Does fennel extract ameliorate oxidative stress frozen-thawed ram sperm?

The aim of this study was to evaluate the quality of ram semen after cryopreservation with different levels of fennel (Foeniculum vulgare) extract (0 (F0), 5 (F5), 10 (F10) and 15 (F15) mg/L) and sperm concentrations (200 (C200) and 400 (C400) × 106 sperm/mL) in a soy lecithin (SL)-based extender. Twenty ejaculates were collected from four ghezel rams and diluted with eight sperm concentrations/fennel combinations: F0C200, F5C200, F10C200, F15C200, F0C400, F5C400, F10C400 and F15C400. Sperm motility, abnormality, plasma membrane, viability, mitochondrial activity, lipid peroxidation (LPO), mitochondrial activity and apoptotic changes were evaluated after freeze-thawing process. It was observed that F10C400 significantly improved total and progressive motility, VSL, membrane integrity of post-thawed ram sperm. MDA level was lower in F5C200 and F10C400 compared to other treatments. The higher percentage of live sperm and the lower percentage of apoptotic sperm were obtained in F10C200 compared to F0C200, F5C200 F15C400, F0C400, F5C400 and F15C400. Extender F10C200 resulted in the highest mitochondria activity compared to the rest of the extenders except F10C400. We conclude that a combination of 10 mg/mL fennel (Foeniculum vulgare) extract and sperm concentration of 200 × 106 sperm/mL can improve the ram semen quality cryopreserved in a soybean lecithin based extender.

Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen.

The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P < 0.05) higher total motility (MOT) (73.78 ±â€¯2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 ±â€¯0.38, 16.78 ±â€¯0.38) in comparison with the control group (MOT: 58.48 ±â€¯2.92; PROG: 9.08 ±â€¯0.38). Sperm viability (Vi) was higher (P < 0.05) in n1t3 (74.62 ±â€¯1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 ±â€¯1.91, 63.73 ±â€¯1.91, respectively) compared to the control groups (Vi: 66.85 ±â€¯1.55; Mi: 53.18 ±â€¯1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 ±â€¯0.31). There was no significant difference for SOD and CAT values with the use of various extenders. In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen.

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l-1 pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l-1) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.

Effect of green tea (Camellia sinensis) extract and pre-freezing equilibration time on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender.

The aim of this study was to determine the effect of Camellia sinensis extract as antioxidant supplement and pre-freezing equilibration times in a soybean lecithin extender for freezing ram semen. In this study, a total of 20 ejaculates were collected from four Ghezel rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and Camellia sinensis extract (5, 10, and 15 mg/L) and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 4 h after equilibration. Sperm motility characteristics, membrane integrity, abnormal morphology, mitochondria activity, apoptotic status, MDA and antioxidant activities (GPx, SOD and total antioxidant capacity (TAC)) were evaluated following freeze-thawing. Camellia sinensis extract at level 10 mg/L led to the highest total and progressive motilities percentages, in comparison to other treatments (P < 0.05). Our results showed that Camellia sinensis extract at level of 5 and 10 mg/L led to higher plasma membrane integrity, mitochondria activity and Total antioxidant capacity (TAC) in comparison to the level of 15 mg/L and control group (P < 0.05). Camellia sinensis extract at 10 mg/L level produced the highest percentage of live spermatozoa and the lowest apoptotic spermatozoa in comparison to all treatments (P < 0.05). In addition, level of MDA formation significantly decreased at this concentration, 10 mg/L, compared to all treatments (P < 0.05). No differences (P > 0.05) were observed between equilibration times (0 h vs. 4 h) for sperm samples incubated with or without different concentrations of Camellia sinensis extract. In conclusion, addition of Camellia sinensis extract at level of 10 mg/L can improve post-thawing quality of ram semen cryopreserved in a soybean lecithin extender. However, further research is needed to standardize the process of Camellia sinensis extraction and specially for identifying which compounds are responsible of its beneficial effect on ram sperm cryopreservation.

Effects of dietary basil (Ocimum basilicum) supplementation on reproductive hormones, semen parameters, and testicular development in Zandi male lambs.

This study investigated the potential impact of feeding whole plant basil on sperm quality and the concentration of certain reproductive hormones in male lambs. A total of 18 Zandi male lambs with an initial weight of 28.8 ± 2.03 kg were included in a completely randomized design with three treatments and six repetitions. The experimental treatments included: 1) control (basal diet without basil), 2) diet containing 12.5 % basil, and 3) diet containing 25 % basil. The results showed that feeding basil to male lambs significantly increased testosterone concentration and decreased blood cortisol levels (P < 0.05). Additionally, feeding high levels of basil significantly improved sperm concentration, motility, and viability in the experimental samples, while reducing the level of complete abnormalities and malondialdehyde concentration (P < 0.05). The findings suggest that dietary supplementation of 25 % whole plant basil could be a useful strategy to improve sperm quality and increase testosterone secretion while reducing cortisol levels in male lambs.

Apigenin supplementation substantially improves rooster sperm freezability and post-thaw function.

This pioneering research investigated apigenin potential to augment rooster sperm cryosurvival in an extender model. Apigenin is a natural antioxidant flavonoid showing promise for improved post-thaw sperm function. However, its effects on avian semen cryopreservation remain unexplored. This first study supplemented rooster sperm Lake extender with 0, 50, 100, 200, 400 µmol/L apigenin to determine the optimal concentrations for post-thaw quality. Supplementation with 100 µmol/L apigenin resulted in significant enhancements in total motility (from 41.5% up to 71.5%), progressive motility (18.1% to 29.1%) (p < 0.05), membrane integrity (40% to 68%), mitochondrial function (p < 0.001), viability (37% to 62%) and total antioxidant capacity (p < 0.001) compared to the control. It also substantially reduced percentages of abnormal morphology, reactive oxygen species and apoptosis (p < 0.001). Although 200 µmol/L apigenin significantly enhanced some attributes, effects were markedly lower than 100 µmol/L. Higher doses did not improve cryoprotective parameters. This indicates 100 µmol/L as the optimal apigenin concentration. This represents the first report of apigenin protecting rooster sperm from cryodamage. The natural antioxidant improved post-thaw sperm quality, likely by suppressing oxidative stress and apoptosis. Apigenin shows promise for enhancing rooster sperm cryosurvival.

Gallic acid supplementation partially ameliorates reproductive aging in rooster breeders by improving semen quality, sperm kinetics, hormones, and antioxidant status.

Aging leads to decreased fertility in roosters, which is likely due to increased oxidative stress. This study evaluated the antioxidant effects of gallic acid (GA) supplementation on sperm quality and fertility of aged roosters. This study evaluated whether GA supplementation can mitigate age-related fertility decline. Roosters were randomly assigned to: control, 100 mg/kg GA, or 200 mg/kg GA. Semen parameters, sperm kinetics, hormone levels, fertility rate, and hatchability were assessed. GA increased semen concentration, membrane integrity and viability while decreasing defects versus control (P < 0.01). Testosterone was higher in GA groups (P<0.01) without affecting gonadotropins. Furthermore, 200 mg/kg GA optimized motility, velocity, linearity, and beat cross frequency versus control and 100 mg/kg GA (P < 0.01). Fertility and hatchability were higher in both GA groups. In conclusion, GA supplementation in aged roosters improves sperm quality, antioxidant status, testosterone, and fertility outcomes, likely by mitigating oxidative stress. The 200 mg/kg dose elicited optimal effects on motion parameters.

Effects of dietary Moringa oleifera leaf extract on semen characteristics, fertility, and hatchability in aged broiler breeder roosters.

Declining semen quality will have a negative impact on the fertility of aged roosters. Various factors influence this decrease in quality. This study was conducted to investigate the effects of different levels of Moringa plant extract on semen characteristics, fertility, and hatchability in aged broiler breeder roosters. A total of 24 roosters were fed 1 of 4 dietary supplements for 10 wk: Control, 100 µL/kg (Moringa oleifera leaf extract [MOLE]-100), 200 µL/kg (MOLE-200), or 400 µL/kg body weight (MOLE-400) of Moringa oleifera extract. Results showed supplementation with MOLE-200 significantly improved (P < 0.05) semen concentration, total motility, progressive motility, sperm membrane integrity compared to other treatments. However, semen volume and body weight were unaffected (P > 0.05). Sperm lipid peroxidation, as indicated by malondialdehyde concentration, was lowest in MOLE-200. There was a significant difference observed among the treatments in terms of total antioxidant capacity (TAC) results. The testosterone concentration in the MOLE-200 treatment was significantly higher than the other treatments (P < 0.05). However, no significant differences were observed in the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) hormones among the experimental treatments. Fertility and hatchability rates were measured at the end of the trial. Fertility, defined as the number of fertilized eggs, was greatest in the MOLE-200 treatment compared to the other treatments. Similarly, hatchability (hatched chicks/fertilized eggs %) was highest at 88.02% for MOLE-200. In conclusion, dietary supplementation with M. oleifera extract improved semen quality, fertility, and hatchability in aged broiler breeder roosters.

The histopathological changes of liver and testis of Japanese quail chicks fed different levels of dietary L-valine.

This experiment was carried out to investigate the histological changes of liver and testis of Japanese quail fed different levels of dietary valine (Val) in low protein diet. A total of 1000 one-day-old Japanese quail chicks (mixed sex) were assigned to five experimental diets including diets containing 7.5, 8.5, 9.5, 10.5 and 11.5 g digestible (dig.) Val/kg diet in a completely randomized design, with 5 replicates of 40 quail chicks per pen. Experimental diets were formulated to be isoenergetic and isonitrogenous (170 g crude protein/kg) to meet nutrients recommendation of growing quails suggested by Brazilian tables. At d 42, quail chicks were slaughtered, and tissue samples were collected and fixed to evaluate the histological indices of liver and testis. High levels of Val, increased (P < 0.05) diameter of liver cell nucleus and liver hepatocytes in both male and female. While 11.5 g Val showed mild hepatosteatosis, bile duct hyperplasia was observed in 10.5 g Val. In 7.5 and 8.5 g Val groups, there was no negative effects on the liver histology. The male quail chicks which fed on diets containing 8.5 g Val had better significant (P < 0.05) reproductive indexes [Tubular differentiation (TDI) and spermatic index (SI)]. In conclusion, the use of high levels of Val (≥ 9.5 g dig. Val/kg diet) during d 0 - 42 of age can lead to histological damage in liver and testis of quail chicks.

Effect of different sources of dietary zinc on sperm quality and oxidative parameters.

Zinc has a critical physiological role in sperm function. The purpose of this study was to investigate the effect of different sources of zinc on sperm quality. For this purpose, 18 Zandi lambs with an average weight of 32 ± 1.2 kg were subjected to three treatments in a completely randomized design. Experimental treatments include (1) control treatment of basal diet without zinc supplementation, (2) basal diet with 40 mg/kg of zinc supplementation from zinc sulfate source and (3) basal diet with 40 mg/kg of zinc supplementation with organic source. At the end of feeding period, lambs were slaughtered. To determine the effect of experimental treatments on sperm quality, the testes were transferred to the laboratory. After that, epididymal spermatozoa were evaluated for sperm motility parameters, abnormal morphology, viability, membrane functionality, malondialdehyde (MDA) and antioxidant activity [glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC)], sperm concentration and testosterone level. Zinc sulfate administration decreased MDA levels compared to other treatments and increased GPx and TAC activity compared to the control (P < 0.05), although SOD activity was not affected by any supplementation. Also, the use of zinc sulfate supplementation increased the percentage of total and progressive motility compared to the control group (P < 0.05). Membrane integrity and sperm viability were also affected by zinc sulfate supplementation (P < 0.05). Therefore, the results of this study showed that the use of zinc sulfate, can improve sperm motility and survival indices and its antioxidant capacity.

Enhancing post-thaw quality of ram epididymal sperm by supplementation of rutin in cryopreservation extender.

The purpose of this study was to examine the effect of different rutin concentrations on rams epididymal sperm. A local slaughterhouse provided 50 pair of testes from 25 rams. The testes were sent to the lab at room temperature. Spermatozoa were extracted by suspending portions of cauda epididymis in tris solution. Ram sperm was cryopreserved (in liquid nitrogen) in a tris extender containing rutin at 0, 0.5, 0.75, 1, and 1.25 mM. Rutin showed superior sperm total and progressive motility, beat cross frequency, straight line velocity, velocity average pathway and membrane integrity values at 0.75 and 1 mM. The morphology of the sperm and the superoxide dismutase levels did not significantly change with different treatments. Moreover, rutin at 0.75 and 1 mM was also shown to have the highest level of mitochondrial activity. The results showed ATP, total antioxidant capacity, and glutathione peroxidase levels were significantly greater in the rutin 0.75 and 1 mM groups (P < 0.05). Rutin at 0.75 and 1 mM levels had the lowest reactive oxygen species concentrations. Rutin at 0.75 and 1 mM substantially increased the proportion of viable sperm (P < 0.05). The lowest amount of apoptosis was observed in 0.75 and 1 mM rutin. Rutin at 0.75 and 1 mM yielded the least significant percentage of dead sperm. It may be inferred that adding 0.75 and 1 mM to the sperm extender can enhance the quality of the epididymal sperm in rams after the cryopreservation process.

Comparison of the performance of targeted mitochondrial antioxidant mitoquinone and non-targeted antioxidant pentoxifylline in improving rooster sperm parameters during freezing and thawing.

Oxidative stress is associated with impaired sperm quality after thawing. Since mitochondria are the main source of reactive oxygen species (ROS) in sperm, the aim of this study was to investigate the effects of targeted mitochondrial antioxidant mitoquinone (MitoQ) and non-targeted mitochondrial antioxidant pentoxifylline (PTX) during cooling and cryopreservation of rooster sperm. Sperm samples were collected from 15 roosters aged 28 wk and diluted with Beltsville extender. After dilution and addition of treatments (50, 100, and 200 pMol MitoQ and 0.5, 0.75, and 1 µM PTX), samples were cooled for 2 h to 4°C and they were first analyzed at this stage and were frozen and re-evaluated after thawing. After the freezing and thawing, level of 100 pMol MitoQ significantly increased total motility (TM), progressive motility (PGM), curvilinear velocity (VCL), membrane integrity, viability, total antioxidant capacity (TAC) and the glutathione peroxidase (GPx), as well as the level of 50 pMol significantly increased TM, PGM, average path velocity (VAP), straight-line velocity (VSL), membrane integrity, viability, and mitochondrial activity. Moreover, these 2 levels (50 and 100 PMol) decreased malondialdehyde and sperm with abnormal morphology. Addition of 0.75 µM PTX also increased total motility compared to the control group and levels of 0.5 and 0.75 µM decreased sperm with abnormal morphology. It could be concluded the addition of MitoQ and PTX can be useful for sperm cryopreservation industry and reduce the harmful effects of freeze-thawing.

Protective effect of rosiglitazone on microscopic and oxidative stress parameters of ram sperm after freeze-thawing.

The purpose of this study was to investigate the effects of rosiglitazone on ram semen after cryopreservation on the quality of thawed sperm. Sperm motility, membrane functionality, viability, total abnormality, acrosome membrane integrity, mitochondrial activity, reactive oxygen species production, ATP content and apoptotic features were assessed after thawing. Rosiglitazone at concentration of 60 µM resulted in the highest (P < 0.05) total motility, progressive motility and straight-line velocity. The percentages of average path velocity and curvilinear velocity were greater in the 60 µM group. Different concentrations of rosiglitazone did not have significant effects on amplitude of the lateral head displacement, linearity and straightness. The highest amounts of membrane functionality and mitochondrial activity after freeze-thawing were observed in groups containing 60 µM. By increasing the rosiglitazone level to 80 µM, no positive effect was observed in most of the evaluated parameters. The lowest ROS concentration was recorded in 60 µM rosiglitazone group (P < 0.05). The group containing 60 µM rosiglitazone also produced the lowest significant percentage of apoptosis-like changes and dead sperm. A greater (P < 0.05) percentage of acrosome integrity in frozen-thawed spermatozoa was observed in the 60 µM rosiglitazone group. There was no significant difference between 40 and 60 µM rosiglitazone in intact acrosome of ram thawed semen. The result showed that supplementation in ram semen extender with rosiglitazone had a positive role in the regulation of ram sperm motility and had strong protective effect on the sperm membrane and acrosome integrity.

Comparing the effect of rooster semen extender supplemented with gamma-oryzanol and its nano form on post-thaw sperm quality and fertility.

Antioxidant nanoparticles include the potential for improving sperm cryopreservation. The aim of performing this study was to evaluate the effects of gamma-oryzanol (GO) at 0 (C) (control group), 20 (GO20), 40 (GO40), 60 (GO60), 80 (GO80), and 100 (GO100) µM and gamma-oryzanol nanoparticles (GON) at 0 (CN), 20 (GON20), 40 (GON40), 60 (GO60), 80 (GON80), and 100 (GON100) µM on post-thawed sperm quality and fertility of rooster sperm. Sperm motility, plasma membrane integrity, total abnormality, mitochondrial activity (Rhodamine 123), apoptotic features (Annexin V/Propidium iodide), reactive oxygen species (ROS) production, ATP content and the fertility and hatchability were evaluated after thawing. Total motility in GON60 and GON80 were significantly higher compared to control groups (C and CN). GON80 showed the greatest percentages of progressive motilities. When GO80, GON60, and GON80 were added to the cryopreservation medium, the plasma membrane functionality of the semen samples improved. The minimum abnormality of spermatozoa is observed in the group treated with GON80. The groups treated with GON60 and GON80 had greater (P < 0.05) mitochondrial activity. The level of sperm ROS after cryopreservation was significantly lower in GON60 and GON80 groups. Live sperm was significantly higher (P < 0.05) in GON60 and GON80 group compared to other groups. GON60 and GON80 groups also led to the lowest significant percentage of apoptosis-like change sperm. Greater fertility percentages were observed (P < 0.05) when sperm were stored in extenders treated with GON60 and GON80. GON80 resulted in significantly improved hatched eggs compared to C, GO60, GO180 and CN. In conclusion, supplementation of Lake extender with 60 and 80 µM gamma-oryzanol nanoparticles could be a proper process to improve freeze-thawing rooster sperm quality leading to better freeze/thaw characteristics.

Effect of tempol and straw size on rooster sperm quality and fertility after post-thawing.

The aim of the present study was to investigate the effects of tempol and straw size on rooster sperm post-thaw quality and fertility. Rooster semen was cryopreserved in Lake extender containing 0 (control), 5, 10, 15 and 20 µM tempol (in two different straw size, 0.25 and 0.5). The percentage of total and progressive sperm motility, VAP and VSL increased in the 10 µM tempol group. Moreover, 10 µM tempol led to lower ROS compared to other groups. The lowest percentage of apoptotic-like changes was detected when the extender was treated with 10 µM of tempol. The minimum ROS was observed in the group treated with 0.5 straw size. Straw size did not have any significantly effect on GPx and SOD activities and TAC of frozen-thawed sperm. The highest significant percentage of fertility and hatching rate were observed in 10 µM of tempol. The results of the present study showed that supplementation of the Lake cryopreservation medium with 10 µM tempol improved cryo-survival. Also, the results of the present study suggested that Lake cryopreservation medium with 0.5-ml straw may perhaps be an appropriate method to improve the quality and fertility post-thawed rooster sperm.

Poloxamer 188 and hydroxyethyl starch have a cryoprotective synergic effect improving post-thawing quality and fertility of rooster spermatozoa.

Poloxamer and hydroxyethyl starch have cytoprotective effects. In the present study, effectiveness was evaluated of these compounds as a cryoprotectant for rooster semen. In Experiment 1 (E1), poloxamer 188 (1%, P188), poloxamer 407 (1%, P407), and control groups were compared after sperm cryopreservation. Experiment 2 (E2) was conducted with 3%, 5%, and 7% of hydroxyethyl starch (H3, H5, H7), also combined with P188 (H3P188, H5P188, H7P188), based on results from E1. Sperm motility was assessed using CASA, abnormal forms and hypo-osmotic swelling (HOS) were evaluated using microscopy, and viability, apoptotic-like changes, and mitochondrial activity were determined using flow cytometry. In E2, there were assessments of fertility and hatching capacity. Results from E1 indicated total and progressive motility, velocity, membrane functionality, viability, and mitochondrial activity were greater with inclusion of P188 in semen extender, with less apoptotic-like changes (P < 0.05). In E2, HES inclusion in semen extender improved total motility, membrane functionality, and mitochondrial activity (P < 0.05), especially H5, which also markedly increased sperm viability and decreased apoptotic-like changes. The combination of P188 with HES increased sperm quality overall, with inclusion of H5P188 resulting in increases of progressive motility and VSL (P < 0.05). The H5 inclusion also increased proportion of fertilized eggs (P < 0.05). Furthermore, the combination of HES and P188 increased proportions of fertilized and hatched eggs compared with the control, with inclusion of H5P188 having the greatest effects. In conclusion, supplementation of semen extender with H5P188 increases post-thawing quality and fertility of rooster sperm, being a safe and effective method for the poultry industry.

Does alpha-lipoic acid-loaded nanostructured lipid carriers improve post-thawed sperm quality and ameliorate apoptosis-related genes of rooster sperm?

Oxidative stress could be prevented by antioxidant-loaded nanoparticles. The purpose of this study was to assess the effects of 10 (A10), 20 (A20), 30 (A30), 40 (A40), and 50 (A50) µM alpha-lipoic acid and alpha-lipoic acid nanostructured lipid carriers (ALN) at 10 (ALN10), 20 (ALN20), 30 (ALN30), 40 (ALN40), and 50 (ALN50) µM on post-thawed sperm quality, fertility, and apoptosis-related genes of rooster sperm. The extender supplemented with ALN30 led to higher total and progressive motility, straight-line velocity, and linearity in comparison to the control group. The ALN30 resulted in higher percentage of mitochondria activity and glutathione peroxidase level compared with control (P < 0.05). The extender supplemented with ALN30 led to lower percentage of apoptotic sperm, when compared with the control. CASPASE 3 expression in ALN30 was lower (P < 0.05) than the other groups. The results showed that BCL-2 mRNA expression of sperm was significantly (P < 0.05) higher in ALN30 compared with the other groups (P < 0.05). Higher percentages of fertility and hatchability rates were observed in ALN30 group. The results indicate that ALN30 could be regarded as a novel potential cryoprotectant for the cryopreservation of rooster semen. Therefore, nanostructured lipid carriers improve not only the active compound (such as alpha-lipoic acid) of biomedical applicability but also the potential for industrial application in sperm cryopreservation.

Does ergothioneine and thawing temperatures improve rooster semen post-thawed quality?

The present study focuses on the effect of different levels of ergothioneine and thawing temperature on rooster semen cryopreservation. Semen was diluted in Lake extender containing ergothioneine at 5, 10, 15, and 20 µM and cryopreserved. Two thawing temperatures (37°C for 30 s and 60°C for 5 s) were consequently examined. Sperm motility parameter, membrane integrity, abnormal morphology, viability, apoptotic status, mitochondria activity, and lipid peroxidation were determined after freeze-thaw process. Ergothioneine levels of 5 and 10 µM led to higher (P < 0.05) total motility (66.58 ± 1.44 and 72.11±1.44, respectively) and average path velocity (VAP) (34.54 ± 0.89, 37.28 ± 0.89, respectively). Higher (P < 0.05) significant membrane integrity and mitochondria activity after freeze-thawing were observed in the groups supplemented with 10 µM ergothioneine (68.62 ± 1.24 and 69.12 ± 1.26, respectively). Also, 5 and 10 µM of ergothioneine led to the lowest significant percentage of apoptotic and dead sperm. The total motility and progressive motility resulted in significantly (P < 0.05) higher amount when sperm were thawed with 60°C (60.58 ± 0.91 and 24.76 ± 0.53, respectively) compared to thawed sperm in 37°C. The membrane integrity, viability and mitochondria activity led to significantly (P < 0.05) higher when sperm were thawed with 60°C (58.2 ± 0.78, 63.21 ± 0.80 and 56.85 ± 0.79, respectively). It could be concluded the addition of 5 and 10 µM ergothioneine in the semen extender and thawing temperature at 60˚C in 5 s can be an efficient strategy to preserve rooster cryopreserved semen quality.

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa.

Antifreeze proteins (AFP) have the potential for improving sperm cryopreservation. We have applied Type III antifreeze protein (AFP3) on the cryopreservation of spermatozoa from broiler breeder roosters, aiming to enhance post-thawing quality and fertility. Semen was extended at 37°C in Lake's extender containing AFP3 at 0.01, 0.1, 1, 5, and 10 µg/mL (no AFP3 as control). Post-thawing sperm assessment included sperm motility (CASA), morphology, membrane functionality by hypoosmotic swelling test (HOST), lipoperoxidation as malondialdehyde (MDA) production, and sperm viability, early apoptosis (phosphatidylserine exposure as annexin V-positive staining in viable spermatozoa), and mitochondrial activity by flow cytometry. Fertility was assessed after artificial insemination (30 hens/treatment). Total and progressive motility, membrane functionality, and mitochondrial activity increased in 0.1 and 1 µg/mL AFP, compared to control and other concentrations, whereas apoptosis was significantly lower. VAP, VSL, and viability were significantly higher for 1 µg/mL AFP3 than with the other treatments except for 0.1 µg/mL (which was not always significantly different from the control or other concentrations), and with abnormal forms being significantly lower. The proportion of fertilized and hatched eggs was also higher for 1 µg/mL AFP3, with 0.1 µg/mL also showing significantly higher results than the control, and no differences with other concentrations). In conclusion, 1 µg/mL AFP3 could improve the post-thawing results of rooster spermatozoa frozen in Lake's extender. According to our results, concentrations between 1 and 0.1 µg/mL could be similarly efficient.

Effect of astaxanthin nanoparticles in protecting the post-thawing quality of rooster sperm challenged by cadmium administration.

The protective role of astaxanthin nanoparticles (Ast NPs, 25 mg/kg p.o) against cadmium (Cd, 1 mg/100 g b.w. SC), a known inductor of lipid peroxidation and changes in the antioxidant defense system in the Ross 308 breeder roosters sperm, was examined. Sperm motility (computer-assisted sperm motility analysis), membrane integrity (hypoosmotic swelling test), viability, total abnormality, and enzymatic parameters were assessed after thawing. The testis/body weight (mg/kg) ratio and HE staining results of testis were also performed. The obtained results showed that Cd induced detrimental effects on testis and sperm, while Cd treated by Ast NPs (Cd Ast) diminished this change compared to the Cd group. Cd-treated group resulted in significantly (P < 0.05) lowest total (37.29 ± 2.46) and progressive (5.84 ± 0.47) motility and decreased antioxidant enzyme activity (CAT, TAC, and GPx), as well as producing a significant (P < 0.05) decrease in testis weight (mg) compared to the control group. Treatment with Ast NPs (Ast NPs + Cd) had reversed Cd-induced changes in the antioxidant defense system and significantly prevented Cd-induced testis damage. In conclusion, the results of our work suggest that Ast NPs at 25 mg/kg act as a potent antioxidant in protecting rooster testes against oxidative stress induced by Cd.