Hypo-osmotic Swelling Test Research Articles

Sperm Cryopreservation in Canaries to Protect Endangered Songbird Species: Comparison of Different Cryoprotectants.

Sperm cryopreservation is a rather complex process that needs to be adapted to wild and domestic bird species to ensure adequate efficiency. This study aimed to determine the usability of different cryoprotectants in the cryopreservation of Gloster canary sperm. For this purpose, sperm samples were collected from 12 2-year-old male Gloster canaries three times a week using cloacal massage for 4 weeks. After individual evaluation, sperm samples from the canaries were combined. Mixed sperm were divided into two groups in the study. Overall, 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were used as cryoprotectants. Sperm samples were drawn into straws after adding Dulbecco's Modified Eagle Medium (DMEM) extender with high glucose ratio and two different cryoprotectants in a 1:1 ratio and frozen to -80°C with liquid nitrogen vapour and then stored in liquid nitrogen at -196°C. Frozen-thawed semen samples were evaluated regarding motility, vitality, plasma membrane integrity (hypoosmotic swelling test [HOST]), density and abnormal spermatozoa rate. The highest motility value after freezing and thawing was determined in the EG group with 31.667%±4.773%. In addition, vitality, plasma membrane integrity and normal sperm morphology were statistically significantly higher in the EG-frozen group, whereas head and tail abnormality was low (p<0.05). This study determined that a DMEM extender containing 8% EG was more advantageous than a DMEM containing DMSO regarding spermatological parameters and could be used for long-term storage of canary sperm.

Piceatannol Protects Sperm from Cryopreservation Damage by Modulating the Keap1-Nrf2/ARE Signaling Pathway.

The purpose of this study was to explore the mechanism of action of Piceatannol (PIC) in attenuating oxidative damage to sperm during cryopreservation. Semen samples were collected and homogenized into six equal parts for freeze-thawing experiments. Four different concentrations of PIC were utilized as cryoprotectants during the freeze-thawing process, maintaing a semen to PIC ratio of 1:1, while sperm motility after freezing and thawing was analyzed using computer-assisted sperm analysis (CASA). Sperm plasma membrane integrity was assessed via the hypo-osmotic swelling (HOS) test. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, long with the ability to scavenge sperm malondialdehyde (MDA), were examined in sperm following the addition of PIC. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of Keap1, Nrf2, GCLC, GCLM, and HMOX1 in sperm. The mechanism by which PIC protects sperm during cryopreservation from oxidative stress damage was further verified. Treatment with PIC at a dose of 5.0 μmol/L significantly improved both sperm motility and viability while effectively reducing ROS levels in frozen sperm. Additionally, the integrity of the sperm plasma membrane was significantly enhanced. Furthermore, the expression level of Keap1 was significantly reduced, whereas the expression levels of GCLC, GCLM, HMOX1, and Nrf2 were significantly increased (p < 0.05) after the addition of PIC. Notably, a significant attenuation of sperm motility and viability was observed in this treatment group when PIC treatment was accompanied by the addition of an Nrf2 inhibitor, resulting in a significant elevation of ROS levels. The finding that PIC modulates ROS in frozen sperm via the Keap1-Nrf2/ARE pathway thereby enhancing sperm viability levels after freezing and thawing provides a novel approach to optimize semen cryopreservation.

Effects of Supplementation of Different Antioxidants to Cryopreservation Extender on the Post-Thaw Quality of Rooster Semen-A Meta-Analysis.

The standardization of the semen cryopreservation technique could be an effective tool in poultry for the conservation of genetic resources. During this process, the production of reactive oxygen species increases, leading to oxidative stress that causes damage to the spermatozoa. To reduce this effect, the addition of exogenous antioxidants in the cryopreservation diluent has been reported to be effective. Multiple antioxidants such as catalase, vitamin E, cysteamine, ergothioneine, and serine have been studied in roosters. Therefore, the present investigation aims to perform a meta-analysis to determine if the use of the aforementioned antioxidants added to the cryopreservation extender produces an improvement in semen quality parameters in roosters after thawing. After collecting the data, a discriminant canonical analysis was performed to determine which of the selected semen quality traits provided the most information, with hypo-osmotic swelling test (HOST), viability, and total motility variables showing the highest discriminatory power. However, according to the descriptive statistics, catalase and serine are the antioxidants that improve a greater number of seminal quality parameters, and since catalase gives the most favorable results for most of the discriminating variables, it could be the antioxidant of choice.

Cyclodextrin-Cholesterol Alone or in Combination With Cyclodextrin-Vitamin E Improves Bull Sperm Cryopreservation in a Soybean Lecithin Extender.

Combining cholesterol-loaded methyl-β-cyclodextrin (CD-CHL) with vitamin E-loaded methyl-β-cyclodextrin (CD-Vit E) to combat cold shock and oxidative stress during sperm cryopreservation in soybean lecithin extenders remains unexplored. Thus, the current study aimed to investigate the effect of treating bull sperm with CD-CHL and CD-Vit E prior to cryopreservation in a soybean lecithin extender. Sperm collected from eight fertile bulls were pooled and split into six aliquots. Five aliquots were treated, in a Tris-based extender, with CD-CHL (2 mg/120 × 106 cells/mL) and either 0, 0.5, 1.0, 1.5 or 2 mg CD-Vit E/120 × 106 cells/mL. The control aliquot was diluted in a Tris-based extender without further supplementation. After incubation at 22°C for 15 min and addition of a soybean lecithin extender, all aliquots were equilibrated for 2 h at 4°C and then cryopreserved in liquid nitrogen. Computer-assisted sperm analysis (CASA) was used to explore the different sperm motility parameters, hypo-osmotic swelling test to determine membrane functionality and fluorescein isothiocyanate-conjugated Aeachis hypogaea (peanut) agglutinin (FITC-PNA) to quantify acrosome integrity. The effect of oxidative stress on the sperm membrane was assessed through lipid peroxidation measurement. Compared to control, CD-CHL alone improved significantly (p < 0.05) all CASA motility parameters, membrane functionality and acrosome integrity of thawed sperm. The membrane functionality was more significantly (p < 0.05) improved when 0.5 mg CD-Vit E was combined with CD-CHL. Concerning lipid peroxidation, no significant differences (p > 0.05) in malondialdehyde (MDA) levels were registered between groups. In conclusion, the combination of CD-CHL and CD-Vit E demonstrated a significant positive effect on the cryopreservation of bull sperm in a soybean lecithin extender.

Effects of Resveratrol-Loaded Cyclodextrin on the Quality Characteristics of Ram Spermatozoa Following Cryopreservation.

This study investigated the effects of pure and methyl-β-cyclodextrin loaded forms of resveratrol (10 µg/mL, 20 µg/mL, and 40 µg/mL) on ram sperm functions post-thawing. Semen samples were pooled and divided into ten groups: Control, RES10, RES20, RES40, CD10, CD20, CD40, RLC10, RLC20, and RLC40. The groups were pre-diluted with media containing the group-specific chemicals, followed by 15 min of incubation, dilution, and freezing. To assess the effects of the chemicals, a post-thaw sperm quality assessment was conducted. Motility and other velocity parameters were evaluated using computer-assisted semen analysis. The functional integrity of spermatozoa membranes was assessed with the hypo-osmotic swelling test, and the capacitation status of spermatozoa was determined through fluorescent microscopic evaluation. Additionally, flow cytometry was used to evaluate mitochondrial activity, oxidative stress, and the integrity of the sperm membrane and acrosome. The results indicated that cyclodextrin adversely affected sperm functions following freezing-thawing, notably increasing the rate of spermatozoa exhibiting pre-capacitation and mitochondrial activity by approximately 34% and 16%, respectively (p < 0.05). It was found that 20 µg/mL resveratrol prevented pre-capacitation (p < 0.05). Both resveratrol and resveratrol-loaded cyclodextrin groups improved post-thaw sperm qualities overall, demonstrating their utility for freezing ram semen. However, higher concentrations of resveratrol were found to negatively impact sperm functions.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration.

Effect of Supplementation of a Cryopreservation Extender with Pectoliv30 on Post-Thawing Semen Quality Parameters in Rooster Species.

Sperm cryopreservation is a fundamental tool for the conservation of avian genetic resources; however, avian spermatozoa are susceptible to this process. To cope with the high production of reactive oxygen species (ROS), the addition of exogenous antioxidants is beneficial. Pectoliv30 is a substance derived from alperujo, and in this study, its effect was analyzed on seminal quality after its addition to the cryopreservation extender of roosters at different concentrations. For this purpose, 16 Utrerana breed roosters were used, and seminal collection was performed in six replicates, creating a pool for each working day with ejaculates of quality. After cryopreservation, one sample per treatment and replicate was thawed, and several seminal quality parameters were evaluated. Statistical analysis revealed numerous correlations between these variables, both positive and negative according to the correlation matrix obtained. Furthermore, the chi-squared automatic interaction detection (CHAID) decision tree (DT) reported significant differences in the hypo-osmotic swelling test (HOST) variable between groups. Moreover, results for this parameter were more desirable at high concentrations of Pectoliv30. The application of this substance extracted from the by-product alperujo as an antioxidant allows the improvement of the post-thawing seminal quality in roosters and facilitates optimization of the cryopreservation process as a way to improve the conservation programs of different endangered poultry breeds.

The influence of rutin and chlorogenic acid on oxidative stress and in vivo fertility: Evaluation of the quality and antioxidant status of post-thaw semen from Azari water buffalo bulls.

The vulnerability of buffalo sperm to cryoinjury necessitates the improvement of sperm cryo-resistance as a critical strategy for the widespread use of assisted reproductive technologies in buffalo. The main aim of the present study was to evaluate the effects of different concentrations of rutin and chlorogenic acid (CGA) on buffalo semen quality, antioxidant activity and fertility during cryopreservation. The semen was collected and pooled from the 3 buffaloes using an artificial vagina (18 ejaculations). The pooled sperm were divided into nine different groups: control (Tris-based extender); 0.4, 0.6, 0.8 and 1mM rutin (rutin+Tris-based extender); and 50, 100, 150 and 200µM CGG (CGA+Tris-based extender). Sperm kinematics, viability, hypo-osmotic swelling test, mitochondrial activity, antioxidant activities and malondialdehyde (MDA) concentration of frozen and thawed buffalo sperm were evaluated. In addition, 48 buffalo were finally inseminated, and pregnancy was rectally determined 1 month after insemination. Compared to the control group, adding R-0.4, R-0.6, CGA-100 and CGA-150 can improve total and progressive motility, motility characteristics, viability, PMF and DNA damage in buffalo sperm. In addition, the results showed that R-0.4, R-0.6, CGA-50, CGA-100 and CGA-150 increased total antioxidant capacity, catalase, glutathione peroxidase and glutathione activities and decreased MDA levels compared to the control group. Furthermore, it has been shown that adding 150µM CGA and 0.6mM rutin to an extender can increase in vivo fertility compared to the control group. In conclusion, adding rutin and CGA to the extender improves membrane stability and in vivo fertility of buffalo sperm by reducing oxidative stress.

Characteristics of Bachaur bull semen.

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR-National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5-5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0-5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo-osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds.

A comparative study between curcumin and curcumin nanoparticles on reproductive performance and antioxidant system of aged roosters

Increased lipid peroxidation and decreased antioxidant status can result in reduced reproductive activity and fertility in aged male broiler breeders. This study was conducted to evaluate the effects of curcumin supplements (natural or nanoparticles) on the sperm characteristics, antioxidant system, fertility, and hatchability of aged roosters (54-64 wk), and to estimate the relative bioavailability value (RBV) of nano-curcumin on the measured parameters including the hypo-osmotic swelling test (HOST), motility, viability, sperm count, volume, the concentration of testosterone, glutathione peroxidase (GPx), and superoxide dismutase (SOD), diameter of the spermatogenic tube (DST), epithelium thickness (EpiTh), spermatogonia count (SPcount), fertility, hatchability, and relative weight of testis (RW-testis). A total of 30 roosters were individually caged and randomly assigned to 5 treatments comprising control (without curcumin as the basal diet), basal diet + 15 mg/kg curcumin (CUR15), basal diet + 30 mg/kg curcumin (CUR30), basal diet + 15 mg/kg nano-curcumin (Nano15), and basal diet + 30 mg/kg nano-curcumin (Nano30) for 10 wk. The slope ratio method was used to estimate the bioavailability of nano-curcumin by regressing each response on supplemental curcumin intake. Increasing dietary curcumin (P < 0.001) elicited a linear response to all studied traits. The RBV for volume, viability, motility, HOST, RW-testis, and GPx were estimated as 135 (CI: 115-156%), 143 (CI: 114-173%), 159 (CI: 122-196%), 132 (CI: 107-157%), 195 (CI: 126-264%), 176 (CI: 103-249%), and 178% (28-328%), respectively. Our findings revealed that curcumin nanoparticles enhance the reproductive efficiency of aged breeder roosters. In addition, the curcumin nanoparticles RBV exceeded that of natural curcumin, suggesting that lower concentrations of curcumin nanoparticles could have a significant effect on reproductive characteristics.

Novel insights into necrozoospermia from a single-center study: reference ranges, possible etiology, and impact on male fertility.

Necrozoospermia is a poorly documented condition with a low incidence, and its definition and clinical significance are unclear. Herein, we provide a reference range for necrozoospermia and discuss its possible etiology and impact on male fertility and assisted reproductive outcomes. We extracted relevant information from 650 Chinese male partners of infertile couples and statistically analyzed sperm vitality. Necrozoospermia was present in 3.4% (22/650) of our study population, and the lower cut-off value for sperm vitality was 75.3%. We compared two methods for assessing sperm vitality (eosin-nigrosin head staining and hypo-osmotic swelling test [HOST]), for which the percentage in the eosin-nigrosin group (mean ± standard deviation [s.d.]: 77.5% ± 10.5%) was significantly higher than that in the HOST group (mean ± s.d.: 58.1% ± 6.7% [5-10 min after incubation] and 55.6% ± 8.2% [25-30 min after incubation]; both P < 0.001). The incidence of necrozoospermia increased with age (odds ratio [OR] = 1.116, 95% confidence interval [CI]: 1.048-1.189, P = 0.001), while the percentage of normal sperm morphology and DNA fragmentation index (DFI) were significantly associated with necrozoospermia, with ORs of 0.691 (95% CI: 0.511-0.935, P = 0.017) and 1.281 (95% CI: 1.180-1.390, P < 0.001), respectively. In the following 6 months, we recruited 166 patients in the nonnecrozoospermia group and 87 patients in the necrozoospermia group to compare intracytoplasmic sperm injection (ICSI) and pregnancy outcomes between the two groups. The necrozoospermia group had a significantly lower normal fertilization rate (74.7% vs 78.2%, P = 0.041; OR = 0.822; 95% CI: 0.682-0.992) than that in the nonnecrozoospermia group. This study presents substantial information on necrozoospermia to establish comprehensive and applicable reference values for sperm vitality for spontaneous conception and artificially assisted reproductive management.

Evaluation of Genetic and Non-genetic Factors Influencing Semen Production Potential in Sahiwal Bulls

Background: Understanding genetic and non-genetic factors that influence the quality and quantity of semen production plays a crucial role in determining the reproductive efficiency and genetic progress within the Sahiwal breed. The objective of this study was to assess the impact of genetic and non-genetic factors on the semen production potential of Sahiwal bulls. Methods: Semen ejaculate data, including volume (VOL), sperm concentration per milliliter (CONC), total sperm count per ejaculate (TS), initial motility (INIT) and post-thaw motility (PT), as well as semen quality traits such as hypo-osmotic swelling test (HOST) and acrosome integrity of frozen semen (AIFS), were obtained from 52 Sahiwal bulls. The data spanned from January 2011 to December 2018 and were collected at BAIF’s frozen semen station in Jind, Haryana, India. To ensure data reliability, observations beyond the mean±4 standard deviations (S.D.) were identified as outliers and subsequently excluded from the study. Two key factors, namely the season of semen collection (winter, summer and monsoon) and the age of bulls at collection (categorized into 12-month intervals ranging from less than 36 months to 156 months), were considered influential factors affecting semen characteristics. To investigate the correlation between bulls (genetic + permanent environmental correlation) and semen parameters, a repeatability animal model was employed, treating the bull as a random effect. The statistical analysis was conducted using WOMBAT software. Result: The findings revealed that the season of semen collection had a highly significant effect (P less than 0.01) on VOL, CONC, TS and PT. However, INIT, HOST and AIFS were not influenced (P greater than 0.05) by the season of collection. Furthermore, age at semen collection significantly affected all semen characteristics. VOL and TS exhibited an increasing trend until 72 to 84 months, followed by a decline until 96 to 108 months.

Effects of shilajit addition to honey bee diet on semen freezing

This study aimed to investigate the effects of adding shilajit to the diet of honey bees on semen freezing. A total of 5 groups were formed in the research, one of which was a control group (SH-0) and the other four were an experimental group (SH-1, SH-2, SH-3, SH-4). A total of 25 study colonies were used, 5 in each group. While the SH-0 group was formed without using any additives, the experimental groups were fed with the addition of shilajit (1/1 sugar/water) in different doses (5, 10, 15, 20 mg/L) to the honey bee diet. The collected semen samples were frozen in liquid nitrogen vapor and then stored in liquid nitrogen at -196°C. Then, semen samples were thawed at 37°C and evaluated to determine spermatological parameters (motility, acrosome integrity, plasma membrane integrity, spermatozoa concentration). It was also examined in terms of total oxidant and total antioxidant. Compared to the control group, it was determined that all shilajit doses significantly increased spermatological parameters such as motility, hypoosmotic swelling test (HOST), acrosome integrity (p&amp;lt;0.001), but did not significantly affect the spermatozoa concentration value (p &amp;gt;0.05). Although there is no statistical difference between the groups in terms of semen TAS (Total Antioxidant Status) and TOS (Total Oxidant Status) values, which are oxidative stress parameters, the numerical increase in TAS values in SH-2 and SH-3 groups; striking. As a result, it was determined that adding shijajit to the honey bee diet positively affected the post-thawing spermatological parameters of frozen bee semen.

Effect of quercetin, L-ergothioneine and H89 on sperm motility and kinematic pattern, plasma membrane functionality and in vitro heterologous fertilizing capacity of cryopreserved equine semen

Semen cryopreservation causes extensive chemical and physical damage to sperm structure, which generates premature aging and reduces viability and fertility of spermatozoa. The addition of antioxidants to freezing extenders can reduce the oxidative damage caused by excessive generation of reactive oxygen species (ROS), and the premature aging could be reduced by adding an enzyme inhibitor that prevents an anticipated capacitation. The aim of this study was to evaluate the in vitro effect of quercetin (Q), L-ergothioneine (E) and H89 addition to cryopreserved equine spermatozoa. Six experimental groups were stablished: control, Q, E, H89, H89Q and H89E. The analyzed parameters were sperm motility and kinematic using computer assisted sperm analysis (CASA), plasma membrane functionality with the hypoosmotic swelling test (HOST) and fertilizing capability with in vitro heterologous fertilization. Quercetin reduced curvilinear velocity (VCL) and increased beat-cross frequency (BCF), while its combination with H89 (H89Q) reduced total motility, progressive motility, VCL and hyperactive sperm (HA). Likewise, H89 and its combination with E (H89E) decreased VCL and amplitude of lateral head displacement (ALH). No significant differences were observed among treatments for membrane functionality and fertilizing capacity of sperm. In conclusion H89 in combination with Q and E reduced sperm motility or some kinematic parameters. However, they did not influence plasma membrane functionality and in vitro fertilizing capacity of frozen-thawed equine semen.

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Correlation of sperm motility, acrosome integrity, protamine deficiency, and DNA fragmentation in proven and unproven Friesian Holstein bulls

Objective: The evaluation of frozen semen quality is an essential aspect in determining male fertility for artificial insemination programs. This study aims to evaluate the characteristics of Friesian Holstein (FH) bull-frozen semen in different classes (proven and unproven) based on protein profiling and molecular evaluation. Materials and Methods: This study used frozen semen straws from FH bulls selected according to criteria for proven (6 individuals) and unproven (6 individuals) bulls produced by the Singosari AI Center (AIC). Sperm motility parameters were assessed using Computer Assisted Semen Analysis (CASA Supervision®, Germany), while sperm viability and abnormality were evaluated through eosin-nigrosin staining under a microscope at 400´ magnifications. The integrity of the sperm plasma membrane was determined using the hypoosmotic swelling test, and acrosome integrity was analyzed using the fluorescein isothiocyanate PNA-propidium iodide staining method. Protamine deficiency was quantified using Chromomycin A3 fluorescence staining, while DNA fragmentation was assessed using the acridine orange technique. Results: The findings demonstrated that there were no statistically significant differences (p &gt; 0.05) in the assessed parameters of frozen semen quality between FH-proven and unproven bulls. Furthermore, in FH-proven bulls, a negative correlation was observed between protamine deficiency and acrosome integrity (r = −0.528) and between protamine deficiency and sperm DNA fragmentation (r = −0.467). The parameters of protamine deficiency in unproven bulls exhibited a positive correlation with sperm progressive motility. Conclusion: The frozen semen quality of FH bulls in different classes (proven and unproven) was found to be equally good. Molecular-based analysis allows for a more accurate determination of semen quality. These findings are significant for bull breeding stations when comprehensively evaluating semen quality.

Integrity of Sperm Cell Bali Bull Preserved at 5°C Using a Diluent Formulation of Extracted Red Fruit and Coconut Water

This study aims to determine the role of the best diluent in protecting the integrity of the spermatozoa membrane after storage using an alternative diluent formulation of red fruit extract (Pandanus Conoideus Lamk) and coconut water. An alternative diluent formulation featuring red fruit extract (Pandanus Conoideus Lamk) and coconut water was utilized. Fresh semen from Bali Bull was evaluated in the Integrated Field Laboratory, Department of Animal Science of Universitas Muhammadiyah Gorontalo. Bali bull sperm of high quality was diluted using a combination diluent containing red fruit extract (Pandanus Conoideus Lamk), green coconut water, and CEP-2 mixed with 10% egg yolk. Technical term abbreviations were explained at their initial use. Spermatozoa membrane integrity was assessed using the hypoosmotic swelling test (HOST), while chlortetracycline (CTC) fluorescent dye was used to evaluate spermatozoa capacitation and acrosome reaction. The randomized group design (RAK) was used in the research, consisting of four conditions and 10 replications. P0 served as the control group, which received CEP-2 plus 10% egg yolk. P1 was administered EBM diluent AK enriched with 15% KT, while P2 received EBM diluent AK plus 15% KT and fructose 1 mg/ml. P3 was given EBM diluent AK plus 15% KT and fructose 2 mg/ml. Analysis of variance was used to examine the resulting data. The findings indicated that CEP-2 plus 10% egg yolk diluent was more effective than the diluent formulations containing red fruit extract and coconut water. Spermatozoa with intact membranes and no capacitation remain high, while those with capacitation and acrosome reaction remain low.

Reliability of hypo-osmotic swelling test on fresh and frozen-thawed ejaculated or testicular immotile sperm: A sibling oocyte study

The viability of sperm is a crucial factor for achieving a successful pregnancy in intracytoplasmic sperm injection (ICSI) cycles. The aim of this study was to evaluate the accuracy of the hypo-osmotic swelling test (HOST) in fresh and frozen-thawed sperm samples of different origins (ejaculated/testicular). A retrospective analysis was conducted on the outcomes of 2167 oocytes subjected to ICSI using motile and immotile-HOST-positive sperm from 2011 to 2023. We evaluated embryonic development, as well as clinical, obstetric, and neonatal outcomes in four groups based on different sperm origins (ejaculated/testicular) and processing (fresh/frozen). When comparing the results of ICSI between motile and immotile-HOST-positive sperm within each group, it was observed that there were no significant differences in the outcomes for fresh samples. However, for frozen-thawed samples, fertilization rates and blastocyst development rates were significantly lower when ICSI was performed with immotile-HOST-positive sperm compared to motile sperm. Of note, clinical, obstetric, and neonatal outcomes were statistically similar across all groups. Our findings indicate that HOST is more reliable in fresh samples than in those subjected to the freeze–thaw process. Nonetheless, HOST is considered a safe method for selecting viable sperm in all subgroups.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Germinated Wheat as a Potential Natural Source of Antioxidants to Improve Sperm Quality: A Canary Trial.

The present study was carried out to determine the effects of germinated wheat on some spermatological parameters (motility, abnormal spermatozoa, hypo-osmotic swelling test (HOST), and viability) in male Gloster canaries. For this purpose, the canaries were divided into two groups, each consisting of six canaries, one control (C), and the other experimental group (GW). Group C was fed commercial canary feed. The GW group was fed germinated wheat (germinated for 5 days) and commercial canary feed. The canaries were kept individually in four-storey cages (60 cm × 50 cm × 40 cm). In this study, which lasted 2 months, feed/water was provided ad libitum and lighting was applied daily for 16 h (turned on at 05:30 and turned off at 21:30). At the end of the experiment, the sperm samples obtained from the canaries were examined in terms of the aforementioned parameters. The effects of germination time × concentration, germination time, and solvent rate on DPPH radical scavenging activity and phenolic compounds in germinated wheat were significant (p < 0.001). The highest DPPH (2, 2-diphenyl-1-picryl hydrazyl) radical scavenging activity (86.06%) and phenolic content (316.25 mg GAE/g) in germinated wheats were obtained on the fifth day (90% solvent rate) of germination. It was determined that germinated wheat did not significantly affect abnormal sperm and HOST values (p > 0.05). However, it significantly affected the motility and viable sperm rates (p < 0.05). As a result, it is thought that germinated wheat can be used as a natural antioxidant source to increase motility and vitality in canary sperm.