechanoporation Damage under Deformation Simulations.

Damage induced by transient disruption and mechanoporation in an intact cell membrane is a vital nanoscale biomechanical mechanism that critically affects cell viability. To complement experimental studies of mechanical membrane damage and disruption, molecular dynamics (MD) simulations have been performed at different force field resolutions, each of which follows different parameterization strategies and thus may influence the properties and dynamics of membrane systems. Therefore, the current study performed tensile deformation MD simulations of bilayer membranes using all-atom (AA), united-atom (UA), and coarse-grained Martini (CG-M) models to investigate how the damage biomechanics differs across atomistic and coarse-grained (CG) simulations. The mechanical response and mechanoporation damage were qualitatively similar but quantitatively different in the three models, including some progressive changes based on the coarse-graining level. The membranes yielded and reached ultimate strength at similar strains; however, the coarser systems exhibited lower average yield stresses and failure strains. The average failure strain in the UA model was approximately 7% lower than the AA, and the CG-M was 20% lower than UA and 27% lower than AA. The CG systems also nucleated a higher number of pores and larger pores, which resulted in higher damage during the deformation process. Overall, the study provides insight on the impact of force field-a critical factor in modeling biomolecular systems and their interactions-in inspecting membrane mechanosensitive responses and serves as a reference for justifying the appropriate force field for future studies of more complex membranes and more diverse biomolecular assemblies.

Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen

Background and Aim: The Kacang goat (Capra hircus) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods: Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (IPM), viability and motility analyses. Results: Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm living cells, sperm motility, MPI, and the highest levels of MDA. The addition of EGCG CNPs in the T3 and T4 groups was increased (p &lt; 0.05) post-thawed catalase, DPPH, living cells, and sperm motility and decreased (p &lt; 0.05) MDA levels than those of the T0 group. Meanwhile, sperm MPI was higher (p &lt; 0.05) in the T4 group than the sperm MPI of the T0 group. Conclusion: This study was the first in using of EGCG CNPs in the SM–EY extender, in which adding 1.5 or 2.0 μg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.

SP22 sperm protein as a potential biomarker of fertility in humans: A preliminary study.

Infertility affects approximately 15% of couples of reproductive age, and 50% of the cases are directly related to men. The evaluation of male fertility is based on analyses of routine seminal parameters and the use of more advanced techniques can help identify fertility biomarkers. SP22 sperm protein is considered a biomarker in murine species since its concentration is highly correlated with sperm fertility. As the role of this protein as a biomarker is already well-established in other species, we hypothesized that this same correlation could apply to human. Thus, the present study aimed to investigate possible correlations between SP22 concentration and sperm parameters in fertile and infertile men. For this, a study was carried out on 21 volunteers' seminal samples who were grouped according to fertility as fertile (n=10) or infertile (n=11). Conventional and functional sperm analyses, membrane protein extraction, quantification and immunolocalization of SP22 were performed. The infertile volunteers showed an increase in the percentage of sperm with abnormalities in head morphology and a decrease in the percentage of sperm with intact plasma membrane and damaged acrosomal membrane. Serum concentration of the hormone SHBG was also decreased in infertile volunteers. The damage to the plasma membrane was positively correlated with the superoxide anion production. Although none of the functional parameters were correlated with SP22 concentration, type D sperm motility was negatively correlated and type A+B sperm motility was positively correlated. This preliminary study opens new paths in the characterization of SP22 as a non-invasive biomarker for predicting fertility/infertility.

Bacteria and Virus Inactivation: Relative Efficacy and Mechanisms of Peroxyacids and Chlor(am)ine.

Peroxyacids (POAs) are a promising alternative to chlorine for reducing the formation of disinfection byproducts. However, their capacity for microbial inactivation and mechanisms of action require further investigation. We evaluated the efficacy of three POAs (performic acid (PFA), peracetic acid (PAA), and perpropionic acid (PPA)) and chlor(am)ine for inactivation of four representative microorganisms (Escherichia coli (Gram-negative bacteria), Staphylococcus epidermidis (Gram-positive bacteria), MS2 bacteriophage (nonenveloped virus), and Φ6 (enveloped virus)) and for reaction rates with biomolecules (amino acids and nucleotides). Bacterial inactivation efficacy (in anaerobic membrane bioreactor (AnMBR) effluent) followed the order of PFA > chlorine > PAA ≈ PPA. Fluorescence microscopic analysis indicated that free chlorine induced surface damage and cell lysis rapidly, whereas POAs led to intracellular oxidative stress through penetrating the intact cell membrane. However, POAs (50 μM) were less effective than chlorine at inactivating viruses, achieving only ∼1-log PFU removal for MS2 and Φ6 after 30 min of reaction in phosphate buffer without genome damage. Results suggest that POAs' unique interaction with bacteria and ineffective viral inactivation could be attributed to their selectivity toward cysteine and methionine through oxygen-transfer reactions and limited reactivity for other biomolecules. These mechanistic insights could inform the application of POAs in water and wastewater treatment.

Effect of Different Equilibration Times and Freezing Rates on Bull Spermatozoa Cryotolerance

Sperm cryopreservation is a complex process consisting of several steps, the details of which are not yet universally agreed upon. The objective of this study was to evaluate the effects of different equilibration times and freezing rates on post-thaw resilience and functional integrity. Collected semen from five Holstein bulls (30 samples in total) were split into 4 aliquots to produce frozen straws after 2 different equilibration times (2 h and 4 h) and freezing rates (2-phase and 3-phase). After thawing, the sperm characteristics were measured by flow cytometry. A linear model procedure was used to determine the effects of bull, equilibration time, freezing rate and interaction between them, on sperm variables. The evaluated sperm characteristics included spermatozoa with intact plasma and acrosomal membrane (PMAI), PMAI with high mitochondrial membrane potential (HMMP), spermatozoa with plasma membrane damage (PMD), and spermatozoa with acrosome damage (AD) were analysed immediately, 1 h and 2 h after thawing. In conclusion, the extension of the equilibration time from to 4 h in combination with 2-phase freezing rate led to a significant improvement in all measured parameters of frozen-thawed spermatozoa (P &lt; 0.01).

Low-density lipoprotein supplementation improves the quality of Holstein bulls' insemination doses

Despite the routine use of cryopreservation in dairy cattle, the subsequent deterioration in the quality of bull insemination doses (IDs) is an incentive to refine cryopreservation protocols, including the composition of the ID. The aim of this study was to examine, using a flow cytometry assay, whether the selected concentrations of low density lipoproteins (LDLs) extracted from hen-egg yolk may improve the post-thaw quality of the ID diluted with a plant-based extender. In total, 30 ejaculates from five sires (six trials per animal) were collected and processed at the Artificial Insemination Centre. The effect of the low density lipoprotein supplementation to the AndroMed® diluent at 4 and 8% (v/v) on the cryopreservation efficiency was tested. The effects of the modified extenders were determined on different sperm subpopulations by simultaneous quadruple staining for flow cytometry as the percentage of sperm with intact plasma membrane and acrosome (PMAI), PMAI sperm showing high mitochondrial membrane potential (HMMP), sperm with plasma membrane damage (PMD), and sperm with acrosome damage (AD). It was observed that the 8% LDL treatment was more effective (P < 0.05) in preserving all the analysed parameters than AndroMed® without any supplementation. The supplementation of the AndroMed® extender with 8% LDL resulted in the most optimal values of the PMAI (30.61 ± 1.13), HMMP (68.81 ± 1.25), PMD (68.69 ± 1.14), and AD (38.36 ± 1.13) compared to the control treatment (24.86 ± 1.13 for PMAI, 47.79 ± 1.25 for HMMP, 74.77 ± 1.14 for PMD, and 42.83 ± 1.13 for AD. The results of the study also demonstrated a synergistic positive effect of the LDL and soybean lecithin-based diluent on the spermatozoa post-thaw quality and resilience, based on 2 h long incubation. In conclusion, the soybean based semen extender treatment with LDL represents a beneficial tool to mitigate the detrimental effect of cryopreservation.

Quantification of Age-Related Changes in the Lateral Organization of the Lipid Portion of the Intact Membranes Isolated from the Left and Right Eye Lenses of the Same Human Donor.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a "threshold" for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

Omecamtiv mecarbil augments cardiomyocyte contractile activity both at resting and systolic Ca2+ levels.

Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1Hz in the presence of different OM, EMD, or Levo concentrations. SL was reduced by about 23% or 9% in the presence of 1μM OM or 1μM EMD in the absence of electrical stimulation, whereas 1μM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1μM EMD or 1μM Levo to about 152%, but only to about 128% in the presence of 0.03μM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1μM Levo had no effects on contraction durations. One-micromole Levo, but not 1μM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+ -sensitizing effect.

Frozen Semen Quality of Simmental Cattle in Various Commercial Diluents

This study determines the effect of different commercial diluents on the quality of frozen Simmental semen. The extraction of fresh semen from 3 Simmental bulls using an artificial vaginal method was performed twice a week. Fresh semen with motility of &gt;70% was further processed to frozen using a one-step dilution method, and the commercial diluents used were Andromed®, Optixcell®, and Steridyl®. The diluted semen was packed into mini-straws, equilibrated, frozen, and stored for further testing. Data were analyzed by analysis of variance (ANOVA), followed by Duncan Multiple Range Test (DMRT) analysis with a 5% confidence level. The results showed that commercial diluents significantly affected motility, viability, intact plasma membrane, abnormalities, and recovery rate of frozen semen (P&lt;0.05). Commercial diluents with different sources of lecithin reported different results on the quality of frozen semen. Steridyl® has a more optimal ability to maintain the quality of frozen semen of Simmental cattle, followed by Optixcell® and Andromed®.

Progressive Motility, DNA Fragmentation, Intact Plasma Membrane, and Acrosome Status of Frozen Semen Bali and Simmental Bulls

In increasing the population of bulls in Indonesia, it is necessary to utilize Artificial Insemination (AI) technology. AI reproductive technology aims to increase reproductive efficiency and use superior semen in livestock and can prevent the spread of reproductive diseases. Frozen semen is one of the factors that influence the success of AI. Thus, the quality of frozen semen must be maintained so fertility remains good. This study aimed to determine the quality of frozen semen of Bali and Simmental bulls at the Artificial Insemination Center, South Sulawesi. This study used frozen semen from two Bali and Simmental bulls. Parameters observed were progressive motility, deoxyribonucleic acid fragmentation (DNA), acrosome status, and intact plasma membrane. The progressive motility was tested using Computer-assisted sperm analysis (CASA Sperm Vision Minitube), the DNA fragmentation using acridine orange dye, the acrosome status using peanut agglutinin, and the intact plasma membrane staining using HOS solution. This study gives a result that the progressive motility, DNA fragmentation, and acrosome status of spermatozoa in frozen semen of Bali and Simmental bulls were not significantly different (P&gt;0.05). In contrast, the whole plasma membrane of spermatozoa in Bali bulls' frozen semen was significantly higher (P&lt;0.05) than in Simmental bulls. The results indicate that based on the parameters of progressive motility, DNA fragmentation, and acrosome status of spermatozoa, both bull breeds had the same quality of frozen semen. Meanwhile, according to the spermatozoa intact plasma membrane, the frozen semen of Bali bulls had better quality than that of Simmental bulls. Keywords: Artificial Insemination, frozen semen quality, Bali and Simmental bulls

Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen

Background and Aim: The Kacang goat (Capra hircus) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods: Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (IPM), viability and motility analyses. Results: Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm viability, sperm motility, IPM, and the highest levels of MDA. However, the addition of EGCG CNPs at doses of 1.5 µg/mL extender increased post-thawed catalase, DPPH, sperm IPM, viability, and sperm motility and decreased MDA levels (p &lt; 0.05) than those of control group. Conclusion: This study was the first in which EGCG CNPs were used in SM–EY extender, and the addition of only 1.0 µg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.

Development of a novel fluorescence assay for studying lipid bilayer perturbation induced by amyloidogenic peptides using cell plasma membrane vesicles

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and β-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells.

Response to Zhang et al., 'do egg cell-secreted aspartic proteases promote gamete attachment?'

In angiosperms, the molecular mechanism controlling polytubey/polyspermy block and fertilization recovery was not unraveled until recently (Zhong et al., 2022), which can be described as follows: Five pollen tube-expressed RALF peptide ligands, perceived by the receptor complex composed of three receptors FER/ANJ/HERK1 at the septum, establish the polytubey block at the septum so that only one pollen tube is allowed to emerge from the septum to target the ovule for fertilization. Pollen tube rupture releases the two sperm cells and fertilization is completed within 20 minutes. This article is protected by copyright. All rights reserved.

Calcitriol induces post-thawed bovine sperm capacitation

<b>Background:</b> Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity. <i>In vivo</i>, this process is directly associated with high calcium levels in sperm cytoplasm. Calcitriol, the vitamin D hypercalcemic metabolite, is related to human sperm motility, capacitation, and acrosome reaction. This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation. <b>Methods:</b> One million freeze-thawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min. Untreated cells (negative control) and treated ones with calcitriol or heparin (100 µg/mL, positive capacitation control) were evaluated for motility, viability, and functional parameters. Menadione (70 µM, 30 min) treatment was included as a reactive oxygen species (ROS) positive sperm agent. <b>Results:</b> The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability, vigor, and capacitation than their positive and negative controls. The percentage of sperm with intact plasma and acrosome membranes, mitochondrial membrane potential (ΔΨm), and phosphatidylserine externalization was similar in all the conditions evaluated, while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control. <b>Conclusion:</b> Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility, viability, and vigor without altering key biological parameters such as redox status, ΔΨm, and cell death.

Fluorescence Resonance Energy Transfer to Detect Plasma Membrane Perturbations in Giant Plasma Membrane Vesicles.

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Frozen Semen Quality of Kolbroek Boars Concerning Glutathione Concentrations

Glutathione(GSH) is an antioxidant that is being studied for its ability to improvecellular responses to semen preservation challenges. The research goal aimed toevaluate the impact of introducing different glutathioneconcentrations to boar semen freezing extender (0, 1, 5, and 10 mM) onthe Kolbroek boar sperm's quality after being frozen and thawed. For 2 h, thecollected sperm-rich fraction was chilled at 17°C. Following the 1 h ofequilibration, fraction B: Control (egg yolk 20% + BTS 72% + glycerol 8%), 1 mM(egg yolk 20% + BTS 70% + glycerol 8% + GSH 2%), 5 mM (egg yolk 20% + BTS 62 + 8%glycerol + GSH 10%) and 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%)was added into the semen samples. Following that, 0.5 mL straws with thediluted, cooled semen samples were submerged in liquid nitrogen (LN2)vapor and then stored inside the LN2 tank (-196°C). After thawing(37°C), parameters of boar sperm motility, velocity, morphology and viability,membrane permeability, and Malondialdehyde content were assessed and recorded.Using the analysis of variance, the data were examined. The results revealed adifference in total motility (%) on fresh semen as compared to post-thawedcontrol and GSH treatments. A significant difference was revealed in spermprogressive motility (%) in fresh semen (27.07±4.5). Significant differenceswere recorded in live sperm morphology and viability on fresh semen (81.8±2.8%)compared to control, 1, 5, and 10 mM. In the10 mM diluted semen (78.5±6.8), the proportion of sperm with an intact plasmamembrane integrity was highest (P<0.05), while the percentage in the1 mM diluted semen (73.0±2.7) was lower. Malondialdehyde levels were lowest (P<0.05)in the group receiving 5 mM treatment. In conclusion, 5 mM of GSH is therequired amount to be added to the freezing extender while cryopreserving semenfrom Kolbroek boars.

Deterioration of Frozen Semen of Bali Cattle after Cooling at 5°C

Frozen semen is produced through several stages, which deteriorate spermatozoa. This research aimed to evaluate the deterioration degree of frozen semen after 5 °C cooling and freezing of Bali cattle. The samples included 10 male Bali cattle with a body weight of 542-668 kg, from which semen was collected once a week for five weeks. The deterioration of each individual’s sperm was determined by observing two distinct straws. The parameters observed included viability, abnormalities, intact plasma membrane, and intact acrosome cap. Initial observations of the parameters were conducted following the addition of semen to diluent A1 (AD) as much as the volume of fresh semen. The semen in the AD group was not cooled and frozen. The A1 semen was then divided into two, namely, those with cooling at 5 °C for 4 hours (PT1) and at 5°C for 22 hours (PT2). The results showed that individual variations in Bali cattle caused significant differences in viability and intact plasma membrane of AD and PT1 groups, while PT2 did not differ in viability and intact plasma membrane spermatozoa. Abnormalities were significantly different between AD and PT2 groups, however PT1 did not differ in abnormalities spermatozoa. Intact acrosomal cap was significantly different in the AD, PT1, and PT2 groups. In conclusion, individual variations, including viability, abnormalities, intact plasma membrane, and acrosome cap of spermatozoa, were better at 4 hours compared to cooling at 5°C for 22 hours. A Cooling time of 4 hours at 5°C can be recommended for frozen semen processing.

Effects of Environmental Stress on the Pollen Viability of Ornamental Tree-Species in the City of Granada (South-Eastern Spain)

Atmospheric conditions, as well as pollutants, can induce changes in the viability and germinability of the pollen grain. This process frequently occurs in cities due to the high rate of air pollution that can alter the quality of pollen, affecting its biological functions. In this work, the effect of different environmental stress factors, mainly UV-B radiation and polluting gases (CO, NO2 and SO2), on the viability and maturity of the pollen of four ornamental tree-species present in the green infrastructure of Granada, namely Acer negundo, Carpinus betulus, Olea europaea and Cupressus spp. is analyzed. Differential staining techniques were used with fresh pollen collected in areas with different exposure to environmental stress to detect intact cell membranes (Trypan blue) and the state of maturity (Pyrogallol red). It was observed that the species from sectors more exposed to environmental stress registered a low viability and were affected by factors such as UV-B radiation and atmospheric pollutants. On the contrary, the pollen from tree species growing in peri-urban forests presented a higher rate of viability and less effect of pollutant factors. Differences were also observed according to the species/genus and according to the sampling area. This modification in the morphological and/or organic composition of the pollen wall may cause a loss of quality in the reproductive processes of plants, and it may be bioindicator of the process of progressive degradation that plant species can experience in urban environments under conditions of environmental stress, and prevent the impacts that can affect other species.

Differential Expressions of Protamine 1 (PRM1) and Protamine 2 (PRM2) Genes as Markers of Semen Quality in Pasundan Bulls

Several gene expressions are related to the success of spermatogenesis. Protamine plays an important role in sperm DNA packaging, spermatogenesis, and sperm quality. This study aimed to isolate RNA from spermatozoa and determine the gene expression profiles of protamine 1 (PRM1) and protamine 2 (PRM2). Six Pasundan bulls aged 5-8 years with a body weight of 380 kg-430 kg were used for this study. The Pasundan bulls were classified into group A (70%-79%) and group B (80%-89%) based on their sperm motility in fresh semen. In this study, correlation analysis was performed between fresh semen characteristics (volume, motility, concentration, abnormality, viability, intact plasma membrane (IPM), DNA integrity) and frozen semen characteristics after thawing (motility, viability, IPM, and DNA integrity). Total RNA was isolated from the frozen sperm pellet, and cDNA was synthesized. Specific PCR primers were used for the transcription of PRM1 and PRM2 from sperm. PRM1 and PRM2 gene expressions were evaluated by qRT-PCR, and ACTB was used as a control. The results showed that progressive sperm motility in the fresh sperm positively correlated with sperm viability and IPM. PRM1 and PRM2 were higher (p<0.05) in group B than in group A. This condition indicates the existence and influence of protamine on the parameters of progressive sperm motility. The expression of PRM1 and PRM2 genes could use as markers of semen quality in Pasundan bulls.