Egg yolk plasma enriched with ß-carotene through the diet of laying hens and adding it to the extender improves the quality of frozen semen in Arabic stallions.

Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with ß-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, ß-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with ß-carotene. Subsequently, various variants of enriched extender (INRA-96 + 2.5% glycerol [G]) were gained by adding 2% EYP from four treatment groups. The sperm characteristics, including motility, viability, morphology, plasma membrane integrity (HOS test), lipid peroxidation (MDA) and DNA fragmentation, were evaluated after thawing. According to the results obtained in this study, the addition of EYP from T2 and T4 (500 and 2000 mg/kg of ß-carotene in hens' diet) to the extender (INRA-96 + 2.5% G) leads to an increase in total motility (50.50% and 49.49%, respectively), progressive motility (32.6% and 31.8%, respectively), viability (68.7% and 66.1%, respectively) and plasma membrane integrity (57.7% and 50.6%, respectively). Moreover, lipid peroxidation (1.3 and 1.4 nmol/mL, respectively) and DNA fragmentation (8.6% and 9.9%, respectively) were diminished using the mentioned treatments. However, sperm morphology was not affected by the treatments. In the current study, we concluded that the optimal concentration of ß-carotene in the laying hen's diet (500 mg/kg) could reveal the best results about sperm quality. So, EYP enriched with ß-carotene acts as a valuable natural and safe supplementary material that could be exploited for enhancing stallion sperm quality in cryopreservation conditions.

L-carnitine improves quality parameters and epigenetic patterns of buck's frozen-thawed semen.

Buck sperm cryopreservation is an effective method to distribute qualified sperm for reproductive purposes, but this procedure reduces sperm quality. The current study was conducted to investigate the effect of L-carnitine (LC) on the quality and epigenetic patterns of buck's post-thawed semen. Semen samples were collected from five male goats twice a week and diluted in extenders supplemented with 0 (LC-0), 1 (LC-1), 5 (LC-5) and 10 (LC-10) mM LC. Samples were cryopreserved according to standard protocol. After thawing, motility characteristics, lipid peroxidation, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, epigenetic modifications, viability, apoptotic-like changes and DNA fragmentation were assessed. Samples supplemented with 5 mM LC showed greater (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, DNA methylation, viability, and lower (P ≤ 0.05) apoptotic-like changes. Lipid peroxidation was lower (P ≤ 0.05) in LC-5 and LC-10 compared to the control group. Addition of LC to the cryopreservation extender had no effect (P > 0.05) on velocity parameters, abnormal morphology, histone modifications, or DNA fragmentation. In conclusion, supplementing the cryopreservation extender with 5 mM LC significantly preserves the quality of buck sperm after the cryopreservation process.

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.

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.

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.

Supplementation of ram's semen extender with Mito-TEMPO II: Quality evaluation and flow cytometry study of post-thawed spermatozoa.

Cryopreservation is an effective method to spread qualified ram spermatozoa for reproductive goals in different farms, but cryopreservation's shocks reduce sperm quality. This study investigated the efficacy of the new mitochondria-targeted antioxidant Mito-TEMPO on post-thawed quality of spermatozoa in sheep. Collected samples were divided into five groups and after dilution, received different doses of Mito-TEMPO (0, 0.5, 5, 50 and 500 µM), and frozen. Thawed sperm motility parameters, malondialdehyde content, membrane functionality, abnormal morphology, mitochondria activity, acrosome integrity, DNA fragmentation, ROS concentration, viability and apoptotic-like changes, were evaluated. According to the results, Mito-TEMPO (5 and 50 µM) improved (p ≤ 0.05) motility parameters, average path velocity, membrane functionality, mitochondria activity and viability compared with the other groups. Moreover, apoptotic-like changes, lipid peroxidation and ROS concentration were lower (p ≤ 0.05) in groups received 5 and 50 µM Mito-TEMPO. Mito-TEMPO showed no effect (p > 0.05) on sperm acrosome integrity, morphology and DNA fragmentation. In conclusion, Mito-TEMPO as a targeted antioxidant could be an efficient cryo-additive to enhance quality parameters of post-thawed ram semen.

Conjugated linoleic acid (CLA) supplementation effects on performance, metabolic parameters and reproductive traits in lactating Holstein dairy cows.

The objective of the study was to determine the effects of conjugated linoleic acid (CLA) supplement on milk yield and composition, blood metabolites and reproductive parameters in lactating Holstein dairy cows. Twenty Holstein dairy cows were randomly assigned to one of two dietary treatments: 1) supplementing 110 g per day of fat (control), 2) supplementing 120 g per day of rumen-protected CLA. The diets were formulated to be nutritionally isocaloric and isonitrogenous. The experimental period started 21 days pre-calving and continued until 60 days in milk (DIM). Treatments had no effect on dry matter intake (DMI), body weight (BW) and body condition score (BCS). The CLA treatment increased milk yield (3.04 kg per day and milk lactose concentration, but decreased milk fat concentration and, short and medium chain fatty acids concentrations. No treatment differences were observed in milk protein concentration, milk energy output and net energy balance. Serum concentrations of glucose, cholesterol, triglyceride (TG), insulin, insulin-like growth factor-1(IGF-1), estradiol and progesterone were higher in CLA treated cows when compared to cows fed on the control diet. Serum beta-hydroxybutyric acid (BHBA) concentration was reduced in cows fed on the CLA treatment. Days to first insemination and days open were not different between the two treatment groups. Cows fed on the CLA supplement had increased conception rate from the first service. The results indicated that cows fed on diets supplemented with CLA produced milk with decreased milk fat concentration whereas some related cow blood serum metabolic parameters associated with reproductive response were increased and resulted in an increased conception rate from the first service.

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.

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.

Poloxamer 188 exerts a cryoprotective effect on rooster sperm and allows decreasing glycerol concentration in the freezing extender.

Glycerol is the most widely used cryoprotectant for rooster sperm because it declines the mechanical damage to sperm during the freezing process. Despite its high molecular weight and viscosity, which may be cytotoxic, glycerol can cause damage to cells during the cryopreservation process, resulting in less fertility. Poloxamer 188 (P188) is an embryo cryopreservation supplement effective in many species and also for cell lines and plant cells. We tested the suitability of P188 in the cryopreservation of rooster sperm, considering post-thawing motility, abnormalities, membrane functionality (hypo-osmotic swelling test), mitochondrial activity, viability, apoptosis status, reactive oxygen species production, and ATP content after thawing and the fertility and hatchability after AI. We carried out a factorial experiment with glycerol concentrations of 2% glycerol (G2) and 8% glycerol (G8) and P188 concentrations of 0% (P0), 0.1% (P0.1), 0.5% (P0.5), and 1% (P1) as fixed effects, with replicate (seven) as a random effect. Interactions between glycerol and P188 were found, with G2P1 yielding higher quality and fertility. G8P0.5 yielded better in most parameters, however, not reaching G2P1. G2P1 showed significantly higher results for total and progressive motility, kinetic parameters (average path velocity, straight-line velocity, and linearity), membrane functionality, viability, mitochondrial activity, and ATP content and lower apoptosis, dead sperm, and reactive oxygen species production. G2P1 resulted in the highest percentages of fertilized and hatched eggs, with no effects in the hatched eggs ratio. Interestingly, G2 was less efficient in many parameters than G8 when combined with P0 and P0.1, being equivalent to G8 with P0.5 and superior to any G8 treatment as G2P1. In conclusion, P188 could improve rooster semen cryopreservation and allow reduction of glycerol in extenders, with a consequent impact in the poultry industry.

Effect of crocin and naringenin supplementation in cryopreservation medium on post-thaw rooster sperm quality and expression of apoptosis associated genes.

The aim of our study was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender for freezing rooster semen. Sperm motility, viability, abnormalities, membrane functionality, active mitochondria, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertile eggs, hatched eggs and hatching rate were investigated following freeze-thawing. C1 and N100 resulted in higher (P < 0.05) total motility and progressive motility in comparison to the control group. The C1 and N100 groups improved viability, membrane functionality and reduced lipid peroxidation. We found higher values for active mitochondria with C1 and N100 compared to control group. The C1 and N100 groups showed lower percentages of early apoptosis when compared with control group. Also, C1 and N100 had higher TAC, compared to the control group. The mRNA expressions of BCL-2 in the C1 and N100 groups were significantly higher than that of other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when compared to control group. Significantly higher percentages of fertile eggs, hatched eggs and hatching rate were observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and could protect the sperm by reducing the pro-apoptotic (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes.

Effects of new synthetic cryoprotectant agents on histological characteristics of various classes of vitrified bovine pre-antral follicles.

Previous studies have reported many discrepancies about the best type and concentration of cryoprotective agents (CPAs) and biological variability among various pre-antral follicle classes after cryopreservation of ovarian tissue. The aim of this study was to investigate the impacts of some synthetic polymers on histological characteristics of different types of pre-antral follicles after bovine ovarian tissue vitrification. From each bovine ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control group) or submitted to vitrification (sucrose, X-1000, Z-1000 and polyvinylpyrrolidone groups), either followed by in vitro culture for 1 or 5 days. In this case, although, the addition of X-1000 resulted in greater percentages of normal follicles for almost all pre-antral follicle classes compared to those of other groups, there are some exceptions. These results indicate that the inclusion of polyvinylpyrrolidone in the freezing media can improve the morphology of the post-warmed transitional follicles and cultured primordial follicles on day five more than other CPAs. According to the results of this study, it can be concluded that although ovarian tissue cryopreservation is often performed to preserve the primordial follicles, by choosing the best combination of permeating and non-permeating CPAs (synthetic polymers), more advanced stages of bovine pre-antral follicles, transitional, primary and secondary follicles, may also survive the cryopreservation process.

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 resveratrol-loaded nanostructured lipid carriers supplementation in cryopreservation medium on post-thawed sperm quality and fertility of roosters.

The objective of this study was to assess the effect of resveratrol and resveratrol-loaded nanostructured lipid carriers (NLC) supplementation of semen extender on values for fertility variables of cryopreserved rooster semen. Rooster semen was cryopreserved in modified Beltsville extender containing 0 (control group), resveratrol at 20, 40 and 60 µM and resveratrol-loaded NLC at 20, 40 and 60 µM. After thawing, motility properties, abnormal morphology, viability, membrane functionality, mitochondrial activity, apoptotic status, malondialdehyde (MDA) and antioxidant activities (glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (TAC)) and fertility potential (fertility and hatchability rates) were assessed. Using 40 µM resveratrol and resveratrol-loaded NLC improved total motility. The results indicated that sperm fertility and hatching rate, viability and membrane functionality were greater in the 40 µM resveratrol and resveratrol-loaded NLC compared to control group. The percentage of apoptotic spermatozoa in 40 µM resveratrol and resveratrol-loaded NLC group was less compared with the 60 µM resveratrol, 60 resveratrol-loaded NLC and control groups. Mitochondria activity was greater in the 40 µM resveratrol and resveratrol-loaded NLC extender group compared to 20 and 60 µM resveratrol, 60 resveratrol-loaded, and control groups. Also, the 40 µM resveratrol and resveratrol-loaded NLC extender group had a greater TAC and reduced MDA. Morphology and SOD were not affected by dose of resveratrol. The results indicate supplementation of the modified Beltsville extender with 40 µM resveratrol and resveratrol-loaded NLC resulted in a greater quality of frozen-thawed rooster sperm.

Effect of egg yolk plasma and soybean lecithin on rooster frozen-thawed sperm quality and fertility.

This experiment was conducted to study the effects of egg yolk plasma (10%, 15% and 20%), soybean lecithin (0.5%, 1% and 1.5%) and whole egg yolk (WEY) (control) on post-thawed sperm quality, hatchability and fertility outcomes. In experiment 1, sperm motility, abnormalities, membrane integrity, viability, apoptosis status, mitochondrial activity were studied following freeze-thawing. The best quality of frozen-thawed rooster sperm was chosen to be used for the assessment of the hatchability and fertility rate in experiment 2. The significantly higher percentages of post-thawing sperm total and progressive sperm motilities, membrane integrity, viability were observed in 1% soybean lecithin and 20% egg yolk plasma in comparison with 0.5 and 1% soybean lecithin, 10% egg yolk plasma and control, except for 15% egg yolk plasma (P < 0.05). Using 20% egg yolk plasma in the extender improved mitochondrial activity. Supplementation of 1% soybean lecithin and 20% egg yolk plasma into the extender resulted in the least percentages of dead sperm (P < 0.05). Sperm abnormalities and early apoptosis did not differ in various extender supplementations. In experiment 2, higher percentages of hatchability and fertility rate were observed in semen containing 1% soybean lecithin and 20% egg yolk plasma compared with the WEY group. The results showed that supplementation of the rooster sperm extender with 1% soybean lecithin and 20% egg yolk plasma resulted in higher quality of frozen-thawed sperm.

Effect of supplementing a diet with monensin sodium and Saccharomyces Cerevisiae on reproductive performance of Ghezel ewes.

Effect of supplementing a diet, in an attempt to enhance reproduction, with monensin sodium and Saccharomyces cerevisiae yeast on reproductive performance was investigated during the breeding season using 44 Ghezel ewes (body weight 56.97±7.47kg, age 2-5 years and body condition score (BCS) 2.5) which were allocated randomly in equal numbers to the four dietary treatments as follows: 1) Basal diet plus supplemental feed (450g/ewe/d) plus monensin sodium (30mg/ewe/d) (MS); 2) Basal diet plus supplemental feed (450 g/ewe/d) plus Saccharomyces cerevisiae yeast (4×109CFU/ewe/d) (SC); 3) Basal diet plus supplemental feed (450g/ewe/d) (FG); 4) Basal diet (only grazing on pasture, Control; G). Estrous synchronization of all ewes was done using controlled internal drug release (CIDR) and all ewes were mated with purebred Ghezel rams after CIDR removal. The results indicated that MS and SC treatments with 15 lambs had greater number of lambs than ewes of the other two treatment groups. Ewes in MS group with 50% twining rate had the greatest value followed by the FG, SC and G treatment groups (P<0.05). The lambs from ewes in MS and SC groups were heavier in weight than those in FG and G treatments (P<0.01). Blood sample analysis provided evidence that ewes in MS and SC groups had greater concentrations of 17ß-estradiol (E2), progesterone (P4), blood urea nitrogen (P<0.05), insulin, glucose, cholesterol and total protein (P<0.01) than ewes of the other groups. These results indicated that using a diet for enhancing reproduction, including monensin sodium and Saccharomyces cerevisiae yeast in the breeding season could have beneficial effects on reproductive performance of Ghezel ewes.

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.

Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.

The aim of this study was to evaluate the effects of glycerol, ethylene glycol or DMSO in a soybean lecithin extender for freezing ram semen. In this study, 20 ejaculates were collected from four Ghezel rams and diluted with soybean lecithin extender with glycerol (7%), ethylene glycol (3%, 5% and 7%) or DMSO (3%, 5% and 7%). Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality, mitochondrial activity (Rhodamine 123) and apoptotic features (Annexin V/Propidium iodide) were assessed after thawing. There was no significant difference between glycerol and ethylene glycol at different concentrations (3% and 5%) regarding sperm total and progressive motility, viability, and membrane integrity. The least percentages of mitochondrial functionality were observed in samples frozen with all different DMSO concentrations tested (P<0.05). Moreover, the percentage of post-thawed dead sperm was the greatest for all the DMSO concentrations compared with other groups (P<0.05). Thus, DMSO had an adverse effect on the post thaw ram sperm parameters. In contrast, ethylene glycol could be a desirable substitute of glycerol in the freezing extender, in view of similar results obtained in post-thaw quality of ram semen cryopreserved in a soybean lecithin extender. We propose that glycerol in a soybean lecithin based extender could be replaced by ethylene glycol at 3% or 5% concentrations.