Soybean Lecithin-based Extender Research Articles

A soy lecithin nanoparticles-based extender effectively cryopreserves Holstein bull sperm

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL.

The mitochondria-targeted antioxidant “MitoQ” preserves quality and reproductive performance of ram spermatozoa cryopreserved in soybean lecithin-based extender

Cryopreservation of ram semen is helpful for distributing proved spermatozoa for reproductive goals, but cold shock has destructive effects on fertility ability of frozen sperm cells. This study was performed to investigate the effect of the novel mitochondria-targeted antioxidant “MitoQ” on ram sperm quality and fertility potential during cryopreservation process. Semen samples were diluted in extenders supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and then frozen according to the standard protocol. Motility and velocity characteristics, lipid peroxidation, acrosome integrity, membrane functionality, mitochondria active potential, viability, apoptosis status, DNA fragmentation, ROS concentration and reproductive performance were evaluated after thawing. In results, 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, acrosome integrity, membrane functionality, mitochondria active potential and viability as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis status, DNA fragmentation and ROS concentration compared to the control group and the other treatments. Moreover, after fertility trial, 10 and 100 nM MitoQ resulted in higher (P ≤ 0.05) pregnancy, parturition and lambing rates than control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in sheep and it could be an effective additive for supplementation of ram's semen cryopreservation medium during reproductive programs.

Procyanidins supplemented soybean lecithin-based extenders improve post-thaw quality of goat spermatozoa

Procyanidins supplemented soybean lecithin-based extenders improve post-thaw quality of goat spermatozoa

PUFA-rich phospholipid classes and subclasses of ram spermatozoa are unevenly affected by cryopreservation with a soybean lecithin-based extender

Cryopreservation is known to affect spermatozoa structure and function. Ram sperm are among the most highly sensitive mammalian gametes to freezing, due to their lipid composition, which limit their efficiency in artificial insemination programs. The aim of this study was to investigate the effects of cryopreservation with a chemically defined soybean lecithin-based extender on ram spermatozoa functionality on the one hand, and quantifiable changes in lipid and fatty acid profile on the other. Freeze-thawing decreased sperm quality, as indicated by post-thaw parameters related to membrane integrity, mitochondrial viability and sperm motility. The most relevant lipid change after cryopreservation was a remarkable loss of all glycerophospholipids containing 22:6n-3. Species of sphingomyelin with very long chain polyunsaturated fatty acids (VLC-PUFA), that are exclusively located in the sperm head, where responsible of its reduction after cryostorage. Freezing caused a reduction in mitochondrial function, which was confirmed by significantly decreased of mitochondrial membrane potential and by the generation of 4-HNE. Mitochondria damage was accompanied by a loss in cardiolipin with 18:2n-6 and phosphatidylethanolamine with 20:4n-6, two well-known lipids that are critical components for mitochondrial membrane functionality. Loss of sterols after cryopreservation occurred along with a decrease in the order of sperm membrane lipids. Our research provides new insights on deleterious effects of cryopreservation on PUFA-rich phospholipids of ram sperm and highlight their importance as biomarkers of ultrastructural, biochemical and functional damage that ram spermatozoa undergo after freezing-thawing.

Dimethylformamide Preserves the Integrity of Cryopreserved Goat Semen in a Soybean Lecithin-Based Extender.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein‐free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II‐S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris‐based extender, containing either 20% egg yolk, 1% soybean lecithin Type II‐S or 1% soybean lecithin Type IV‐S. The samples were then frozen. Sperm motility was evaluated by computer‐assisted sperm analysis (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (SYBR‐14/PI) post‐thaw, as well as after 2 and 4 hr incubation at 37°C. Post‐thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin‐based extenders. Individual motility post‐thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean‐based extenders. The number of high green stained spermatozoa were significantly higher in Type IV‐S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin‐based extenders to cryopreserve canine semen.

Extenders modify the seminal plasma ability to minimize freeze-thaw damage on ram sperm.

Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze-thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk-based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen-thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin-based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p<.05), there was no effect of the extender. SP treatment was applied after freeze-thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p<.05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p<.05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non-capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p<.05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.

Cryopreservation and egg yolk extender components modify the interaction between seminal plasma proteins and the sperm surface

It is known that the addition of seminal plasma (SP) or SP proteins either before freezing or post thawing show contradictory results on sperm quality and fertility due to the interference between SP and the extender. Thus, the aim of this study was to determine whether egg yolk (EY) interferes with SP ability to protect the functionality and fertility of ram sperm during freeze-thawing by modifying the interaction between seminal plasma proteins and the sperm plasma membrane.Ejaculated or epididymal ram sperm collected during the breeding season were incubated with SP in the presence or absence of EY or soybean lecithin-based extenders before cryopreservation. No significant differences were observed after thawing in sperm quality (total and progressive sperm motility, membrane integrity, plasma membrane functionality, percentage of non-capacitated sperm) between the extenders, either in presence or absence of seminal plasma (P ≥ 0.05). The amount of proteins retained by the sperm surface normalized to number of cells was diminished after freeze-thawing compared to their fresh counterparts for all the treatments (P < 0.05), demonstrating that cryopreservation weakens the interaction between external proteins and the sperm surface. The electrophoretic analysis of sperm-bound proteins showed that the retention of several SP peptides onto the sperm surface (based on densitometry estimation) was affected by the presence of the diluents on both ejaculated and epididymal sperm (P < 0.05). Moreover, variation was observed in the protein pattern after thawing compared to the corresponding fresh samples, suggesting that freezing affects surface protein profile. Pregnancy rate after artificial insemination at fixed time was higher (P < 0.05) for samples treated with reconstituted with heterologous SP compared to those supplemented with 20% additional seminal plasma or control samples despite the presence of EY.In conclusion, both freeze-thawing and EY components affected the interaction among seminal plasma proteins and the sperm surface, although these changes were not reflected on different sperm quality parameters under our experimental conditions. In vivo fertility of sperm reconstituted with exogenous SP before freezing was improved even in the presence of EY components considering an optimal ratio SP:sperm.

Low Density Lipoprotein - important player in increasing cryoprotective efficiency of soybean lecithin-based bull semen extenders.

Currently, considering cryopreservation of bull semen, there is no clear consensus over the comparability of cryoprotective efficacy of extenders with soybean lecithin and those based on egg yolk. The objective of this study was to prove the use of Low Density Lipoprotein (LDL) extracted from hen-egg yolk as an enhancing factor for soybean lecithin-based extenders. In total, 35 ejaculates of (seven bulls x five ejaculates per bull) were collected and cryopreserved at a commercial insemination centre. The effect of the LDL addition to the extenders AndroMed® and Bioxcell® was tested in a 6% (v/v) concentration on spermatozoa after thawing. Modified extender composition effects were assessed on sperm functional parameters motility, plasma membrane, mitochondrial membrane potential and acrosomal integrity after thawing by CASA, flow cytometry and fluorescent microscopy, respectively. Based on kinematic parameters determined from CASA, k-means cluster analysis was used to classify individual spermatozoon into specific subpopulations (fast, medium fast and slow). A subpopulation of fast spermatozoa was increased in the presence of LDL in both selected extenders (P < 0.05). Moreover, the positive effect of LDL on sperm motility was confirmed by decreasing the percentage of sperm in slow subpopulation (P < 0.05). The effect of LDL addition on the incidence of spermatozoa with intact plasma membrane was not demonstrated in any case of extender used (P > 0.05). The percentage of sperm with intact acrosome was improved when LDL was added to Bioxcell® extender (P < 0.05). On the other hand, addition of LDL to AndroMed® extender improved mitochondrial intactness after thawing (P < 0.05). In conclusion, our results showed that adding LDL to selected soybean lecithin-based extenders considerably ameliorated the functional parameters of spermatozoa after thawing and thus this lipoprotein could represent an improving agent for soybean lecithin-based extender for bull semen cryopreservation.

Concentration of soybean lecithin affects short-term storage success of goat semen related with seminal plasma removal.

The objective of this study was to investigate the need of seminal plasma removal for short-term cooling of buck semen in soybean lecithin (SL) based extender. Each pool was divided equally, and one half was subjected to centrifugation to remove seminal plasma (SP-), while the other half remained with seminal plasma (SP+). Then, both SP+ and SP- samples were diluted in two SL extenders (extender A = 1% SL; extender B = 2% SL), cooled to 5ºC and stored for 48 hours. The sperm kinetics, evaluated by CASA, and plasma membrane integrity (PMI), acrosomal integrity (ACI) and high mitochondrial membrane potential (HMMP), evaluated by epifluorescence microscopy, were determined within five minutes after reaching 5°C (T0), as well as after 24 (T24) and 48 (T48) hours of storage. Interactions (seminal plasma vs. extender vs. time;) were observed for all variables assessed. Total and progressive motility and other variables of sperm kinetics decreased after 24 hours of cooling in the SP+ group, and after 48 hours of storage, these same variables were lower in SP+/B compared to SP-/B groups. Furthermore, SP+ reduced PMI (extender B, T48), HMMP (A and B extenders, T48) and ACI (extender A, T0) compared to SP- samples. The interactions between seminal plasma and soybean lecithin phospholipids seemed to occur in a time-dependent manner. It was concluded that the removal of seminal plasma improves the quality of goat semen that was cooled in a soybean lecithin-based extender, especially when using 2% soybean lecithin.

Attenuation of cryopreservation-induced oxidative stress by antioxidant: Impact of Coenzyme Q10 on the quality of post-thawed buck spermatozoa

The aim of this study was to determine the quality of post-thawed buck spermatozoa by attenuation of cryopreservation-induced oxidative stress using CoQ10, a lipophilic antioxidant. Ejaculates at every sampling period were collected from four Mahabadi bucks, pooled and diluted in soybean lecithin-based extenders containing 0 (negative control, NC), 0.5 (CQ0.05), 1 (CQ1), and 1.5 (CQ1.5) μM CoQ10 and 0.9% (v/v) DMSO (positive control, PC). The diluted semen was gradually cooled to 4 °C, then frozen and stored in liquid nitrogen. After thawing, total motility although was significantly higher in CQ1 (53.40 ± 1.83) than control groups (43.60 ± 1.83% and 42.20 ± 1.83%; P < 0.05), but this parameter did not differ between CQ1 and CQ1.5. Sperm viability was significantly higher in CQ1 (54.20 ± 2.03%) than that of control and CQ0.5. The CQ1 and CQ1.5 led to significantly higher the plasma membrane functionality compared to control groups. Sperm abnormality was significantly lower in CQ1 than that of NC. The results also showed that MDA level was significantly lower in CQ1 and CQ1.5 compared with control and CQ0.5. The CQ1 (59.43 ± 3.93%) was significantly increased mitochondrial activity compared to control groups. Although a greater value for %DFI was found in NC (10.24 ± 0.48%) and PC (9.77 ± 0.48%) groups compared to others, it was lower in CQ1 group (4.26 ± 0.48%). In conclusion, based on our research results, 1 μM CoQ10 could protect buck spermatozoa from cryoinjury.

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.

Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 106 spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze–thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05).In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation.

Effect of low-density lipoprotein addition to soybean lecithin-based extenders on bull spermatozoa following freezing-thawing - preliminary results

Effect of low-density lipoprotein addition to soybean lecithin-based extenders on bull spermatozoa following freezing-thawing - preliminary results

Preservation of honey bee spermatozoa using egg yolk and soybean lecithin-based semen extenders and a modified cryopreservation protocol

Better protocols and suitable extenders for semen handling and cryopreservation are required for honey bees to enhance sperm quality and fertility similar to that of other species. The purpose of the present research is the improvement of honey bee sperm quality using different semen extenders and a modified cryopreservation protocol. Three different extenders were used: (1) buffer and egg yolk (EY); (2) buffer and 0.5% soybean lecithin (SL0.5); and (3) buffer and 2% soybean lecithin (SL2). Semen was collected and then diluted with the experimental extenders. The diluted semen was gradually cooled in a refrigerator to 5 °C and immediately loaded into straws and frozen with liquid nitrogen. Motility and viability data analyzed using the GENMOD and GLM procedure of SAS software. The results demonstrate that the mean fresh sperm motility in EY (4.75 ± 0.14) and SL2 (4.5 ± 0.2) were significantly (p < 0.05) higher than the SL0.5 (4.12 ± 0.12). Also, the mean of cooled motile spermatozoa in EY and SL2 were significantly higher than the SL0.5 (3.87 ± 0.24). Post-thawed sperm motility in EY (3.6 ± 0.24) was significantly (p < 0.05) higher than the other extenders. Furthermore, percentage of viable spermatozoa in EY (69.75 ± 2.32%) was significantly higher than SL0.5 and SL2 (38.5 ± 2.32 and 45 ± 2.32% respectively. Therefore, according to the results of this study, use of an egg yolk-based extender could better maintain honey bee semen motility and viability after freeze-thawing process, compared to SL-based extenders. However, more advanced tests and fertility evaluation are needed to reveal the exact effects of egg yolk and lecithin based semen extenders on post-thawed drone sperm quality.

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.

Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience

The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3–5 on a 0–5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Semen samples also incubated for 6 h in humidified air with 5% CO2 at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups.

Protective role of glutathione in buck semen cryopreservation.

The aim of this study was to determine the effect of low levels of glutathione on post-thawed buck sperm quality. In this experiment, different concentrations of glutathione [0 (LG-0), 0.5 (LG-0.5), 1 (LG-1), 1.5 (LG-1.5), and 2 (LG-2) mM] were added in a soybean lecithin-based extender. A total of 16 ejaculates from four bucks were collected and pooled. Each pooled sample was divided into five equal parts and each part was diluted by one of the above mentioned groups. After freeze-thawing process, motility and velocity, plasma membrane integrity and functionality, and apoptosis features of spermatozoa were evaluated. The results of this experiment showed that total motility (50.75 ± 2.33), plasma membrane integrity (55.75 ± 3.01) and functionality (46.75 ± 2.79) were higher in LG-1 extender compared to other extenders (P<0.05). The percentage of live spermatozoa (53.23 ± 3.26) was higher in LG-1 extender compared to other extenders, with the exception of LG-1.5 extender (P<0.05). Also, the percentage of late apoptotic spermatozoa (21.33 ± 1.63) was lower in LG-1 extender compared to other extenders (P<0.05). In conclusion, our results showed that GL-1 extender resulted in higher post-thawed buck sperm quality compared to other extenders.

Cryopreservation of ram semen in extenders containing soybean lecithin as cryoprotectant and hyaluronic acid as antioxidant.

A soybean lecithin-based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen-thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1mgml(-1) in a Tris-based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH-PX) activity, lipid peroxidation and acrosomal status after freezing-thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity-related parameters, ALH, BCF, lipid peroxidation, GSH-PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1mgml(-1) HA), total motility (only with 1.5% lecithin), viability (1mgml(-1) HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality-related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze–thawing process of ram sperm

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p<0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p<0.05) viable and lowest (p<0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p>0.05) by different levels of rosemary aqueous extract. Lower (p<0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner.