Sperm Plasma Membrane Integrity Research Articles

Effect of sperm treatment with lysolecithin on in vitro outcomes of equine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) in horses is currently employed for clinical and commercial uses, but the protocol could be optimized to improve its efficiency. We have hypothesized that destabilization of plasma and acrosomal membranes prior to injection would positively impact the developmental potential of equine zygotes generated by ICSI. This study evaluated effects of the sperm treatment with lysolecithin on plasma and acrosomal membranes and on oocyte activation ability, initially following heterologous ICSI on bovine oocytes and subsequently employing equine oocytes. The effects of the lysolecithin -treatment on the efficiency of conventional and piezo-assisted equine ICSI were evaluated. To do this, the equine sperm were treated with different concentrations of lysolecithin and the sperm plasma membrane, acrosome and DNA integrity were evaluated by flow cytometry. The results showed that a lysolecithin concentration of 0.08 % destabilized the membranes of all sperm and affected DNA integrity within the range described for the species (8–30 %). In addition, the heterologous ICSI assay showed that lysolecithin treatment was detrimental to the sperm's ability to activate the oocyte, therefore, chemical oocyte activation was used after equine ICSI after injection with lysolecithin -treated sperm. This group showed similar developmental rate to the control group with and without exogenous activation. In conclusion, lysolecithin pre-treatment is not necessary when using ICSI to produce equine embryos in vitro. The results from the current study provide additional insight regarding the factors impacting ICSI in horses.

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Effect of the addition of soursop juice (Annona muricata L., 1753) to egg yolk citrate extender on the quality of bull semen

The present study was carried out to investigate the effect of the addition of soursop (Annona muricata L., 1753) juice to the citrate-egg yolk extender (EYC) on the quality of cryopreserved bull semen. After pre-evaluation, the semen was distributed in five extenders T1, T2, T3, T4 and T5 containing respectively 0% soursop juice (SJ) (T1), 25% SJ (T2), 50% SJ (T3), 75% SJ (T4), 100% SJ (T5) and microscopic evaluation was performed daily to assess plasma membrane integrity (PMI), progressive motility of spermatozoa (MP) and sperm pH until all sperm were dead. The results indicate a significant increase in spermatozoa PMI and MP (p<0.05) in the case of treatment with 50% SJ (T3). Spermatozoa showed a motility of 50.78% 9 days later in the citrate-egg yolk extender containing 50% SJ (T3), whereas at day 9, spermatozoa in the other extenders had a progressive motility of less than 50%. Similar results were obtained for PMI with 50.78% of spermatozoa having an intact plasma membrane 9 days later in the citrate-egg yolk extender containing 50% SJ whereas the PMI of cells in the other stents was less than 50%. However, the pH of the extender containing 50% SJ was closest to 7 during the experimental period. The results of this experiment suggest that the addition of 50% soursop juice (T3) to the citrate-egg yolk extender could be adopted as an acceptable alternative to traditional egg yolk-based extenders for the cryopreservation of bull semen.

β-Nicotinamide mononucleotide improves chilled ram sperm quality in vitro by reducing oxidative stress damage.

The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4°C in vitro. Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 μM) was used to dilute semen collected from rams and it was stored at 4°C. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4°C. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4°C. Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 μM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 μM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4°C. Ram sperm quality can be maintained during storage at 4°C with the addition of NMN at 60 μM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4°C in vitro.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO₄ (0, 12.5, 25, 50, and 100 &micro;M) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 &micro;M ZnSO₄ was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 &micro;M ZnSO₄. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 &micro;M ZnSO₄ increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO₄.

Kojic acid inhibits pig sperm apoptosis and improves capacitated sperm state during liquid preservation at 17°C.

The parameters of sperm apoptosis and capacitation during liquid storage at 17°C can indicate the quality of pig sperm and the potential development of early embryos. However, the effect of kojic acid (KA) on semen preservation and its mechanism has not been fully understood. In this study, we discovered that adding KA to the diluent improved the antioxidant capacity of sperm mitochondria, maintained the normal structure of sperm mitochondria, and reduced sperm apoptosis. Western blot analysis revealed that KA prevented the release of Cytochrome c from mitochondria to the cytoplasm, reduced the expression of pro-apoptosis proteins cleaved Caspase-3 and cleaved Caspase-9, and increased the expression of the antiapoptosis protein Bcl-XL. Furthermore, KA also enhanced the motility parameters, oxidative phosphorylation level, adenosine triphosphate level, and protein tyrosine phosphorylation of capacitated sperm, while preserving the acrosome integrity and plasma membrane integrity of capacitated sperm. In conclusion, this study offers new insights into the molecular mechanism of how KA inhibits porcine sperm apoptosis and improves capacitated sperm parameters. Additionally, it suggests that KA can serve as an alternative to antibiotics.

Effect of asiatic acid supplementation in tris-extender on post-thaw functional competence, antioxidant enzyme activity and in vivo fertility of bull sperm.

Reactive oxygen species at supra-physiological levels trigger oxidative stress during cryopreservation, which can be neutralised by incorporating suitable antioxidants into the semen extender medium. This study was intended to explore the effect of asiatic acid (AA) as an antioxidant in semen extender on frozen-thawed sperm quality and in vivo fertility of bull sperm. Semen was collected from Holstein Friesian bulls for 10 consecutive weeks (total ejaculates = 60). Semen was cryopreserved with a Tris citric acid egg yolk-based extender supplemented with 0 (control), 20, 40, 60, and 100 μM AA. The supplementation of the extender with 40 and 60 μM AA improved (p < 0.05) post-thaw motility kinematics, plasma membrane integrity, acrosome integrity, sperm viability, and DNA integrity of bull sperm. Mitochondrial membrane potential was high (p < 0.05) with 60 μM of AA concentration in extender media. The catalase activity in seminal plasma was maintained (p < 0.05) when semen was added with 20, 40, and 60 μM of AA. The in vivo fertility was found to be significantly high with the semen extended with 60 μM AA. Conclusively, this study showed that AA supplementation in semen extender significantly improved sperm motility kinematics and cell integrity, conserved antioxidant enzyme activity, and improved in vivo fertility.

Effect of reduced glutathione on quality, lipid peroxidation and antioxidant potential of frozen‐thawed ring‐necked pheasant semen

AbstractReduced glutathione (GSH) is a natural redox force immediately consumed by cells to combat toxins. Today's world is shifting towards clean farming practices whence ring‐necked pheasants dependent on croplands are facing threats and sperm‐assisted reproductive techniques become indispensable in preserving this valuable species for game and meat purposes. This study aims at evaluating the cryosurvivability (motility, viability, plasma membrane and acrosomal integrity, DNA fragmentation) and antioxidant capacity (Free radical scavenging activity, LPO detection, Ferric reducing antioxidant power, mitochondrial status) of ring‐necked pheasant semen with GSH supplementation in the extender. Semen collected from ten males was pooled, divided and diluted with Beltsville poultry semen extender (1:5) containing 0.0, 0.2, 0.4, 0.6 and 0.8 mM GSH. Samples were cryopreserved, and sperm quality was evaluated post‐dilution, cooling, equilibration, and freeze–thaw. Free radical scavenging activity, total antioxidant potential, mitochondrial action and lipid peroxidation were assessed at post‐thawing. Sperm motility, plasma membrane and acrosomal integrity, viability and DNA integrity were recorded highest (p &lt; .05) at 0.4 mM GSH compared to other concentrations and control at all the stages of cryopreservation. Similarly, sperm mitochondrial action, antioxidant potential and free radical scavenging capacity were recorded higher (p &lt; .05) with 0.4 mM GSH at post‐thaw compared to other concentrations of GSH and control. Lipid peroxidation was recorded as highest (p &lt; .05) in the control compared to GSH‐supplemented groups at post‐thaw. Hence, it is concluded that GSH at 0.4 mM preserves the quality and ROS scavenging capacity of cryopreserved ring‐necked pheasant sperm as an antioxidant.

Fresh Seminal Characteristics of Marwari Stallions in Breeding Season

Preserving the sperm of stallions with significant genetic worth is a widely accepted procedure. Thus, the species' typical reproductive behaviour is altered to facilitate the retrieval of sperm for the use of assisted reproductive biotechnologies, like artificial insemination and cryopreservation of sperm. As the Marwari horse breed is a native breed of our country, the present experiment was designed to define the basic semen parameters of Marwari stallions. The experiment was conducted on six healthy Marwari horses aged between 50 and 140 months for three weeks with a semen collection frequency of twice a week. A total of thirty-six semen samples (six from each horse) were collected using an artificial vagina. Following a macroscopic examination, fresh semen samples underwent a microscopic evaluation. The appearance of fresh semen was milky white or creamy white and the consistency was thin to thick. Fresh seminal pH was 7.54±0.03. Total ejaculated semen volume, gel volume and gel-free semen volume were 67.22±5.77 ml, 14.08±1.98 ml and 53.14±4.16 ml, respectively. Progressive sperm motility of sperm was 78.99±0.65%. Sperm concentration was 195.50±5.67 million/ml. Viability and total morphological abnormalities of sperm were 85.82±0.48% and 4.42±0.23%, respectively. Plasma membrane integrity (HOST), acrosome integrity and DNA integrity of sperm were 53.65±0.85%, 87.36±0.36% and 95.83±0.24%, respectively. A significant difference was observed among Marwari stallions for semen volume (total ejaculated, gel and gel-free) (P&lt;0.05), progressive motility (P&lt;0.05), viability (P&lt;0.05), concentration (P&lt;0.05), plasma membrane integrity (HOST) (P&lt;0.05), acrosome integrity (P&lt;0.05), and DNA integrity (P&lt;0.05) of sperm. Non-significant difference was observed for seminal pH (P&gt;0.05) and total sperm morphological abnormalities (P&gt;0.05).

Effect of Adenosine 5´ Triphosphate (ATP) and Incubation Time on Frozen Thawed Buffalo Bull Spermatozoa

The present study was aimed to determine the effect of ATP dose rate on longevity, motility, and viability of frozen-thawed buffalo bull’s sperm. For this purpose, 20 frozen semen straws of four different buffalo bulls were obtained from a private semen production unit (Al-Hiawan Sires, Sahiwal). The experiment was replicated four times. In each replicate, four frozen straws of the same bulls were thawed and pooled. After evaluating the motility, the pooled frozen-thawed sample was aliquoted into three separate tubes containing 0 (control), 1-, or 2-mM concentrations of ATP. Afterward, each aliquot was incubated at 37oC for 4 hrs. The sperm motility, viability, and membrane integrity were observed at various time points (10 min., 1hr, 2hr, and 4hr) during the incubation period of four hrs. It was observed that progressive motility and most of the motion kinematic parameters were greater (P&lt;0.05) in control. A significant (P&lt;0.05) interaction was observed between ATP dose and incubation time. The progressive motility, total motility, VCL, VAP, ALH, and BCF decreased (P&lt;0.05) both by incorporation of ATP (1mM or 2mM) and increasing incubation time (from 10 min to 4hrs) which indicate the ATP and incubation time of 4 hours negatively affect the motion kinetics of buffalo bull sperm. In conclusion, it was witnessed that the motility and plasma membrane integrity of sperm declined by the supplementation of ATP in frozen-thawed buffalo bull semen.

Metabolomics Analysis of Sodium Salicylate Improving the Preservation Quality of Ram Sperm.

The aim of this study was to investigate the effects of sodium salicylate (SS) on the preservation and metabolic regulation of sheep sperm. Under 4 °C low-temperature conditions, SS (at 10 µM, 20 µM, 30 µM, and 50 µM) was added to the semen diluent to detect sperm motility, plasma membrane, and acrosome integrity. Based on the selected optimal concentration of SS (20 µM), the effects of 20 µM of SS on sperms' antioxidant capacity and mitochondrial membrane potential (MMP) were evaluated, and metabolomics analysis was conducted. The results showed that on the 20th day of low-temperature storage, the sperm motility of the 20 µM SS group was 62.80%, and the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher than those of the control group (p < 0.01). The content of Ca2+, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly lower than those of the control group (p < 0.01), and the total antioxidant capacity (T-AOC) was significantly higher than that of the control group (p < 0.05); mitochondrial activity and the total cholesterol (TC) content were significantly higher than those in the control group (p < 0.01). An ultrastructural examination showed that in the SS group, the sperm plasma membrane and acrosome were intact, the fibrous sheath and axoneme morphology of the outer dense fibers were normal, and the mitochondria were arranged neatly. In the control group, there was significant swelling of the sperm plasma membrane, rupture of the acrosome, and vacuolization of mitochondria. Using metabolomics analysis, 20 of the most significant differential metabolic markers were screened, mainly involving 6 metabolic pathways, with the amino acid biosynthesis pathway being the most abundant. In summary, 20 µM of SS significantly improved the preservation quality of sheep sperm under low-temperature conditions of 4 °C.

Supplementing semen extenders with a combination of phosphorus and vitamin B12 Improves post-thawed cryopreserved rooster semen quality.

Semen cryopreservation is an important technique for preserving the genetic material of numerous species. However, frozen semen is highly susceptible to sperm DNA damage and reduced motility, resulting in decreased fertility. The standard method for cryopreservation and several approaches have not been elucidated. This study aimed to determine the effects of supplementing rooster semen extender with a combination of phosphorus and vitamin B12 on cryopreserved semen quality. Semen was collected weekly via dorso-abdominal massage from 57 Burmese × Vietnam-crossbred Thai native roosters aged 1-3 years. In total, 139 semen samples were collected, pooled, and diluted to 200 million sperm per dose. The pooled sample was divided into six experimental groups: a control group (0.00%) diluted with modified Beltville Poultry Semen Extender (BPSE) and five treatment groups diluted with modified BPSE supplemented with phosphorus and vitamin B12 at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%, respectively. The semen samples were frozen and evaluated at 0, 15, and 30 min after thawing. Sperm kinematic parameters were determined using a computer-assisted sperm analysis system. Sperm quality was evaluated by measuring sperm viability, mitochondrial activity, acrosome integrity, and plasma membrane integrity. Statistical analyses were performed using a general linear mixed model (MIXED) in SAS. Factors in the statistical model were experimental groups, time after thawing, and interaction between experimental groups and time after thawing. Total and progressive motilities were greater in semen supplemented with 0.04% phosphorus and vitamin B12 compared with those in the control (p < 0.05). At 15 min post-thawing, VCL, VAP, and HPA in the 0.04% phosphorus and vitamin B12 supplementation group was greater than that in the control (p < 0.05). Phosphorus and vitamin B12 supplementation did not affect sperm kinematics at 0 and 30 min after thawing (p > 0.05). All the sperm parameters that were tested for the 0.04% phosphorus and vitamin B12 supplementation group in modified BPSE were the highest at all the timepoints after thawing. Thus, supplementing frozen semen extender with 0.04% phosphorus and vitamin B12 increased sperm motility, sperm kinematic parameters, and sperm quality.

Protein Profiles in Seminal Plasma of Iraqi Buffalo Bulls (Bubalus bubalis) Associated with Fresh and Cryopreserved Semen Quality

This study was undertaken to identify some protein profiles in the seminal plasma of Iraqi buffalo bulls and their correlation to the quality of fresh and cryopreserved semen. Semen was collected from eight buffalo bulls and divided according to the percentage of sperm individual motility into two groups. The first group (n=3) included good semen quality, while the second group (n=5) included poor semen quality. Seminal plasma was extracted from each semen sample by centrifugation and seminal proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nine types of protein bands were observed in the seminal plasma of the buffalo bulls belonging to good semen quality, ranging from 10-68 kDa. The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (100%), and 43 (37.5%) kDa. On the other hand, 14 types of protein bands resolved for the poor-quality semen, ranging from 10-155 kDa. The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (87.5%), 130 (50%) and 155 (50%) kDa. A significant negative correlation was noticed among proteins with molecular weights of 28. 88 ± 3.26, 22.50 ± 2.85, and 12.00 ± 1.15 kDa, and the percentage of sperm individual motility and plasma membrane integrity. Moreover, there was a positive significant correlation between proteins whose molecular weight 22.50 ± 2.85 kDa and the percentages of total abnormalities and normal sperms. In conclusion, the identification of seminal proteins may be a useful tool in the future evaluation of spermatozoa and the prediction of fertility in Iraqi buffalo bulls.

Effect of white guava (Psidium guajava L.) fruit juice on the quality of lead acetate induced rats (Rattus norvegicus) spermatozoa

This study aims to determine the effect of the administration of white guava (Psidium guajava L.) fruit juice on spermatozoa plasma membrane integrity (PMI), morphological abnormality, viability, and motility of lead acetate induced rats (Rattus norvegicus). Twenty-five male rats were divided into five groups: NC (negative control) group, rats were administered with distilled water twice daily at four-hour intervals; T0 (positive control) group, rats were administered daily with lead acetate 50 mg/kg bw and distilled water four hours later; T1, T2, and T3 groups, rats were administered daily with lead acetate 50 mg/kg bw and 0.5 mL of 25, 50, and 100% white guava fruit juice four hours later. The treatment of the rats was conducted for 14 days, and on day 15, all rats were sacrificed to assess the spermatozoa quality. Data was analyzed using ANOVA followed by Duncan's multiple range test at a confidence level of 95%. The results showed that exposure to lead acetate (T0) caused lower spermatozoa PMI, viability, and motility as well as higher spermatozoa morphological abnormalities (p &lt;0.05) compared to those of the T0 group. Administration of white guava fruit juice starting at a dose of 25% (T1) resulted in higher spermatozoa motility, viability, and PMI as well as lower spermatozoa morphological abnormalities (p &lt;0.05) compared to rats in the T0 group. It could be concluded that white guava fruit juice maintained the spermatozoa quality of lead acetate induced rats.

Effect of white guava (Psidium guajava L.) fruit juice on the quality of lead acetate induced rats (Rattus norvegicus) spermatozoa

This study aims to determine the effect of the administration of white guava (Psidium guajava L.) fruit juice on spermatozoa plasma membrane integrity (PMI), morphological abnormality, viability, and motility of lead acetate induced rats (Rattus norvegicus). Twenty-five male rats were divided into five groups: NC (negative control) group, rats were administered with distilled water twice daily at four-hour intervals; T0 (positive control) group, rats were administered daily with lead acetate 50 mg/kg bw and distilled water four hours later; T1, T2, and T3 groups, rats were administered daily with lead acetate 50 mg/kg bw and 0.5 mL of 25, 50, and 100% white guava fruit juice four hours later. The treatment of the rats was conducted for 14 days, and on day 15, all rats were sacrificed to assess the spermatozoa quality. Data was analyzed using ANOVA followed by Duncan's multiple range test at a confidence level of 95%. The results showed that exposure to lead acetate (T0) caused lower spermatozoa PMI, viability, and motility as well as higher spermatozoa morphological abnormalities (p &lt;0.05) compared to those of the T0 group. Administration of white guava fruit juice starting at a dose of 25% (T1) resulted in higher spermatozoa motility, viability, and PMI as well as lower spermatozoa morphological abnormalities (p &lt;0.05) compared to rats in the T0 group. It could be concluded that white guava fruit juice maintained the spermatozoa quality of lead acetate induced rats.

The cryoprotective effect of Guar gum co-supplemented with ethylene glycol and glycerol in Simmental bull semen

Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation.

Association between levels of insulin-like growth factor-1 in serum and seminal plasma with fresh and frozen-thawed semen characteristics in Beetal bucks.

The aim of this study was to investigate the relationship between the levels of insulin-like growth factor-1 (IGF-1) in serum and seminal plasma and the characteristics of semen in Beetal bucks (Capra hircus). A total of 12 adult Beetal bucks were involved in the study, with each buck providing six ejaculates collected using a standard artificial vagina (n = 72 total). Only qualified semen samples (volume of 0.7 mL, a mass motility rating of 3+ or higher on a 0-+ scale, and individual progressive motility of 80% or more) divided into three fractions were processed for estimation of IGF-1 and other seminal parameters like motility, viability, acrosome integrity, sperm abnormality and superoxide dismutase (SOD) activity. The first and second fraction were diluted and extended with Optixcell extender (1:15 ratio). The first ejaculate fraction was processed for studying fresh semen parameters and the second fraction was cryopreserved for evaluating frozen semen parameters. French mini straws (0.25 mL) were used for semen filling, and polyvinyl alcohol powder of different colours was used for sealing the extended semen. The third fraction of each ejaculate was centrifuged at room temperature (1100 × g for 7 min) to separate the seminal plasma. Additionally, blood samples were taken from each buck on the same day as semen collection, resulting in a total of 36 blood samples. The results revealed a significant positive correlation (r = .4243; p < .05) between the concentration of IGF-1 in both serum and seminal plasma of the Beetal bucks. Furthermore, the concentration of IGF-1 in serum showed significant positive correlations with sperm viability (r = .554; p < .05), acrosome integrity (r = .527; p < .05), post-thaw sperm motility (r = .407; p < .01), post-thaw sperm viability (r = .426; p < .01) and post-thaw acrosome integrity (r = .333; p < .05). However, it had a significant negative correlation with SOD activity in fresh semen (r = -0.458; p < .01). Moreover, the concentration of IGF-1 in seminal plasma demonstrated significant positive correlations with individual progressive motility (r = .341; p < .05), sperm viability (r = .527; p < .05), acrosome integrity (r = .539; p < .05), sperm plasma membrane integrity (r = .464; p < .05), post-thaw sperm motility (r = .644; p < .01), post-thaw sperm viability (r = .643; p < .01), post-thaw acrosome integrity (r = .487; p < .01) and post-thaw sperm plasma membrane integrity (r = .521; p < .01). Additionally, it showed a significant negative correlation with SOD activity in both fresh semen (r = -0.714; p < .01) and frozen semen (r = -0.558; p < .01) of Beetal bucks. Based on these findings, IGF-1 in seminal plasma can be considered as a potential biomarker for the selection of bucks for breeding purposes.

Effects of vitamins C and E in tris citric acid glucose extender on chilled semen quality of Kail ram during different storage times

Mammalian sperm cells are highly vulnerable to lipid peroxidation by free radicals. Antioxidants such as vitamin E, and vitamin C neutralize the activity of free radicals and protect the sperm from reactive oxygen species. The present study was conducted to investigate the effects of vitamin C, vitamin E, and their combination in a Tris-based extender on the semen quality of Kail Ram. Semen samples from five mature Kail rams were collected in this study. The semen samples were diluted by Tris-glucose-egg yolk. Diluted semen samples were divided into four parts. The first part was added with 1 mg/ml of vitamin C, the second part was added with 1 mg/ml of vitamin E and the third part was added with both vitamin C and E, in combination with a dose of 1 mg/ml. The fourth part was considered as control without any addition. The diluted semen samples were cooled gradually and preserved at 5˚C for three days. Sperms in chilled diluted semen samples were examined for motility, viability, and plasma membrane integrity every 24 h for three days (72 h). Present study results showed significant (P < 0.05) effects of vitamins C, E, and their combination on some parameters such as motility, straightness of average special path, linearity of the curvilinear trajectory, and beat-cross frequency. However, there was no significant (P < 0.05) effect of storage duration and antioxidants (vitamin C, E, and their combination) on viability, sperm plasma membrane integrity, and some CASA parameters. From the present study, it could be concluded that the supplementation of vitamins C, and E, and their combination do not enhance the life span and quality of semen in Kail ram during liquid storage at 5 °C.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P &lt; 0.001) and 3 h (P &lt; 0.0001), the optimum viability index (P &lt; 0.0001), SPMI (P &lt; 0.05), acrosome integrity (P &lt; 0.0001), non-fragmented DNA (P &lt; 0.01), DNA in the comet head (P &lt; 0.05), minimum DNA in the comet tail (P &lt; 0.05), shortest comet tail length (P &lt; 0.0001), and lowest comet tail moment (P &lt; 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Antioxidant Activity of Fermented Red Bean Extract on Sperm Quality of Mice Exposed to Cigarette Smoke

Smoking has a negative effect on fertilization as it decreases sperm quality. The current research aimed to investigate the effect of fermented red bean (Phaseolus vulgaris L.) extract on sperm motility, viability, and plasma membrane integrity of white mice (Mus musculus) exposed to cigarette smoke. The red beans were subjected to a 36-hour fermentation process using Rhizopus spp. Then, methanol was extracted by maceration method for 24 hours until maceration was obtained. In this study, 25 male white mice aged 3 months were randomly divided into 5 groups of 5 mice. Group C (negative control) was given 0.5 mL of carboxymethyl cellulose natrium (CMC Na) 1% solution orally without unfiltered cigarette smoke exposure, and group C+ (positive control) was given 0.5 mL of CMC Na 1% solution orally and unfiltered cigarette smoke exposure. Treatment groups T1, T2, and T3 were orally given fermented red bean extract at doses of 26 mg/kg Body weight (BW), 52 mg/kg BW, and 104 mg/kg BW, respectively, and then were exposed to unfiltered cigarette smoke. For 36 days, treatment groups (except the negative control) were subjected to the inhalation of smoke from an unfiltered cigarette containing a nicotine dose of 2.2 mg. The exposure period lasted for 20 minutes each day. Each group was put into a cigarette smoke-exposing box. The sperm motility (observing the forward movement of spermatozoa), the sperm viability (examining the color of the sperm head), and the sperm plasma membrane integrity (observing the tail shape using the hypoosmotic swelling test) were then evaluated. The findings indicated significant differences in sperm motility, viability, and plasma membrane integrity of each group with positive control. A dose of 104 mg/kg BW of fermented red bean extract had the best potential to maintain sperm motility (70%), viability (82.13%), and plasma membrane integrity (61.93%) of mice exposed to unfiltered cigarette smoke.