Intact Plasma Research Articles

The Quality of Buffalo Sperm in Tris Egg Yolk Diluent with Addition of Different Levels of Mangosteen Peel Extract

This study aimed to determine the quality of buffalo sperm in tris egg yolk diluent added with mangosteen peel extract at different levels. This study was conducted using an experimental method using a completely randomized design consisting of four treatment concentrations of mangosteen peel extract (T1: 0%, T2: 5%, T3: 10%, and T4: 15%) and four replications, respectively. Parameters observed were motility, abnormalities, intact plasma membrane, and acrosome intact. In the results, the addition of mangosteen peel extract had a significant effect (p > 0.05) on the motility of buffalo sperm that had been diluted in egg yolk tris diluent. This study concluded that the addition of 5–10% mangosteen peel extract in egg yolk tris diluent has a significant effect on sperm motility, could minimize the abnormality, and minimize the decrease in the intact plasma membrane of the buffalo sperm.

Effects of Adding Glutathione to AndroMed Diluent on Intact Plasma and Acrosome Membranes, and Progressive Motility of Cattle Spermatozoa During Freezing Processes

Adding endogenous antioxidants to the diluent is significantly associated with semen quality during the freezing process. This study aimed to investigate the effects of adding glutathione to AndroMed diluent on the preservation of crucial sperm attributes, namely, intact plasma membrane (IPM), intact acrosome membrane (IAM), and progressive motility of Bali cattle spermatozoa. A completely randomized design was used, and spermatozoa samples were obtained from a Bali cattle and divided into two diluent treatment groups (36 diluent samples in each group with six replications), namely pure AndroMed as the control and a group with the addition of glutathione (1 mmol) to AndroMed. Each treatment was replicated six times and evaluated at three freezing stages, including post-dilution, post-equilibration, and post-thawing, for crucial sperm properties. The results indicated that fresh Bali cattle spermatozoa had progressive motility, IAM, and IPM of 75%, 89%, and 88%, respectively. During the freezing process, there was a significant decrease in semen quality, including progressive motility, IAM, and IPM of spermatozoa after dilution to post-equilibration and post-equilibration to post-thawing in both treatment groups. Meanwhile, the addition of 1 mmol of glutathione to AndroMed diluent had a significant difference in increasing progressive motility, IAM, and IPM of Bali cattle spermatozoa at each stage of semen freezing, including post-dilution, post-equilibration, and post thawing when compared with controls. Based on the results, it can be concluded that adding 1 mmol of glutathione to the AndroMed diluent enhanced the quality and integrity of Bali cattle semen, including progressive motility, IAM, and IPM during the freezing process.

Molecular Engineering of Plasma Membrane and Mitochondria Dual-Targeted NIR-II AIE Photosensitizer Evoking Synergetic Pyroptosis and Apoptosis.

Phototherapy provides a noninvasive and spatiotemporal controllable paradigm to inhibit the evasion of the programmed cell death (PCD) of tumors. However, conventional photosensitizers (PSs) often induce a single PCD process, resulting in insufficient photodamage and severely impeding their application scopes. In this study, molecular engineering is conducted by adjusting electron donors to develop an aggregation-induced NIR-II emissive PS (DPITQ) for plasma membrane and mitochondria dual-targeted tumor therapy by evoking synergetic pyroptosis and apoptosis. DPITQ displays boosted type I and II reactive oxygen species generation as well as a high photothermal conversion efficacy (43%) after laser irradiation of 635 nm. The excellent biocompatibility and appropriate lipophilicity help the DPITQ to specifically anchor in the plasma membrane and mitochondria of cancer cells. Furthermore, the photosensitized DPITQ can disrupt the intact plasma membrane and cause mitochondrial dysfunction, ultimately causing concurrent pyroptosis and apoptosis to suppress cancer cell proliferation even under hypoxia. It is noteworthy that the DPITQ nanoparticles (NPs) present clear NIR-II fluorescence imaging capability on the venous vessels of nude mice. Notably, the DPITQ NPs exert efficient NIR-II fluorescence imaging-guided phototherapy both in multicellular tumor spheroids and in vivo, causing maximum destruction to tumors but minimum adverse effects to normal tissue.

Inclusion of 1,3-dimethylxanthine improved quality parameters of bull semen

Methylxanthines are a unique class of drug, derived from the purine base xanthine and 1,3-dimethylxanthine (Theophylline) is the most commonly used methyl xanthine. The present study was conducted to study the effect of supplementation of 1,3-dimethylxanthine in semen extender on sperm motility and morphological parameters of semen. A total of 15 semen ejaculates were collected from three adult cattle bulls. Each ejaculate was divided into five groups. First group was kept as control (C) in which samples were diluted with Tris-egg yolk-glycerol extender and samples of other 4 groups (T-5, T-10, T-20 and T-40) were supplemented with 1,3-dimethylxanthine at a dose rate of 5, 10, 20 and 40 mM respectively. The diluted semen samples were incubated at 37°C in water bath and evaluated for various seminal parameters at 15 and 30 min of incubations. Sperm motility, sperm kinetic parameters and sperm abnormalities of semen of all groups were assessed using computer assisted sperm analyser (CASA). Morphological parameters of spermatozoa of control and supplemented groups were also evaluated. The results revealed that 1,3-dimethylxanthine supplementation resulted in significant increase in motile sperm concentration, kinematic parameters, sperm viability, sperms with intact plasma membrane as compared to other groups. However, sperm abnormalities viz., bent tail, coiled tail, distal droplet and distal midpiece reflex varied non-significantly in all groups. Thus, it can be concluded that 1,3-dimethylxanthine (Theophylline) inclusion at 10 mM concentration in semen extender improved the quality of semen.

Inhibition of phospholipase C reduces the capacitation of cryopreserved ovine sperm

This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 μM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the “Computer-assisted Sperm Analysis” system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 μM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 μM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 μM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes.

Effectiveness equilibration time on the quality of sperm buffalo in diluent tris yolk eggs with 4 % extract skin mangosteen (Garcinia mangostana L.)

A comfortable livestock environment will produce good sperm productivity. Sperm dilution is done so that rural communities can feel the benefits. This study aims to determine the best equilibration time to produce high motility, intact plasma membrane values with low abnormality values in buffalo spermatozoa. This research used a completely randomized design (CRD) with four treatments and ten replications. Sperm came from three buffalo kept at the Ciawi Balitnak. The research treatment consisted of equilibration times of two, three, four and five hours. The parameters measured were sperm motility, abnormality, and intact plasma membrane (IPM). Equilibrium time does not affect buffalo sperm motility, abnormalities, and IPM. The range of motility, abnormalities, and IPM was 43.75%-55.00%; 20.25%-29.00%; 71.75%-74.75%. Conclusion; An equilibration time of two hours can maintain motility, abnormality, the intact plasma membrane of buffalo sperm with egg yolk tris diluent, and the addition of 4% mangosteen peel extract which was suitable for artificial insemination. The application can improve the standard of living of the community through increased meat production.

Uncoupled respiration stability of isolated pancreatic acini as a novel functional test for cell vitality

Background. Assessment of cell viability is crucial in cell studies. Testing plasma membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial functional capacity closely correlates with plasma membrane integrity and overall cell health. This study aimed to investigate whether any aspect of mitochondrial adaptive capacity in isolated pancreatic acini is associated with the quality of isolated pancreatic acini preparations, as determined by the dye exclusion method. Materials and Methods. Experiments were carried out on male Wistar rats weig­hing 250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase. The rate of oxygen consumption of rat isolated pancreatic acini was measured with Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma membrane integrity was assessed with trypan blue staining. A total of 74 preparations of isolated pancreatic acini were used in this study. Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited negative trypan blue staining, indicating intact plasma membranes. The basal and maximal uncoupled respiration rates were not affected by the fraction of trypan-negative cells. However, acini preparations with <less than 95 % plasma membrane integrity had significantly lower uncoupled respiration rates when exposed to a high concentration of FCCP (2 µM), indicating reduced stability of uncoupled respiration. Conclusions. Results of the study suggest that the stability of uncoupled respiration can serve as a novel metabolic functional test to complement the existing methods for assessing cell vitality.

Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa

BackgroundReactive oxygen species (ROS) are strongly linked with oxidative stress (OS) generated during the process of sperm cryopreservation. Indeed, cellular damage from ROS has been implicated during sperm cryopreservation which causes deterioration in sperm quality and antioxidant nanoparticles (NPs) have been successful in preventing such damage. The interaction of NPs with sperm cells has been less frequently explored in farm animals. ObjectiveThe present study explored the effect of NP supplementation on sperm ultrastructure, potential interaction with sperm membrane (plasma and acrosome membrane), heat shock protein (HSP) gene expression levels and sperm quality in cryopreserved buck semen. Materials and methodsThirty-two (32) ejaculates were collected from four (4) adult male bucks and then diluted in Tris- citric acid- fructose- egg yolk (TCFY) extender containing the Zinc-oxide (ZnO) and Selenium (Se) NP treatments (T0: Control; TZn: 0.1 mg/mL ZnO NPs and TSe: 1 µg/mL Se NPs) after initial evaluation. Diluted semen was packed in 0.25 mL French mini straws and then stored in liquid nitrogen (LN2). Sperm parameters, lipid peroxidation (LPO) profile, sperm head morphology ultrastructural classification under transmission electron microscope (TEM), potential interaction of NPs with sperm membrane and expression of HSP genes were evaluated in the different treatment groups. ResultsWe found a significant (p < 0.05) increase in the percentage of spermatozoa with intact plasma membrane, and intact acrosome in the ZnO (0.1 mg/mL) and Se (1 µg/mL) NP supplemented groups in comparison to the frozen control group. TEM assessment revealed no internalization of both ZnO and Se NPs into the sperm structure. Few occasional contacts of ZnO NPs with the sperm membrane and a few agglomerates of Se NPs around the area of damaged membranes were visualized. HSP70 and HSP90 mRNA levels were significantly (p < 0.001) higher in the NP supplemented groups in comparison to the control. HSP70 and HSP90 mRNA levels had a strong positive association with sperm motility and a weak to moderate association with other sperm parameters. ConclusionsCurrent findings indicated that ZnO NPs are more potent than Se NPs in ameliorating peroxidative damages during sperm cryopreservation, increases semen quality parameters possibly by increasing the expression levels of HSP genes in buck semen. Furthermore, NP supplementation may have a potential role in preserving sperm head ultrastructure by acting as an antioxidant and reducing OS during various degrees of cellular insults, which needs to be further explored.

Egg Yolk-Supplemented Tris-Citric Acid Extender Improves the Prefreezing and Post-Thaw Sperm Quality Indices of Guinea Pig (Cavia porcellus) Epididymal Spermatozoa.

This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.

The effect of addition of melon (Cucumis melo L.) flesh juice into Lactated Ringer’s-egg yolk extender on spermatozoa plasma membrane integrity and spermatozoa morphological abnormalities of semen of native rooster

This study aims to determine the effect of adding melon (Cucumis melo L.) juice to Lactated Ringer’s-egg yolk extender on the intact plasma membrane (IPM) and morphological abnormalities of native rooster spermatozoa. Five ejaculate each from two native roosters (Gallus gallus domesticus) were divided into four equal volumes. Ejaculate was extended 1:10 (v/v) in Lactated Ringer's-egg yolk without or with the addition of of 2, 4, and 8% (v/v) melon flesh juice, respectively for groups T0, T1, T2, and T3. Extended semen was stored in a refrigerator (5°C) and evaluated every two hours. The results showed that IPM of all groups decreased (p &lt;0.05) consistently, lasting more than 40% for six hours in the T0 and T1 groups and four hours in the T2 and T3 groups. The percentage of spermatozoa morphological abnormalities in all groups stored for six hours was higher (p &lt;0.05) than before storage. However, it was not significantly different (p &gt;0.05) from that which had been stored for two and four hours. It was concluded that native rooster semen extended in Lactated Ringer's-egg yolk without (group T0) or with 2% melon flesh juice (group T1) and stored at 5°C retained spermatozoa plasma membrane integrity of more than 40% and morphological abnormalities of less than 20% for six hours. While the addition of 4 % (T2) and 8% (T3) melon flesh juice maintained the percentage of IPM and spermatozoa morphological abnormalities for only four hours.

The effect of addition of melon (Cucumis melo L.) flesh juice into Lactated Ringer’s-egg yolk extender on spermatozoa plasma membrane integrity and spermatozoa morphological abnormalities of semen of native rooster

This study aims to determine the effect of adding melon (Cucumis melo L.) juice to Lactated Ringer’s-egg yolk extender on the intact plasma membrane (IPM) and morphological abnormalities of native rooster spermatozoa. Five ejaculate each from two native roosters (Gallus gallus domesticus) were divided into four equal volumes. Ejaculate was extended 1:10 (v/v) in Lactated Ringer's-egg yolk without or with the addition of of 2, 4, and 8% (v/v) melon flesh juice, respectively for groups T0, T1, T2, and T3. Extended semen was stored in a refrigerator (5°C) and evaluated every two hours. The results showed that IPM of all groups decreased (p &lt;0.05) consistently, lasting more than 40% for six hours in the T0 and T1 groups and four hours in the T2 and T3 groups. The percentage of spermatozoa morphological abnormalities in all groups stored for six hours was higher (p &lt;0.05) than before storage. However, it was not significantly different (p &gt;0.05) from that which had been stored for two and four hours. It was concluded that native rooster semen extended in Lactated Ringer's-egg yolk without (group T0) or with 2% melon flesh juice (group T1) and stored at 5°C retained spermatozoa plasma membrane integrity of more than 40% and morphological abnormalities of less than 20% for six hours. While the addition of 4 % (T2) and 8% (T3) melon flesh juice maintained the percentage of IPM and spermatozoa morphological abnormalities for only four hours.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.

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.

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.

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.

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