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.     

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.     

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.     

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.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

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

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

Influence of extender,temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell); IMV, L'Aigle, France) free from additives of animal origin, containing two different final glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 degrees C or at 15 degrees C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no significant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 degrees C, although the results were not always statistically significant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5-10%) concentrations of egg yolk and the addition of glycerol at 5 degrees C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell containing 6.4% glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk.     

The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R²=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R²=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.     

真鲷仔鱼用微粒子饲料中肽酶与大豆卵磷脂添加效果的研究

本研究以酪蛋白分解物为蛋白源配制三种微粒子饲料MD-S、MD-T和MD-U对真鲷开口仔鱼进行饲养试验。以MD-S的配方为基准,MD-T采用粉状大豆卵磷脂和麸质代替液状大豆卵磷脂;MD-U则另外添加0.1%的肽酶。结果表明,微粒子饲料在水中浸泡15min后,MD-T的溶出率(35.5%)低于MD-S(46.8%)和MD-U(45.8%);实验结束时(20日龄),仔鱼的成活率以生物饵料(轮虫)组为最高(86.3%),其次是MD-T组为20.7%,显著高于(P<0.05)MD-S组(13.3%)和MD-U组(13.6%);生物饵料组的仔鱼全长(6.14±0.49mm)显著大于微粒子饲料组(4.23±0.30mm~4.46±0.30mm),各微粒子饲料组之间仔鱼的全长并不存在显著差异(P>0.05)。在孵化后第12d,微粒子饲料组的仔鱼肠上皮细胞发育良好,但至孵化后第18d,仔鱼肠上皮细胞大部分萎缩、并发生脱落。鱼体的蛋白质、DNA与RNA日间增长率微粒子饲料MD-T组高于MD-S和MD-U组,但都低于生物饵料组。由此可见,微粒子饲料中添加肽酶并无助真鲷仔鱼对其消化吸收;可是,使用粉状大豆卵磷脂与麸质代替液状卵磷脂能增强微粒子饲料的黏合性,可减少其营养成分的溶出率,从而提高微粒子饲料的饲育效果。     

Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Effect of antioxidants on microscopic semen parameters,lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation

The aim of this study was to determine the effects of the antioxidants glutamine and hyaluronan and the inclusion of different levels on microscopic semen parameters, lipid peroxidation and the antioxidant activities following the freeze–thawing of Angora goat semen. Ejaculates collected from three Angora goat bucks, were evaluated and pooled at 37 °C. The semen samples which were diluted with a Tris-based extender containing additives including glutamine (2.5; 5 mM) and hyaluronan (500; 1000 μl/ml), and an extender containing no antioxidants (control) were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually (37 °C) for 20 s in a water bath for microscopic evaluation. Freezing extenders supplemented with 2.5 and 5 mM glutamine led to higher sperm motility and hypo-osmotic swelling test (HOST) values, compared to the control (P < 0.05) following the freeze–thawing process. The addition of 500 μl/ml hyaluronan resulted in a higher HOST percentage, compared to the addition of 1000 μl/ml hyaluronan and the control (P < 0.001). No significant difference was recorded in the percentage acrosome and total sperm abnormalities, following supplementation with antioxidants. The addition of antioxidants did not prevent malondialdehyde (MDA) formation, compared to the controls. Antioxidant treatment however decreased (P < 0.01) the superoxide dismutase (SOD) activity. The maintenance of catalase (CAT) activity was demonstrated to be insignificant following addition of antioxidants. Further studies are required to obtain more repeatable results regarding the characterization of the enzymatic and non-enzymatic antioxidant systems in cryopreserved goat sperm.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

Cryostorage of silver barb (Barbodes gonionotus) semen in dry shipper: Efficacy,risk of bacterial cross-contamination and effects of Aeromonas hydrophila on post-thaw sperm quality

This study was conducted to investigate the efficacy of dry shipper for the cryostorage of silver barb (Barbodes gonionotus) sperm, the subsequent risk of bacterial cross-contamination, and the effects of Aeromonas hydrophila on post-thaw sperm. Semen was diluted with calcium-free Hank's balanced salt solution containing 10% ME₂SO, frozen at −8 °C/min and stored for 14 d in a dry shipper. A significant decline (P < 0.05) in the post-thaw sperm motility and viability of samples kept in the dry shipper for 14 d showed a reverse correlation (P < 0.05) with a slight increase in temperature within the dry shipper. The levels of contaminated bacteria in the compartments of the dry shipper were significantly (P < 0.05) lower than those detected in the liquid nitrogen tank. Bacteria from the atmosphere could recontaminate the chambers of the dry shipper and liquid nitrogen tank after 14 d. Bacillus was the most common bacteria isolated from the dry shipper, liquid nitrogen tank, circulating air, bench surface and outer surface of straws. There was no cross-contamination of A. hydrophila from contaminated straws to pathogen-free straws kept in either cryogenic tank. Post-thaw sperm motility and sperm viability significantly (P < 0.05) declined during cryostorage in the dry shipper and liquid nitrogen tank due to the introduction of A. hydrophila and the interaction effect of A. hydrophila and freezing. This study reports, for the first time, the efficacy of a dry shipper for the cryostorage of fish sperm for at least 14 d without a risk of bacterial cross-contamination.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Effect of GnRH administration, combined with the ram effect, on the occurrence of ovulation and pregnancy during the transition period from anoestrus in crossbred ewes

The effect of a GnRH analogue (buserelin) combined with the ram effect on the reproductive efficiency of ewes was investigated in 105 cross-bred fat tailed ewes, during the transition period from anoestrus to the natural breeding season. Plasma progesterone concentration was used in the assessment with regard to ovulation and pregnancy. Ewes were maintained on natural pastures composed of medium to low quality forages, and received supplementation (40% alfalfa hay: 60% wheat straw) ad libitum, plus 100–300 g barley grain per head per day. Ewes were isolated from the rams for at least two months and then kept in close proximity of the rams for one week, before the introduction of the rams. The ewes were randomly divided into three equal groups (n = 35 per group). With the introduction of the rams into the flock (day 1) one group was considered as the control and the other two groups were treated with 4.2 μg (low dose) and 8.4 μg (high dose) buserelin on days 5 and 19, respectively. Blood samples were collected on days 5, 12, 26 and 120 after ram introduction for determining the plasma progesterone levels. On day 12 (one week after treatment) the high dose GnRH group recorded significantly lower plasma P4 concentrations (P < 0.05), compared with the control group (1.0 ± 0.1 ng/ml versus 2.4 ± 0.4 ng/ml). On the same day the low GnRH dose group recorded intermediate P4 concentrations, recording no significant differences with the other two groups. The high dose group recorded a significantly (P < 0.05) higher proportion of non-ovulated ewes (61.8%), compared to the control (32.3%) and low dose (31.4%) groups on day 12 of the study. At days 5 and 26 these differences were not significant, but the proportion of non-ovulated ewes was higher in the high dose buserelin treatment group. The percentage of pregnant (plasma P4 > 2.5 ng/ml) and non-pregnant (plasma P4 ≤ 2.5 ng/ml) ewes at day 120 of the study was not statistically different between the treatment groups. The pregnancy rate was highest in the control group (97.1%), when compared to the treated ewes (94.3% and 88.6% in low dose and high dose treatment groups, respectively). Treatment with buserelin combined with the male effect during the breeding season negatively affected the plasma P₄ concentration, reducing the reproductive performance of the ewe treatment groups.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.