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.     

Development of extender based on soybean lecithin for its application in liquid ram semen

The soybean lecithin is used as a phospholipids source for the commercial extenders available for freezing bull semen which allows replacing the traditional membrane protective of animal origin (egg yolk). These extenders have been tested for freezing semen in various livestock species but specific adjustments cannot be made due to trade protection. The aim of the present study was to develop a soybean-based extender analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. Its effect on the quality of liquid ram semen was also studied. Different TES-Tris-Fructose-based extenders were prepared using two soybean types (S20 and S95) differentiated by their lipid composition (complex or simple, respectively). These extenders were made up in two temperatures: 20 °C (PT20) or 37 °C (PT37); centrifuged and filtered at 20 °C and stored at 15 °C or 5 °C (ST15 and ST05) for several periods (from 6 hours to 7 days). Three different concentrations of soybean (0.5%, 2%, and 3.5%) were evaluated for each extender. The amount and nature of phospholipids present in the extender were evaluated by high performance liquid chromatography (HPLC) method according to the different parameters applied in their preparation. In general, the highest quantity of phospholipids is observed in S20 extender. Centrifugation-filtration process during the extender preparation reduces by 50% the quantity of phospholipids in medium for different experiments. The quantity of phospholipids was not affected significantly by preparation temperature in S20 extender. Storage temperature affects the phospholipids present in the extender (S20 and S95) with minimum values for the storage at 5 °C. As for the storage time, both extenders (S20 and S95) showed a stable quantity of phospholipids in the course of the time, for 2 days at 15 °C and for 7 days at 5 °C. The extender obtained with a higher concentration of soybean (3.5%) showed a higher content of phospholipids under different conditions tested. Finally, sperm motility and viability in new extenders were analyzed. We observed that the sperm quality is not affected by storage temperature for S20 extender. Sperm motility was higher in S20-2% extender and control (UL). Our results suggest that a soybean lecithin extender obtained from S20 soybean at 20 °C, centrifuged and filtered, preserve the sperm motility and viability at 15 °C and 5 °C as an egg-yolk extender.     

Head morphometric changes in cryopreserved ram spermatozoa are related to sexual maturity

The importance of understanding the sperm changes after the cryopreservation process has been emphasized in human and veterinary andrology. In previous studies, we have shown that the morphometric characteristics assessed by computer-assisted analysis following the freeze-thawing process revealed differences in terms of dimension and shape between individuals that may be related to bio-physiologic factors such as sexual maturity. The purpose of this study was to determine if there are differences associated with cryoresistence and sperm head morphometric dimensions in individuals with different sexual maturity ratings (SMRs; 12, 30 and 96 months of age). Ejaculates from nine normospermic fertile rams with different SMRs were analyzed in an attempt to quantify the morphometric dimensions and the shape of sperm heads from each group after the cryopreservation process. The mean values of sperm concentration among individuals with different SMRs were significantly different (P < 0.01). Cryopreservation substantially reduced sperm motility and plasma membrane integrity irrespective of SMR assessed, with young animals being the most affected (P < 0.01). Sperm quality at thawing for all sperm parameters evaluated was significantly higher for old individuals than for middle-aged or young individuals (P < 0.01). There were no significant differences in the sperm head dimension or shape among middle-aged and old individuals (P > 0.05). However, significant differences were detected in area, perimeter and width (lower values) and length, ellipticity and elongation (higher values) in old or middle-aged individuals compared with young individuals (P < 0.01). In conclusion, this study confirms that ram age is related to sperm morphometric dimensions, and sperm size and shape may affect spermatozoa survival, being good indicators of freezability. Therefore, the present study provides information on the morphometric maturation of ram sperm and supports the idea that the dimensions of spermatozoa may be taken as an approximate indication of its relative maturity.     

In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen

Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02 ± 0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm.     

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Determination of the IgE-binding activity of soy lecithin and refined and non-refined soybean oils

In the present study refined and non-refined soybean oils as well as soy lecithins were investigated for residual allergenicity and compared with extracts from native soybeans. By means of immunoblotting and EAST inhibition experiments no IgE-binding activity was detectable in refined soybean oils, which is probably due to thermal treatment during the refining. The investigated non-refined oils and soy lecithins showed a residual IgE-binding activity. In addition in the lecithin extracts a new IgE-binding structure with a molecular mass of approximately 16 kDa was detectable.     

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 °C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 °C, dilution, equilibration, and thawing) had negative effects on motility (P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

大豆卵磷脂对小鼠的抗疲劳和抗氧化作用研究

目的：研究大豆卵磷脂的抗疲劳及抗氧化作用。方法：小鼠经口给予大豆卵磷脂30天后,采用负重游泳实验,观察记录小鼠游泳死亡时间;检测血清尿素氮、肝糖原;测定血清和肝匀浆超氧化物歧化酶（SOD）活性、谷胱甘肽过氧化物酶（GSH—Px）活力、丙二醛（MDA）含量。结果：给予大豆卵磷脂后,与对照组相比,实验组小鼠负重游泳时间明显延长,肝糖原消耗量减少,降低运动后血清尿素氮水平（P〈0．05）;升高小鼠血清和肝匀浆SOD活性及GSH-Px活力,降低MDA的含量（P〈0．05）。结论：大豆卵磷脂具有抗疲劳和抗氧化作用。     

Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures

Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1 M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 °C) for 30 min and subsequently returning them to 37 °C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm (P < 0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1 M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P > 0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm after exposure to 1 M Gly, DMSO or EG (P > 0.05). Both epididymal and ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity (P < 0.05). However, ejaculated ram sperm was more sensitive to chilling stress than epididymal sperm (P < 0.05). In conclusion, the current data suggest that while epididymal ram sperm is extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These findings should be taken into account when developing cryopreservation protocols for ejaculated and epididymal ram sperm.     

In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen

Substitution of egg yolk with soybean lecithin may reduce hygienic risks in extenders. Though a few studies have been performed on the effect of soybean lecithin in bull, to date evaluation of ram semen in vitro fertility after cryopreservation with use of soybean lecithin has not been studied. This study assessed the effect of 1% or 2% (wt/vol) soybean lecithin (L1 or L2) or 15% or 20% (vol/vol) egg yolk (E15 or E20) supplemented with 5% or 7% glycerol (G5 or G7) in a Tris-based medium for cryopreservation of ram (Oviss arries) semen. Although no significant difference was observed in pattern of capacitation, the best results in terms of sperm motility, viability postthaw, and cleavage rates were observed with L1G7 (51.9 ± 4.8%, 48.1 ± 3.5%, and 79.6 ± 3.9%, respectively) and E20G7 (51.8 ± 2.9%, 46.7 ± 4.0%, and 72.9 ± 6.4%, respectively). Our results also showed that 1% lecithin and 20% egg yolk was superior to 2% lecithin and 15% egg yolk. In terms of cleavage rate, 7% glycerol was superior to 5% glycerol. No significant difference was obtained between groups in terms of blastocysts rate per cleaved embryo. Therefore, we concluded that the optimal concentration of lecithin and egg yolk is 1% and 20%, respectively, along with 7% glycerol. In addition, our results suggest that lecithin can be used as a substitute for egg yolk.