Lipid mixtures (from a liposome kit) and melatonin improve post-thawed Angora goat sperm parameters.

Semen freezing and storing has been widely used in reproductive biotechnology, being applied to certain males of livestock breeds or animal species with economic value such as the Angora goat. The development of a semen extender with the cryoprotective agents can prevent the deterioration of sperm parameters after thawing. This study aimed to investigate lipid mixtures (from a liposome kit, Lps) and melatonin (Mel) at different doses to prevent the deterioration of sperm parameters and to provide the cryoprotective effects on sperm DNA. The Angora goat ejaculates were collected and pooled. They were divided into seven equal volumes, and each of them was diluted with the extenders of the experimental groups with additives (Lps 321.99 µg/mL, Lps 841.33 µg/mL, Mel 0.25 mM, Mel 1 mM, Lps 321.99 µg/mL + Mel 1 mM, Lps 841.33 µg/mL + Mel 0.25 mM) and no additives (control group). After the freeze-thawing process, motility, viability, acrosome integrity, DNA double-strand breaks, and abnormal DNA integrity were assessed for different extender groups. It was determined that the use of Lps alone at low dose or the combination of Lps and Mel had significant cryoprotective effects on motility, viability, acrosome integrity, and DNA damage in Angora goat sperm. This study will help us to understand the effects of Lps and Mel used alone or in combination at different doses and which doses give the optimum spermatological parameter rates following the freeze-thawing process, and hence it will shed light on further studies.

Quantitative proteomics of sperm tail in asthenozoospermic patients: exploring the molecular pathways affecting sperm motility.

Asthenozoospermia, characterized by low sperm motility, is one of the most common causes of male infertility. While many intrinsic and extrinsic factors are involved in the etiology of asthenozoospermia, the molecular basis of this condition remains unclear. Since sperm motility results from a complex flagellar structure, an in-depth proteomic analysis of the sperm tail can uncover mechanisms underlying asthenozoospermia. This study quantified the proteomic profile of 40 asthenozoospermic sperm tails and 40 controls using TMT-LC-MS/MS. Overall, 2140 proteins were identified and quantified where 156 proteins have not been described earlier in sperm tail. There were 409 differentially expressed proteins (250 upregulated and 159 downregulated) in asthenozoospermia which by far is the highest number reported earlier. Further, bioinformatics analysis revealed several biological processes, including mitochondrial-related energy production, oxidative phosphorylation (OXPHOS), citric acid cycle (CAC), cytoskeleton, stress response, and protein metabolism altered in asthenozoospermic sperm tail samples. Collectively, our findings reveal the importance of mitochondrial energy production and induced stress response as potential mechanisms involved in the loss of sperm motility in asthenozoospermia.

The investigation of the effects of vitamin A, vitamin E, and ß-carotene plus vitamin E on some fertility parameters in ewes.

This study was aimed at investigating the effects of vitamin A (VITA), vitamin E (VITE), and combined ß-carotene plus vitamin E (ßCAR+VITE) injections on some fertility parameters in ewes. Estrus synchronization was performed by treating the ewes with intravaginal FGA sponges impregnated with 30 mg of fluorogestone acetate. On the days of the insertion and withdrawal of the intravaginal sponges, groups VITA, VITE, and ßCAR+VITE were administered with 500 000 IU of vitamin A, 50 mg of vitamin E, and a combination of ß-carotene plus vitamin E, respectively. The ewes in the control group (C) were maintained for control purposes. Statistically significant differences were determined between groups VITA and ßCAR+VITE, groups VITE and ßCAR+VITE, and groups C and ßCAR+VITE, as well as groups VITE and C, groups VITA and C for the multiple birth rates. While significant differences were determined between groups VITA and C, groups VITE and C, and groups ßCAR+VITE and C for the lambing rates, it was ascertained that the ratio of newborn lambs to delivered ewes (litter size) significantly differed between groups VITA and ßCAR+VITE, groups VITA and C, groups VITE and ßCAR+VITE, groups VITE and C, and groups ßCAR+VITE and C. The highest MDA level and lowest GSH level were determined on day 20 after mating in the control group. In conclusion, it is suggested that both multiple birth rates and litter size can be increased by the combined administration of ß-carotene and vitamin E.

The Effects of Different Doses of ROCK Inhibitor, Antifreeze Protein III, and Boron Added to Semen Extender on Semen Freezeability of Ankara Bucks.

In the presented study, the effects of ROCK inhibitor Y-27632, antifreeze protein III, and boron at two different doses were investigated on the spermatological parameters of Ankara buck semen after freeze−thawing. Ejaculates were collected from bucks using an electroejaculator during the breeding season. The ejaculates that showed appropriate characteristics were pooled and used in the dilution and freezing of semen. The extender groups were formed by adding two different doses of three different additives (ROCK inhibitor Y-27632, 5 and 20 µM; antifreeze protein III, 1 and 4 µg/mL; boron, 0.25 and 1 mM) to the control extender. The semen was diluted with the different extenders at 35−37 °C and loaded into straws. Sperm samples frozen in liquid nitrogen vapors, following equilibration, were stored in liquid nitrogen. It was observed that extender supplementation improved post-thaw motility of Ankara buck semen after freeze−thawing. Differences were significant (p < 0.01) for 5 and 10 µM doses of ROCK inhibitor (71.82% and 74.04 % motility), as well as for 0.25 and 1 mM doses of boron (76.36% and 72.08% motility), compared to the control group (66.15% motility). With respect to the evaluation of acrosomal integrity and mitochondrial activity after freeze−thawing, although supplementation provided protection at all doses, the efficacy was not statistically significant (p > 0.05). It was observed that DNA damage was improved by antifreeze protein III at 1 µg/mL (1.23% ± 0.23%) and by boron at all doses (0.25 mM: 1.83% and 1 mM: 1.18%) compared to the control group (3.37%) (p < 0.01), following the thawing process. In the present study, it was determined that some additives added to the extender provided significant improvements in buck spermatozoa motility and DNA damage after thawing.

The effects of varying concentrations of glutathione and trehalose in improving microscopic and oxidative stress parameters in Turkey semen during liquid storage at 5 °C.

Since turkey reproduction is mainly through artificial insemination, short-term preservation of turkey semen is one of the most important issues in turkey reproduction management. The present study investigates the effects of glutathione (GSH) and trehalose on lipid peroxidation degree and turkey semen quality while being stored at 5 °C for 72 h. To this end, semen samples were collected from 20 turkeys with a weekly frequency for 12 weeks. A glucose-based extender was used to dilute the pooled semen. It was divided into seven equal parts with varying levels of glutathione [0.5, 1 and 2 mM), trehalose [50, 75 and 100] and control [extender without antioxidant]. Subsequently, the divided semen samples were stored at 5 °C for 72 h. Several sperm parameters such as motility and motion parameters, plasma membrane integrity (PMI), plasma membrane functionality, DNA integrity, and oxidative parameters were assessed following storage for 0, 24, 48, and 72 h. The obtained results indicated an improvement in the plasma membrane functionality and DNA integrity, along with the percentages of PMI in GSH-2 mM group in comparison to the control group following storage at 5 °C for 72 h (P ≤ 0.05). It is also notable that the 2 and 1 mM concentrations of GSH increased the spermatozoa motility and motion parameters in comparison to the control group, respectively (P ≤ 0.05). The study results indicated that GSH-2, 1 mM and trehalose- 100 mM concentrations reduced lipid peroxidase levels and increased total antioxidant activity, catalase, superoxide dismutase, and glutathione peroxidase in comparison to the control group (P ≤ 0.05). Our study's data show that improvement of semen parameters and oxidative stress parameters of turkey semen can be improved by glutathione at 2 and 1 mM and trehalose at 75 mM while storing it 5 °C.

Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen.

Supplementing the extender with antioxidants with low molecular weight can enhance the quality of the post-thaw sperm during the freezing process. This study was aimed at determining the impacts of 3,4-dihydroxyphenyl glycol (DHPG) on the spermatozoa of the canine undergoing freeze-thawing process. In this study, 24 ejaculates were obtained from three mixed-breed dogs and were diluted in a Tris-based extender. The diluted semen was divided into aliquots for supplementation of 10, 30, 50 and 70 µg/ml of DHPG, control (without antioxidant) and control sham (DMSO). After being extended, the semen was equilibrated at a temperature of 4°C and then transferred to the straws and kept 4 cm above the liquid nitrogen for 20 min and was finally immersed in the liquid nitrogen. They were cryopreserved for seven days; then, sperm parameters including sperm motility evaluation, motility characteristics, viability, DNA and plasma membrane integrity, total antioxidant capacity (TAC), reduced glutathione content (GSH), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]) activity malondialdehyde (MDA) levels were evaluated. This study showed that spermatozoa cryopreservation with 50, 30 and 70 µg/ml of DHPG concentrations had better progressive motility, Curvilinear Velocity, Linearity, viability, intact plasma membrane and the levels of TAC, GPx and GSH were higher than the control group. The 50, 30 and 70 µg/ml of DHPG concentrations led to the significant decrease of DNA damage compared to the control group. Total motility, average path velocity, straight-line velocity and CAT activity were significantly improved in 30 and 50 µg/ml of DHPG concentrations, compared to the control group. Also, the 50 and 30 µg/ml of DHPG concentrations, decreased MDA levels compared to the other groups, significantly. In conclusion, our study showed that the addition 50 µg/ml of DHPG to the canine semen extender improved the semen characteristics and oxidative markers in the cryopreservation process.

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process.

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 µM, Caffeic acid 100 µM, and Caffeic acid 200 µM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 µM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 µM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 µM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 µM Caffeic acid enhances the semen quality of water buffalo after thawing.

Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses.

This study aimed to evaluate the comparative effects of taxifolin hydrate and trehalose on the quality of frozen-thawed ram spermatozoa for the first time. Ejaculates collected from six mature rams were pooled, and divided to eight equal aliquots to extend them with different concentrations of glycerol (%5 and %3), taxifolin hydrate (10, 100, and 500 µM), and trehalose (60 mM) as eight groups (G5T0, G5T10, G5T100, G5T500, G3T0, G3T10, G3T100, and G3T500). After freeze-thawing process of cryopreservation, microscopic and oxidative stress parameters, and gene expression levels were investigated for understanding of possible impacts of taxifolin hydrate and trehalose. The study showed that G3T10 resulted in the highest post-thawed viability and mitochondrial activity. Moreover, all extenders with taxifolin hydrate reduced DNA fragmentation in comparison to G5T0, but DNA damage was prevented at the highest rate in presence of G5T10. The level of LPO significantly decreased in the groups G5T500 and G3T100, and the expression levels of NQO1, GCLC, and GSTP1 genes significantly increased in the groups G5T100, G5T500, G3T10, and G3T100 compared to the group G5T0. Finally, co-supplementation of tris-based extender having 3% glycerol with 60 mM trehalose and 10 µM taxifolin hydrate in cryopreservation extender may be recommended to improve the quality of post-thawed ram spermatozoa. However, further in vivo and in vitro studies are suggested to evaluate fertility rates of frozen-thawed ram spermatozoa co-supplemented with trehalose and taxifolin hydrate.

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters.

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05). The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05). In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa.

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.

The protease inhibitor antipain has a beneficial synergistic effect with trehalose for ram semen cryopreservation.

The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0  Tre0 : antipain 0 µM and trehalose 0 mM (Control); A10  Tre0 ; A50  Tre0 ; A0  Tre30 ; A0  Tre60 ; A10  Tre60 ; A10  Tre30 ; A50  Tre30 and A50  Tre60 ). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10  Tre0 , A0  Tre30 and A10  Tre60 significantly (p < 0.05) higher as compared to the control (A10  Tre0 ). A10  Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10  Tre0 , A50  Tre0 , A0  Tre30 and A10  Tre60 , compared to control. Addition of A10  Tre0 , A50  Tre0 and A10  Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10  Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10  Tre0 , A0  Tre60 and A10  Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50  Tre60 group. In conclusion, when combination of antipain (10 µM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.

Effects of arginine and trehalose on post-thawed bovine sperm quality.

The present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 °C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 ± 8.21%), CASA motility (12.2 ± 5.69%) and progressive motility (3.52 ± 2.13%), compared with the controls (43 ± 2.73%, 55.4 ± 6.78% and 33.48 ± 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 ± 3.99% and 44.1 ± 2.18%) compared with the control (13 ± 8.15 and 31.7 ± 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.

Influence of fetuin and hyaluronan on the post-thaw quality and fertilizing ability of Holstein bull semen.

It was determined that fetuin and hyaluronan supplementation did not provide any significant effect on the post-thaw subjective and CASA motility percentages and sperm motion characteristics, in comparison to the controls (P>0.05). Sperm acrosome and total abnormalities were similar in all groups (P>0.05). Groups M (hyaluronan+fetuin) and H (hyaluronan) displayed a higher rate of sperm membrane integrity, compared to that of Group C (control) (P<0.01). According to the results of the comet assay, the lowest percentage of sperm with damaged DNA was achieved in Group H, when compared to all of the experimental groups (P<0.01). Furthermore, all of the additives resulted in a lower rate of sperm with damaged DNA than that of the controls, and thus, reduced DNA damage (P<0.01). For pregnancy rates, there were no significant differences between the extender groups (P>0.05). MDA formation was found to be lower in Groups M and F (P<0.01). In Group M, SOD activity was determined to have significantly increased (23.61±5.62 U/ml) compared to the other groups (P<0.01). All experimental groups had a GSH-Px activity higher than that of the control group (P<0.01).

Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5°C.

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5°C. Thirty-two Merino rams (12-14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 µg/day of total AF. The EG group received commercial feed plus 2g/day of EG. The AF + EG group was given commercial feed plus 250 µg/day of total AF and 2g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5°C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5°C at 0, 24, 48, 72 and 96 h of liquid storage. During the first two weeks of the trial, the groups did not statistically differ from each other for sperm motility or rates of abnormal and nonviable spermatozoa at 0, 24, 48 and 96 h of storage. As from the third week, the short-term storage of semen produced statistically significant differences between the AF group and the other treatment groups for sperm parameters (p<0.05). The administration of aflatoxin was observed to have reduced sperm motility and to have increased the rates of abnormal and nonviable spermatozoa in comparison to the control group (p<0.05), while EG co-administered with AF was determined to have ameliorated the adverse effects of AF on sperm parameters, and this ameliorative effect continued throughout the short-term storage of semen. On the other hand, aflatoxin administration resulted in the deterioration of the sperm parameters in the following weeks, and the combined administration of EG + AF reversed this adverse effect, thus, bringing the sperm parameters closer to the values of the control group. This study demonstrated that, in rams, AF adversely affected sperm, biochemical and testis parameters, and that the combined administration of EG and AF reversibly improved these adverse effects.

Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability.

Supplementation of the semen extender with antioxidants did not produce any significant effect on CASA and progressive motilities and sperm motility characteristics, in comparison to the control group (P > 0.05). For sperm acrosome and total abnormalities, TCM-199 supplemented with cysteine (2.60 ± 0.24% and 4.80 ± 0.20%), glutamine (2.80 ± 0.20% and 6.40 ± 0.40%), carnitine (2.60 ± 0.24% and 6.00 ± 0.63%) and methionine (3.40 ± 0.51% and 9.20 ± 0.86%) at doses of 2 mM provided a better protective effect, compared to that of the controls (8.00 ± 0.44 and 15.60 ± 1.895). As regards sperm membrane integrity, supplementation with 2 mM of glutamine and methionine (56.00 ± 1.70% and 62.40 ± 1.78%, respectively) resulted in higher rates, when compared to the control group (41.40 ± 4.74%). According to the results of the COMET assay, only the use of TCM-199 supplemented with 2 mM of cysteine reduced DNA damage and resulted in percentages of sperm with damaged DNA (2.17 ± 0.18%) lower than those of the control group (3.16 ± 0.32%) (P < 0.001). For pregnancy rates, there were no significant differences among the extender groups (P > 0.05).

Apoptotic-Related MiRNAs Correlated with Functional and Flow Cytometric Parameters in Asthenozoospermic Holstein Bulls After Freeze-Thaw Process.

Many cellular processes in spermatozoa, including apoptosis and motility, are regulated by miRNA. Different miRNAs and molecular pathways are involved in asthenozoospermia (AS) conditions, which are thought to be one of the causes of infertility with reduced sperm motility. Thirty-two semen samples from four Holstein bulls with normozoospermia (NS), total motility ≥ 70%, and progressive motility ≥ 60%, and 32 semen samples from four bulls with AS, total motility ≤ 40%, and progressive motility ≤ 32% were used to investigate the function of apoptosis-related miRNAs in the AS group. Samples were then aspirated into a 0.5 mL straw after dilution with a Tris-egg yolk extender and frozen at -196°C. After freezing, semen samples were thawed for 2 weeks at 37°C and sperm kinematic parameters, plasma membrane integrity, acrosome integrity, DNA fragmentation, apoptosis status, and expression of apoptosis-related miRNAs (miR-2114, miR-296-3p, miR-455-3p, and miR345-3p) were evaluated. Our results showed that the functional and flow cytometric parameters of the NS group were significantly better than those of the AS group. In the NS group, miR-455-3pp and miR-2412 were upregulated, while miR-345-3p was downregulated compared with the AS group. In the AS group, miR-296-39, miR-2412, and miR-345-3p levels were strongly correlated with membrane integrity, DNA fragmentation, and apoptosis status. The findings demonstrated that the selected miRNAs based on bioinformatic analysis in AS and NS samples had a substantial association with functional and flow cytometry indicators and may be involved in regulating apoptosis and motility in AS samples.

Effects of different concentrations of BHT on microscopic and oxidative parameters of Mahabadi goat semen following the freeze-thaw process.

Oxidative damage to sperm is one of the main causes for decline in motility and fertility of frozen-thawed sperm. Thus, it is crucial to use cryoprotectant agents in extender in order to prevent lethal intracellular ice crystal formation. The present study aims to evaluate the effects of different concentrations of the antioxidant butylated hyroxytoluene (BHT) on sperm parameters post-thaw. Semen was diluted into five equal aliquots of extender containing different concentrations of BHT (0, 0.5, 1, 2 and 4mM), aspirated into 0.25 mL straws, and equilibrated at 5°C for 2h. After equilibration, straws were frozen in liquid nitrogen vapor and plunged into liquid nitrogen for storage. Sperm parameters, including motility and progressive motility, viability, membrane integrity, acrosome integrity and capacitation status, were assessed. Malondialdehiyde (MDA) and glutathione peroxidase (GSH-PX) activity were also evaluated after freezing-thawing. Results of this experiment show that addition of 1mM of BHT to the extender for freezing of goat semen can improve motility, progressive motility and viability (P<0.05) and reduce the MDA level (P<0.01). HOST (hypo-osmotic swelling test), acrosome integrity, capacitation status and GSH-PX were not affected by the concentrations of BHT (P>0.05). Therefore, we conclude that the optimum concentration of BHT for cryopreservation of goat semen is 1mM.

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters.

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

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.

Microfluidics: The future of sperm selection in assisted reproduction.

BACKGROUND: Obtaining functional sperm cells is the first step to treat infertility. With the ever-increasing trend in male infertility, clinicians require access to effective solutions that are able to single out the most viable spermatozoa, which would max out the chance for a successful pregnancy. The new generation techniques for sperm selection involve microfluidics, which offers laminar flow and low Reynolds number within the platforms can provide unprecedented opportunities for sperm selection. Previous studies showed that microfluidic platforms can provide a novel approach to this challenge and since then researchers across the globe have attacked this problem from multiple angles. OBJECTIVE: In this review, we seek to provide a much-needed bridge between the technical and medical aspects of microfluidic sperm selection. Here, we provide an up-to-date list on microfluidic sperm selection procedures and its application in assisted reproductive technology laboratories. SEARCH METHOD: A literature search was performed in Web of Science, PubMed, and Scopus to select papers reporting microfluidic sperm selection using the keywords: microfluidic sperm selection, self-motility, non-motile sperm selection, boundary following, rheotaxis, chemotaxis, and thermotaxis. Papers published before March 31, 2023 were selected. OUTCOMES: Our results show that most studies have used motility-based properties for sperm selection. However, microfluidic platforms are ripe for making use of other properties such as chemotaxis and especially rheotaxis. We have identified that low throughput is one of the major hurdles to current microfluidic sperm selection chips, which can be solved via parallelization. CONCLUSION: Future work needs to be performed on numerical simulation of the microfluidics chip prior to fabrication as well as relevant clinical assessment after the selection procedure. This would require a close collaboration and understanding among engineers, biologists, and medical professionals. It is interesting that in spite of two decades of microfluidics sperm selection, numerical simulation and clinical studies are lagging behind. It is expected that microfluidic sperm selection platforms will play a major role in the development of fully integrated start-to-finish assisted reproductive technology systems.