Hypo-osmotic Swelling Test Research Articles

Effect of Aqueous extract of Moringa Olivera leaves( AEMOL) on some characteristics of ram epididymal sperms under cooling conditions

The current study was conducted in the Graduate Studies Laboratory at the College of Agriculture, Al-Muthanna University, for the period from December 3, 2022 to March 9, 2023, to determine the effect of an aqueous extract of Moringa leaves on some characteristics of epididymal sperm of rams under cooling conditions for different periods. The aqueous extract of Moringa leaves was prepared and stored in the refrigerator ( 5 °C) until the time of use, the testicles were transferred to the laboratory less than an hour after the slaughter of the animal, the caudal epididymis was diluted with a Tris extender containing 0.03 concentration of aqueous extract of moringa olivera leaves.The results of this study showed a significant increase (P<0.01) in the percentage of live sperm, individual motility, and the plasma membrane integrity, while the adding of AEMOL leads to a decreasing of the percentage of abnormal sperms. Increasing the cooling hours of sperm leads to a significant decrease in the percentage of live sperm, individual motility, testing the integrity of the plasma membrane with an increase in the percentage of abnormal sperm. Using the aqueous extract of Moringa leaves at different times led to a significant effect on the individual motility, percentage of the plasma membrane integrity represented by the hypo-osmotic swelling test (HOST) , While the treatment did not significantly affect the percentage of live sperm and abnormal sperm. According to the results of this study we conclude that the aqueous extract of Moringa Oleifera leaves improves the characteristics of rams epididymal sperms preserved under cooling conditions for different periods

Effect of Aqueous Extract of Dill leaves (AEDL) on some Traits of Ram Epididymal Sperms under Cooling Conditions

The current study was conducted in the Graduate Studies Laboratory of the College of Agriculture, Al-Muthanna University, for the period from December 3, 2022 to March 9, 2023, to determine the effect of an aqueous extract of dill leaves ( AEDL)on some properties of epididymal sperms of rams under cooling conditions of 5 °C for different periods. The aqueous extract of dill leaves was prepared and kept in the refrigerator until the time of use. The epididymal sperms were obtained from the testis of Rams that slaughters in the local slaughterhouse of Al-Muthanna Governorate, the testicles were transferred to the laboratory less than an hour after the slaughter of the animal, the caudal epididymis was diluted with a Tris diluent containing 3% concentration of aqueous extract of dill leaves. The results of using the aqueous extract of dill leaves showed a significant increase (P<0.01) in the percentage of live sperm, Individual motility, and a decrease in the percentage of abnormal sperms. While the use of the extract did affect the percentage of the integrity of acrosome represented by the hypo osmotic swelling test (HOST) at the 0 time of cooling preservation, The Increasing of the time of sperm cooling.leads to a decreases in the percentage of live sperm, HOST, individual motility, with an increase in the percentage of abnormal sperms. The use of the aqueous extract of dill leaves at different times resulted in a significant effect(P< 0.01) on the percentage of live sperm, individual motility, while it did not significantly affect the percentage of abnormal sperm, and there were no significant differences in the percentage of HOST. according to the results of this study We can conclude to adding the aqueous extract of dill leaves to Improve the physical traits of rams epididymal sperm include the live sperm and individual motility under cooling conditions for different periods.

Assessment of Expression Levels and Localization Patterns of Phospholipase C zeta in Different Grades of HOST in Human Sperm.

Phospholipase C zeta (PLC-ζ) deficiency in sperm can underlie oocyte activation failure after intracytoplasmic sperm injection (ICSI). The aim of this study was to determine PLC-ζ expression and location in individual spermatozoa in each host score so that a hypo-osmotic swelling test (HOST) may be used to help routine sperm selection for ICSI. In this experimental study, fresh semen samples were randomly obtained from 30 men who were referred to the Andrology Unit of the Infertility Center. Samples were processed by density gradient centrifugation (DGC) and exposed to hypotonic conditions. Seven different tail patterns, classified from 'a' to 'g' can be detected according to World Health Organization (WHO) criteria. Then, the PLC-ζ protein localization pattern was assessed by quantitative Immunofluorescence in individual sperm Host grades. Moreover, the sperm content of PLC-ζ protein was evaluated by flow cytometry correlated with semen analysis parameters. In the present study, quantitive immunofluorescence analysis indicated that sperm from different host grades exhibited seven localization patterns of PLC-ζ of acrosomal (A); equatorial (EQ), and postacrosomal (PA) patterns. A+EQ=acrosomal and equatorial, A+PA=acrosomal and post-acrosomal, EQ+PA=equatorial and post-crosomal, and A+EQ+PA. The sperm from HOST grade 'd' exhibited significantly higher PLC-ζ (A+PA) and (A+EQ+PA) staining compared to sperm from other grades (P=0.006). The sperm from grade 'd' exhibited higher PLC-ζ (EQ+PA) compared with other grades (P=0.001). However, grade 'd' was not significantly different from 'c' (P=0.087). Analysis of the combined results confirmed that there was a clear reduction in PLC-ζ immunofluorescence in Host grades 'a', 'f' and 'g' sperms. Our data suggest that HOST may represent a useful diagnostic tool for the selection of sperms exhibiting a higher level of PLC-ζ expression.

Discriminant Analysis and Data Mining CHAID Decision Tree as Tools to Evaluate the Buffering Effect of Hydroxytyrosol on Reactive Oxygen Species in Rooster Sperm Cryopreservation.

Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.

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.

Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway.

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A2 (iPLA2) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA2 activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA2 activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates.

Low-density lipoproteins, resveratrol and quercetin as alternative additives to improve boar semen cooling.

Low-density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer-assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC-1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight-line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high-ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period.

Correlation between fatty acids levels in chicken, duck, goose, pigeon, quail and turkey egg yolks and post-thawed quality of ram semen.

The comparison between adding egg yolks (EY) of chicken, duck, goose, pigeon, Japanese quail or turkey to the Tris glycerol extender on the quality of ram semen before freezing and post-thawing was evaluated. The correlation between fatty acids levels in egg yolks of different avian species and the post-thawed quality of ram semen was studied. The pooled ejaculates collected from five rams were extended with Tris EY glycerol extenders containing the EY of chicken, duck, goose, pigeon, quail or turkey and cryopreserved at -196°C. The straws were evaluated before freezing and post-thawing for sperm motility using a sperm cell analyser, vitality using a FluoVit kit and abnormality using a SpermBlue stain besides plasma-membrane and DNA integrities using a hypo-osmotic swelling test and a Halomax kit, respectively. The moisture, ash, protein and fatty acid (FA) contents of EY of chicken, duck, goose, pigeon, quail and turkey were analysed using a gas chromatograph. The chicken and quail EY extenders significantly improved the total progressive motility (32.05 ± 1.41 and 31.68 ± 1.43, respectively), vitality, plasma membrane and DNA integrities and abnormalities of post-thawing ram semen in comparison with other EY extenders. Pigeon EY had the lowest saturated fatty acids (SFAs) in comparison with the other types of EYs. The chicken and turkey EYs had the lowest percentage of (monounsaturated fatty acids) MUFAs in comparison with the other types of EYs. The highest percentage of polyunsaturated fatty acids (PUFAs) was observed in the turkey, pigeon and chicken EYs which were considered double or triple their percentage in duck and goose EYs, respectively. Significant positive correlations existed between SFAs levels and total motility, vitality, plasma membrane functionality and DNA integrity (0.77, 0.80, 0.67, 0.52, respectively). Significant negative correlations existed between gondoic EY levels and total motility, vitality, plasma membrane functionality and DNA integrity. In conclusion, the EYs of duck, goose, pigeon or turkey cannot substitute the chicken EY in ram semen extenders as they gave lower post-thawing quality. The quail EY can be used as a good replacer for chicken EY in the extender used for cryopreservation of ram semen. The EY composition of FAs can significantly affect the quality of ram semen post-thawing.

Mobile Phones Reduce the Penetration Ability of Human Spermatozoa And Increase the Percentage of Swollen Sperm in the Seminal Fluid of Humans.

Generally, people hold mobile phones in the pocket of their pants, especially near the testes, and for that reason, the possible effects of mobile on reproductive health have become a matter of serious concern. The biological responses of such exposure caused various adverse effects on almost all biological systems. So, the present research has been proposed to study the biological responses of different duration of mobile phone usage on certain sperm functional parameters like penetration ability of spermatozoa and percentage of swollen sperm through an artificial medium (polyacrylamide) and Hypo Osmotic Swelling Test, respectively. For this, semen was collected from 65 men who used a mobile phone (SAR value 2.0 W/Kg averaged over 10gram tissue) at least 3 hours a week and kept mobile in their waist pocket. Semen was also collected from 22 men who never used mobile phones and were regarded as control. Their semen is analyzed according to the method given in the WHO laboratory manual for examining human semen, 5th edition, (2010). The results showed a highly significant (p&lt;0.001) decreased spermatozoa penetration ability and a highly significant increased (p&lt;0.001) level of percentage swollen spermatozoa in hypoosmotic swelling test (HOST) in human subjects using mobile phones in the last 01-05 years, 06-09 years and more than 10 years of duration in comparison to non-users human subjects. Evidence from current studies suggests the potentially harmful effects of mobile phones on certain sperm functional parameters and makes the seminal fluid infertile. A further standardized study is needed to assess the risk of mobile phone use on the reproductive system.

Comparison of the effects of sperm selection methods on assisted reproduction outcomes in male infertility

Objective: To compare physiological intracytoplasmic sperm injection (PICSI) and the hypoosmotic swelling test (HOST) sperm selection techniques in terms of embryo development, implantation rate and live birth rates in cases of severe or moderate oligoastheneospermia. Materials and Methods: The electronic material and files of cases admitted to the Medipol University IVF Centre between 2013 and 2022 were analyzed retrospectively. This research included a total of 143 cases with moderate or severe oligoasthenospermia, 80 cases with PICSI as sperm selection technique and 63 cases with HOST as sperm selection technique. Cases of severe or moderate oligoasthenospermia as defined by the World Health Organisation (WHO) are included in this study. The demographic parameters of both groups, including female age, paternal age, duration of infertility, AMH (anti-Mullerian hormone), body mass index (BMI), endometrial thickness, and the number of prior attempts, were analyzed. Implantation, pregnancy loss, and rates of live births were compared between two groups. Results: Female age, paternal age, AMH, endometrial thickness, BMI, and the number of previous trials were similar between the groups included in the study. There were not any significant differences between the groups in terms of fertilization (.18±6.92 vs 9.11±6.35, p=0.345), TQ-GQ blastocyst development (2.36±2.40 vs 1.64±2.35, p=0.097), and the number of embryos transferred (1.59±0.50 -1.76±0.44 p= 0.141). The live birth rates between the PICSI and HOST groups did not vary statistically (p=0.790), however, the rate of implantation was significantly higher in the HOST group (p=0.043). Sperm selection by PICSI or HOST method did not affect fertilization, TQ-GQ blastocyst development, and the rates of pregnancy loss and live birth. Conclusion: Although there were not any differences in embryo development, pregnancy loss, and live birth rates comparing the two methods, the HOST group had a higher implantation rate. Keywords: PICSI, HOST, Male infertlity, live birth

Hypo-Osmotic Swelling Test and Male Factor

For over 30 years, defects of the functional integrity of the sperm membrane, as evidenced by a low hypo-osmotic swelling test when evaluating the semen analysis, are not only associated with male infertility (even with sperm that otherwise seem normal), but unless corrected, successful intrauterine pregnancies will rarely ensue. This defect, interestingly, does not impair fertilization of the oocyte, but instead, prevents a normal-appearing embryo from successfully implanting. The frequency in infertile couples increases with advancing age of the male, ranging from 5% in younger males to 25% in men in their late forties or early fifties. It seems to be related to a toxic protein added to the sperm as they traverse the ejaculatory ducts. The defect is very correctable, either by treating the sperm with the protein digestive enzyme chymotrypsin prior to intrauterine insemination and avoidance of unprotected sex prior to ovulation, or in vitro fertilization with intracytoplasmic sperm injection. Unfortunately, this very inexpensive, easy-to-perform test is rarely performed by the large majority of physicians treating infertility. The purpose of this manuscript is to hopefully rekindle interest within the infertility community to add this test to the standard semen analysis.

Severe subfertility in Thoroughbred stallions positive for the Impaired Acrosome Reaction susceptible gene (FKBP6) in Argentina

Subfertility in stallions is one of the major causes of reproductive and economic loss in the equine industry. Low pregnancy rates have been reported in stallions with normal semen quality after conventional evaluation. Acrosome reaction (AR) is necessary for fertilization and impaired acrosome reaction (IAR) leads to subfertility or infertility in horses; however, the AR test is not included in routine semen analysis. Failure of AR may affect more than 90% of sperm, although the remaining sperm can perform normal AR during fertilization. Up to date, IAR has been confirmed as a cause of subfertility only in Thoroughbred stallions. A genome-wide association study identified FKBP6 as a strong candidate gene responsible for this failure. The gene encodes for FK506 binding protein 6 (FKBP6) which is involved in sperm development and function. The objective of this study was to evaluate the presence of IAR susceptibility genes in stallions with satisfactory breeding soundness examination (BSE) and acceptable semen parameters but a history of severe subfertility. Thoroughbred stallions (A, B and C), between 6 and 9 years old with satisfactory BSE but with a history of low pregnancy rates during the 2020 (stallion A = 11%) and 2021 breeding seasons (stallion A= 0%; stallion B= 29%; stallion C= 10%) were included. They were referred after several consultations, andrological examinations and non-specific treatments by differentveterinarians. Ejaculates of stallions A, B and C in DSO had the following values of total number of spermatozoa/ejaculate (4.6 × 109, 8.5 × 109, 6.7 × 109); progressive motility (40%, 60%, 60%); hypo-osmotic swelling test (73%, 60%, 72%); and normal sperm morphology (58%, 68%, 76%), respectively. Evaluation of acrosome status by FITC-PSA showed 12%, 84% and 20% intact acrosomes for stallion A, B and C, respectively. Based on history and results of BSE, hair samples of the three stallions were sent to Molecular Cytogenetics Laboratory, Texas A&M University, and all three stallions, tested positive for the IAR susceptibility gene (FKBP6). The IAR is associated with male subfertility in humans and some domestic species including horses. A/A-A/A genotype of FKBP6 gene is considered a susceptibility gene for IAR in thoroughbred stallions with idiopathic subfertility. Hernandez Aviles et al. (Theriogenology. 2022; 186, 40-49) reported a low prevalence (0.7%) of acrosomal dysfunction in the overall population of breeding stallions but a significantly higher rate (38.1%) in idiopathically subfertile stallions. New developments in equine genomics provide tools for mapping fertility traits and discover causes of subfertility by genetic defects. Therefore, we must consider this analysis within the andrological examination in stallions with idiopathic infertility.

Reducing oxidative stress by κ-carrageenan and C60HyFn: The post-thaw quality and antioxidant status of Azari water buffalo bull semen

Azeri water buffalo is a species of great interest due to the high quality of its products such as milk. Due to the decreasing trend of its number and risk of extinction in the future, our attention is directed towards ensuring the preservation of its genetic reserves by keeping its sperm. Using antioxidants in semen extender is one of the ways to reduce the detrimental effects of freezing process on post-thawed quality of spermatozoa. This study was conducted to determine the effect of κ-carrageenan (k-CRG) and C60HyFn supplemented semen extender on the quality of post-thawed Azari water buffalo spermatozoa. A total of 30 semen samples were obtained from three buffaloes using an artificial vagina (twice a week for five weeks = 10 replicates). The samples (n = 3) from each replicate were pooled and divided into equal aliquots to prepare 14 extender groups, including control (C), k-0.2, K-0.4, K-0.6, K-0.8 (containing 0.2, 0.4, 0.8 mg K-CRG/mL, respectively), C-0.1, C-0.2, C-0.4, C-0.8, C-1, C-5, C-10, C-20, and C-40 (containing 0.1, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, 20, 40 μM C60HyFn, respectively), and then frozen. After thawing, motility and velocity parameters, plasma membrane integrity (PMI) and functionality (PMF), DNA damage, Hypo-osmotic swelling (HOS) test, malondialdehyde (MDA), total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase glutathione activities and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging were evaluated. In vivo fertility was compared between k-0.6, C-1 and control groups. 60 buffalo were inseminated 24 h after the onset of estrus. The diagnosis of pregnancy was performed rectally at least 60 days after fertilization. Total and progressive motility and velocity parameters were improved by k-0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups) compared to the other groups. Plasma membranes integrity and PMF were improved by k-0.4, k-0.6, C-0.4, C-0.8, C-1, C-5, and C-10 groups compared to other groups, while in terms of sperm DNA damage K-0.4, K-0.6, K-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups showed better results compared to the control group. The evidence also showed that k- 0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve TAC, and decrease MDA levels. Also, k-0.4, k-0.6, k-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve GPx, CAT, and GSH levels, but no significant difference was found regarding SOD compared to the other groups. DPPH scavengers were tested by K-0.6, K-0.8 and C-1, C-5, C-10, C-0.8, C-0.4 and C-0.2 groups and compared to other groups improved. The fertility rate [70% (14/20)] was higher in C-1 than other groups. To conclude that k-CRG and C60HyFn supplementation can increase the quality parameters of cryopreserved buffalo semen after thawing and that 1 M C60HyFn can increase in vivo fertility of buffalo semen.

Effect of different concentrations of green tea on Cryopreservation of German Shepherd dog

To investigate the Effect of adding different concentrations of green tea extract as an antioxidant on specific physical semen characteristics of German Shepherd dogs during various steps of frozen semen processing this study was carried out. A total (of 20) good Ejaculate semen samples were collected in a plastic bag from German Shepherd dogs aged 3-4 yearsIn different locations (dog breeding).To achieve the current study's main objectives, two experiments were designed through periods extended from the beginning of December 2021 until the end of May 2022. The investigations included studying the physical properties of raw Semen (ejaculate volume, semen color, consistency, motility percentage, progressive motility percentage, dead sperm percentage, sperm abnormalities percentage, and hypo-osmotic swelling test (HOST). After that, semen samples were diluted with Tris supplemented with antioxidants. The physical properties of Semen were followed after cooling and after freezing (post-thawing). In the experiment, ten ejaculates were collected. Dilution of Semen was performed with Tris supplemented with two T1, T2, and T3) were cooled slowly up to 5C and equilibrated, packed into 0.5 ml straws, and put in vapor nitrogen (-196C) for 5-10 minutes after that, stored for 48and 72 hr in liquid Nitrogen (-196C). Progressive motility percentage, dead sperm percentage, sperm abnormalities percentage, and hypo-osmotic swelling test (HOST) were evaluated after cooling and post-thawing (Submerging the straws in the water bath at 37C for 30 seconds). It can be concluded from the present study that adding green tea extract as an antioxidant to the semen extender improves the ability to freeze, especially in the treatment T3 (0.01 mg/5 ml) and the T2 (0.05 mg/5 ml). This additive can decrease the negative Effect of freezing in liquid Nitrogen compared to the control group.

TITLE OF THE ARTICLE: EXOINTERACTIVE AMELIORATION AGAINST METHOTREXATE INTOXICATION IN MOUSE SPERMATOZOA

Objective: To assess the synergistic potential using an exointeractive association between Murraya koenigii hydromethanolic leaf extract and the synthetic compound quercetin to offset methotrexate induced toxicity in mouse cauda epididymal spermatozoa. Methods: Healthy adult male albino mice were divided into eight groups (N= 6/group) and were treated with methotrexate intraperitoneally (10mg/kg body weight) at intervals of every 48 hours for 15 days, to combat this toxic effects of the drug, Murraya koenigii hydromethanolic leaf extract (250mg/kg body weight) and quercetin (15mg and 30mg) alone as well as in combination, were given by oral administration. Various sperm parameters such as sperm count, motility and viability followed by Hypo-osmotic Swelling (HOS) test, apoptosis test, Sperm Toroid Integrity (STI) and Sperm Chromatin Dispersion (SCD) assay. Statistical analysis used: Statistical analysis was carried out using Graphpad Prism Version 7 to verify levels of significance at the P &lt; 0.001 value in one-way ANOVA. Results: Methotrexate induced a substantial reduction in sperm count, motility and viability which induces apoptotic changes, also resulted in disruption of sperm toroid protamine-DNA binding, increased fragmentation of nuclear DNA by decreased sperm chromatin dispersion and low percentage of swollen sperm representing membrane integrity loss. whereas Murraya koenigii hydromethanolic leaf extract and quercetin proved effective in reversing the drug induced toxicity, especially with the 15 mg dose of quercetin along with the plant extract brought about significant amelioration than 30mg dose of quercetin due to its pro-oxidant effects. Conclusions: To reduce the toxic manifestations of methotrexate, this exointeractive approach proved efficient.

Establishment of methods to analyze the structural and functional integrity of the quail (Coturnix coturnix japonica) sperm plasma membrane

ABSTRACT 1. The objectives of this study were to establish the use of the fluorophores Hoechst 33342 and propidium iodide for the evaluation of sperm plasma membrane integrity and to identify an adequate hypoosmotic solution for the evaluation of sperm membrane functionality in quails. 2. Sperm samples were collected from the vas deferens of nine quails. After initial evaluation, the samples were subjected to a flash-frozen assay. Three treatments with the following proportions of fresh sperm and sperm subjected to flash freezing were prepared as follows: 100:0 (T100), 50:50 (T50), and 0:100 (T0). The hypoosmotic swelling test used distilled water (0 mOsm/l) and fructose solutions (50, 100, and 200 mOsm/l). 3. Immediately after recovery, the samples showed 75.6 ± 5.0% motility with vigour of 3.7 ± 0.3 and 96.1 ± 0.5% of the sperm appeared normal. The membrane integrity test showed 62.2 ± 5.2% intact sperm at T100, 29.0 ± 4.1% at T50 and 0.1 ± 0.1% at T0. Moreover, a greater number of reactive sperm (74.7 ± 6.7%) were observed when incubated in distilled water (0 mOsm/l) in comparison to other solutions (P < 0.05). 4. The association of fluorescent probes composed of Hoechst 33342 and propidium iodide provided an efficient assessment of the integrity of the plasmatic membrane of quail spermatozoa. However, the study identified that the hypoosmotic swelling test has little predictive value regarding sperm membrane functionality in this species.

Influence of trans-ferulic acid on the quality of ram semen upon cold preservation.

Due to lower antioxidant capacity and higher amounts of polyunsaturated fatty acids, ram spermatozoa are very susceptible during cooling process. The objective was to examine the effect of the trans-ferulic acid (t-FA) on the ram semen during liquid preservation. Semen samples were collected from the Qezel rams, pooled, and extended with the Tris-based diluent. Pooled samples enriched with different amounts of the t-FA (0, 2.5, 5, 10, and 25mM) and preserved at 4°C for 72h. Spermatozoa's kinematics, membrane functionality, and viability were assessed by CASA system, hypoosmotic swelling test, and eosin-nigrosin staining, respectively. Moreover, biochemical parameters were measured at 0, 24, 48, and 72h. Results showed that 5 and 10mM t-FA improved forward progressive motility (FPM) and curvilinear velocity compared to the other groups at 72h (p < 0.05). Samples treated with 25mM t-FA showed the lowest total motility, FPM, and viability at 24, 48, and 72h of storage (p < 0.05). Higher total antioxidant activity levels were observed in the 10mM t-FA-treated group compared to the negative control at 72h (p < 0.05). Treatment with 25mM t-FA increased malondialdehyde amounts and decreased superoxide dismutase activity compared to other groups at the final time assessment (p < 0.05). Nitrate-nitrite and lipid hydroperoxides values were not affected by treatment. The current study indicates the positive and negative influences of different concentrations of t-FA on the ram semen upon cold storage.

The Quality of Frozen Friesian Holstein Semen after Long-Term Storage

Semen cryopreservation is the long-term storage at very low temperatures in liquid nitrogen for future use. This study investigates the quality of frozen Friesian Holstein (FH) semen after long-term storage. Samples of FH semen stored for 25, 20, 15, 10, 5, and 1 years were collected from one of the national centers for artificial inseminations. Frozen semen was stored in containers with liquid nitrogen at -196 ᵒC in a room with a temperature of 20 ᵒC The variables used after thawing were sperm motility, viability, and abnormalities, as well as plasma membrane integrity (IPM) using computer-assisted sperm analysis (CASA), eosin-nigrosine staining, and hypoosmotic swelling (HOS) test, respectively. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. The result showed no significant differences in sperm viability, abnormalities, and IPM. Furthermore, sperm motility was >40%, consistent with the Indonesian standard for frozen bovine semen. CASA analysis showed that all variables of the motility pattern have no significant difference, except linearity (LIN). The Lin of sperm was lower in frozen semen after one and five years than after 20 and 25 years of storage. The overall quality of semen after 25, 20, 15, 10, 5, and 1 years of storage met the standard and was suitable for artificial insemination.

Effects of dietary supplementation of tomato pomace (Solanum lycopersicum L.) and L-Arg on reproductive performance of aged male broiler breeders

This study aimed to investigate the effects of different dietary supplementation of tomato pomace (TP) and L-arginine (L-Arg) supplementation on sperm characteristics, reproductive performance, and semen biochemical components of aged commercial male broiler breeders. Thirty Ross 308 male broiler breeders (58 wk old) were provided and assigned to 5 dietary treatment groups, including control (CON), 5% TP (TPS-5), 10% TP (TPS-10), 15% TP (TPS-15), and L-Arg supplemented (10% above the recommendation, LAS-10). The results indicated that the semen volume increased in the TPS-15 group compared to that of the LAS-10 (and CON on wk 9) throughout the study (P < 0.05). The sperm concentration significantly increased in TPS-10 and TPS-15 groups in comparison to the other experimental groups. On wk 5 and 7, the sperm viability increased in all TPS groups compared to the CON and LAS-10, while on wk 9, it only increased in the TPS-10 group in comparison to the LAS-10 group (P < 0.05). The hypo-osmotic swelling test decreased in the LAS-10 group compared to the other experimental groups on wk 5 and all TPS groups on wk 7 and 9 (P < 0.05). The sperm total motility and forward progressive motility decreased in the LAS-10 group compared to the other experimental groups (P < 0.05). In contrast, unprogressive motility and immotile sperms were increased in the LAS-10 group compared to the other experimental groups (P < 0.05). In addition, the sperm penetration and fertility rate increased in TPS-10 and TPS-15 groups in comparison to CON and LAS-10 groups (P < 0.05). However, hatchability was reduced in the LAS-10 group (P < 0.05). The semen adenosine triphosphate increased in TPS-10, TPS-15, and LAS-10 groups compared to the CON (P < 0.05). Finally, the semen TAC and superoxidase dismutase decreased in the LAS-10 group (P < 0.05), while the glutathione peroxidase increased in the TPS-15 group (P < 0.05). In conclusion, 15% dietary TPS is recommended to improve the reproductive performance of aged commercial male broiler breeders.

Tribulus terrestris aqueous extract supplementation effects on sperm characteristics and anti-oxidant status during chilled storage of canine semen.

The reduction of spermatozoa survival time is a major problem of canine chilled sperm for artificial insemination. The current study looks at the possible advantages of chilling canine sperm to 4.00 ˚C for three days using Tribulus terrestris aqueous extract (TTAE). Three mixed-breed dogs were utilized to extract 24 ejaculates, which were then diluted in a Tris-based extender. The ejaculates were then divided into five groups including 20.00, 40.00 and 50.00 µg mL-1 of TTAE, sham (distilled water devoid of TTAE) and control (without TTAE) groups. During the three days of experiment, several parameters were measured every 24 hr. It was noticed that after 48 and 72 hr of liquid storage, total and progressive motilities were greater in the group with the 40.00 µg mL-1 TTAE concentration than the control group. Compared to the control group, the group with the 40.00 µg mL-1 TTAE concentration exhibited superior motility and viability. The percentages obtained from the hypo-osmotic swelling test were much greater. In contrast to the control group, DNA integrity was poorer in the 40.00 µg mL-1 TTAE concentration. After 72 hr of storage, the group with 40.00 µg mL-1 TTAE concentration had lower malondialdehyde levels but considerably greater total anti-oxidant capacity, superoxide dismutase, glutathione peroxidase and catalase levels than the control groups. The current study found that supplementing the semen extender with 40.00 µg mL-1 TTAE improves semen parameters after 72 hr of storage at 4.00 ˚C, and therefore can improve fertilization efficiency.