Percentage Of Membrane Integrity Research Articles

Oral administration of royal jelly may improve the preservation of rooster spermatozoa.

The aim of the present study was to investigate the effect of dietary supplementation of royal jelly (RJ) on liquid and frozen storage of rooster spermatozoa. Twenty-five 30-week-old of Mazandaran native breeder roosters were randomly divided into five treatments (n=5 roosters/group). Experimental treatments are designed to include a control group and various levels (0.0 (RJ0), 100 (RJ100), 200 (RJ200), 300 (RJ300) mgkg-1 BW-1 ) of royal jelly (RJ) that were fed to the roosters using force-feed method. The percentage of forward progressive motility, abnormal spermatozoa, membrane integrity and viability of spermatozoa evaluated after 24 and 48hr of cooling (at 4°C) and after the freeze-thawing process. Also, mitochondrial activity and DNA fragmentation in fresh (24hr) and post-thawed spermatozoa were assessed. The result of this study showed that the spermatozoa forward progressive motility, abnormality, membrane integrity, and viability were improved by the RJ100 group compared to the other groups after 24 and 48hr storage period at 4°C. The percentage of membrane integrity and forward progressive motility after freeze-thawing in the RJ100 group was significantly higher than the other groups, and the percentage of abnormal spermatozoa was lower. A significant decrease in semen quality parameters was seen after 24 and 48hr of refrigeration, but there was no observed change between 2 and 24hr in the RJ100. The viability percentage of spermatozoa in both RJ100 and RJ200 groups was not different. Moreover, after freeze-thawing, DNA integrity and mitochondrial activity in the RJ100 group were significantly higher than the other groups. According to our results, feeding of RJ at 100mgkg-1 BW-1 to the roosters was improved spermatozoa characteristics during liquid and cryopreservation conditions.

Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue

The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

Using Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality

Reactive oxygen species levels in semen with low quality increased from 25% to about 40%. Resveratrol (Res) is a natural grape-derived phytoalexin, and epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to have high levels of antioxidant activity. The aim of this study was to investigate the effects of this antioxidant's supplementation on cryopreservation of stallion semen with poor quality. Experiment 1: Experiment design to asses stallions semen quality (n = 3). Arabian stallions were managed for breeding with fresh, extended, or chilled semen during 2 years (in two breeding season) with a resulting per cycle pregnancy rate (103/41 estrus cycle/conception) of 39.80%. A total of 81 ejaculates were collected and evaluated in fresh condition (total and progressive motility, membrane integrity, acrosome defects, morphology, and abnormal DNA condensation). Evaluation of several ejaculates revealed that sperm production and semen quality mostly did not change during the breeding season, maintaining normal sperm production with poor quality. Experiment 2: 18 ejaculates, collected with Missouri model artificial vagina from Arabian stallion (n = 3). Extended semen was centrifuged at 600 × g for 10 minutes. To eliminate individual stallion variability, the resulting sperm pellets were pooled and divided in seven different treatments in a final concentration of 100 × 106 sperm/mL by using semiskimmed milk extender containing no supplements (control), and the following supplements with three different concentrations: Res 5, 10, and 20 μM (Res 1, Res 2, Res 3, respectively) and EGCG 25, 50, 100 μM (EGCG 1, EGCG 2, and EGCG 3, respectively). After thawing, all samples were maintained at 37°C for 10 minutes. Evaluation of motility, plasma membrane integrity, viability, mitochondria activity, DNA fragmentation, and lipid peroxidation was determined. For all semen parameters, the lower concentration of Res (5 μM) and EGCG (25 μM) did not show significant differences compared to control (P > .05). Relative to the control group, Res 2 had better (P < .05) postthaw total and progressive motility, yet the higher concentrations (Res 3) showed a negative effect on total motility. Percentage of membrane integrity, viability, and mitochondrial activity was significantly greater in Res 2 and EGCG 2 extenders in comparison with the control group, respectively (P < .05). Results showed that malondialdehyde concentration was lower in Res 2 and EGCG 2 groups. In conclusion, the present study showed that the antioxidant Res prevents DNA damages in our stallions. The addition of Res and EGCG before cryopreservation can ameliorate mitochondrial activity in stallions with low-quality semen. Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damages, and preserve sperm plasma membrane integrity and mitochondrial activity.

Tolerance of Basil Genotypes to Salinity

Basil (Ocimum basilicum L.) is a medicinal species of Lamiaceae family, popularly known for its multiple benefits and high levels of volatile compounds. The species is considered to be one of the most essential oil producing plants. Also cultivated in Brazil as a condiment plant in home gardens. The objective of this study was to evaluate the effect of salinity on the growth of basil in nutrient solution of Furlani and to identify variables related to the salinity tolerance in this species. The first assay was performed with variation of five saline levels (0 - control, 20, 40, 60 and 80 mM NaCl). In the second assay six genotypes were evaluated in two salinity levels 0 and 80 mM NaCl. The height, stem diameter, number of leaves, dry mass and inorganic solutes in different organs, photosynthetic pigments, absolute membrane integrity and relative water content were evaluated. All biometric variables in basil were significantly reduced by salinity. Dry matter yield and percentage of membrane integrity were the variables that best discriminated the characteristics of salinity tolerance among the studied basil genotypes. Basil genotypes showed a differentiated tolerance among the genotypes, the ‘Toscano folha de alface’ being considered as the most tolerant and ‘Gennaro de menta’ as the most sensitive, among the species studied.

Growth and contents of organic and inorganic solutes in amaranth under salt stress

ABSTRACT Amaranthus cruentus L. is a forage species, with grains that exhibit excellent nutritional characteristics, being the 'BRS Alegria' the first cultivar recommended for cultivation in Brazil. This study aimed at evaluating the effect of salt stress on the growth and concentrations of organic and inorganic solutes in Amaranthus cruentus L. ('BRS Alegria' cultivar). Height, stem diameter, number of leaves, leaf, stem and root dry mass, leaf area, relative water content and membrane integrity percentage, as well as soluble carbohydrate, free amino acid, soluble protein, free proline, Na+, Cl- and K+ contents, were evaluated in different plant organs. Salinity significantly reduced the biomass yield and leaf area from the treatment with 25 mM of NaCl, indicating that the 'BRS Alegria' cultivar is sensitive to salt stress. Soluble carbohydrates in the leaves decreased by 59 %, while the other organic solutes showed no substantial increases. These results, coupled with the reduction in the relative water content and membrane integrity, suggest a low ability of this cultivar to adjust osmotically under salt stress. The K+/Na+ ratio abruptly decreased in 25 mM of NaCl, suggesting an ionic imbalance, which may partially explain the salt-induced growth reduction.

Natural honey as a cryoprotectant to improve Arab stallion post-thawing sperm parameters

ObjectiveTo investigate the effect of extender supplementation with different concentrations of a honey bee on post-thawed sperm motility, viability index, membrane and acrosome integrities in Arab stallion. MethodsFive ejaculates from each of four Arabian stallions were subjected to cryopreservation with a modified INRA-82, without any supplementation (control) or supplemented with 1%, 2%, 3%, 4% and 5% honey bee. After thawing, all samples were maintained at 37 °C, while analyses were performed at 0, 1, and 2 and 3 h. Sperm motility percentage, viability index percentage, membrane integrity percentage and acrosome integrity percentage of each sample were determined by conventional laboratory methods. ResultsRelative to the control group, supplementation with honey (2%, 3% and 4% significantly improved (P < 0.01 at least) post-thaw sperm motility, viability index (P < 0.001 at least) and had a positive effect on membrane integrity and intact acrosome percentage (P < 0.001 at least) at 0, 1, 2 and 3 h post-thawing. For all semen parameters, the lower concentration of honey (1%) and higher concentration (5%) did not show significant differences (P > 0.05) compared with the control. ConclusionHoney bee supplementation in Arab stallion semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

Effect of dietary Satureja khuzistanica powder on semen characteristics and thiobarbituric acid reactive substances concentration in testicular tissue of Iranian native breeder rooster.

Because of a paucity of information on the effect of Satureja khuzistanica in male chickens, this study was undertaken to determine the influence of dietary S. khuzistanica powder (SKP) on seminal characteristics and testes thiobarbituric acid reactive substances (TBARS) content in Iranian native breeder rooster. Thirty-six 40-week-old roosters were randomly allotted to 3 equal groups and received either a basal diet without SKP (T1 or control), or a diet containing 20 g/kg (T2) and 40 g/kg (T3) of SKP for 8-week-long experimental period. Semen samples were obtained weekly by abdominal massage to evaluate the seminal characteristics. At the end of the eighth week 18 birds (6 birds per each group) were randomly slaughtered, and sample was taken from right testes for TBARS evaluation. Administration of SKP improved all semen traits, except for sperm concentration. Likewise, TBARS content in SKP treatments did not significantly differ from the control (P>0.05). Seminal volume, live sperm percentage and plasma membrane integrity percentage in SKP-treated groups were higher than the control. Conversely, abnormal sperm percentages reduced in SKP-treated groups (P<0.05). Plasma membrane integrity in experimental treatments was significantly higher than the control in 2nd, 3rd and 7th weeks. However, at 6th and 8th weeks only T3 treatment was significantly different from the control. Notably, there was an increase in total sperm concentration in SKP-treated groups in compared to the control birds. In conclusion, this study indicated that addition of SKP in rooster diet improves sperm quality and also reduces their sperm membrane lipid peroxidation, which may lead to higher fertilization rate.

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.

The quality of Garut ram liquid semen in Tris egg yolk extender to the sucrose supplementation

The successful program of artificial insemination in sheep is determined by the quality of sperm that are used. Therefore, it is important to maintain the quality of sperm during and after storage in low temperature. The research was conducted to study the influence of sucrose in the Tris-yolk extender in maintaining the quality of Garut ram sperm during preservation in reaction tube for four days at 5°C. Ejaculated sperm was collected once a week for five weeks using artificial vagina from same ram. Semen was divided into four groups of extenders, i.e. Tris egg yolk 20% (TKT), TKT + 0,1% sucrose, TKT + 0,3% sucrose and TKT + 0,5% w/v sucrose. The results showed that the percentage of motility on day four (D-4) of storage in TKT (41.00 ± 2.0%) was lower (P 0.05) from TKT + 0.1% (45.00 ± 3.16%). Whereas the percentage of live cells (%H) on D-4 in TKT, TKT + 0.1%, TKT + 0.3% and TKT + 0.5% were 55.00 ± 2.19%; 57.20 ± 2.79%; 59.20 ± 3.25% and 61.20 ± 3.60%, respectively. Meanwhile, the percentage of membrane integrity (MPU) in TKT (51.80 ± 1.94%) was significantly different (P 0.05) from TKT + 0.1% (54.80 ± 2.86%). In conclusion, the addition of sucrose 0.3% w/v into TKT extender could maintain the quality of Garut ram sperm more efficiently and better than TKT extender. Key Words : Sucrose, Tris Egg Yolk, Preservation, Garut Sheep

80 CHOLESTEROL-LOADED METHYL-BETA-CYCLODEXTRIN IMPROVES BOAR SPERMATOZOA CRYOPROTECTANT ADDITION AND REMOVAL TOLERANCE

Difficulties associated with the cryopreservation of boar sperm include their sensitivities to osmotic stresses and chilling sensitivity. We investigated the effects of cholesterol-loaded cyclodextrin (CLC) on boar sperm motility and membrane integrity following exposure to various osmolalities. Samples were collected using the gloved hand method from crossbred boars, and ejaculates having greater than 75% motility were extended 1:3 with Androhep (Minitube Inc., Verona, WI) for this study. Samples were centrifuged at 700g for 5 min, and the resulting pellets were resuspended to 1.2 × 108 cells mL–1 in Androhep. Samples were then treated with 0, 1.5, or 3.0 mg of CLC/1.2 × 108 cells mL–1 for 10 min at room temperature. In experiment 1, samples were aliquoted into 1.5-mL centrifuge tubes, centrifuged at 700g for 5 min and the sperm exposed to Dulbecco’s PBS at different osmolalities for 5 min before being returned to 300 mOsm by adding Dulbecco’s PBS solutions at differing osmolalities. After returning the sperm to isosmotic conditions, sperm motility was analyzed. In experiment 2, samples were treated as in experiment 1 and following exposure to the various osmolalities, sperm were stained with Alexa 488-PNA and propidium iodide to determine sperm membrane integrity. Ten thousand sperm per treatment were analyzed by flow cytometry. Data were analyzed by standard ANOVA. The CLC-treated sperm (normalized means ± SEM; 33 ± 16, 80 ± 8, 86 ± 5, 100, 64 ± 4, 7 ± 3, 0 ± 0, respectively) exhibited greater percentages of motile cells following hypo-isosmotic exposure than control sperm (4 ± 1.6, 33 ± 9.6, 84 ± 7.1, 100, 37 ± 5.5, 3 ± 1.6, 0 ± 0, respectively), and there was a tendency for CLC-treated sperm (P = 0.0225) to maintain motility following hyper-isosmotic exposure. In addition, CLC-treated sperm (87 ± 4, 93 ± 1, 95 ± 1, 93 ± 2, 88 ± 4, 83 ± 3, 41 ± 9, respectively; P &lt; 0.05) maintained greater percentages of membrane integrity following treatment with anisosmotic solutions compared with controls (29 ± 8, 63 ± 10, 81 ± 7, 92 ± 3, 73 ± 8, 44 ± 5, 21 ± 9, respectively). Using a combination of these osmotic tolerance data with previously published boar sperm membrane permeability characteristics, we mathematically modeled the number of steps needed for the addition or removal of cryoprotectants. Computer simulations indicate that an abrupt addition of 1 m glycerol will cause boar sperm to exceed their osmotic tolerance limits unless they are treated with 3 mg of CLC. Moreover, the addition of 1 m EG causes boar sperm to exceed all osmotic tolerance limits and therefore, the addition and removal of EG requires multiple-step protocols. However, the addition and removal of 1 m DMSO maintains volume excursions well within the osmotic tolerance limits with the addition of cholesterol (1.5 and 3 mg). Empirical data for addition of CPA have shown similar results as seen with the computer simulation. These data support the hypothesis that adding cholesterol to porcine sperm broadens their osmotic tolerance limits and potentially provide a mechanism to increase post-thaw survival of porcine sperm.

The quality of spotted buffalo epididymal sperm with addition of raffinose as external cryoprotectant

The aims of this research was to obtain the quality of Spotted buffalo epididymal sperm in different kind of extender in the three stages of cryopreservation (after dilution, post equilibration and post thawing). Spermatozoa was collected with combination of slicing and pressure method into the epididymal tissue in Andromed â extender. Soon after diluted and equilibrated, epididymal spermatozoa was cryopreserved in liquid nitrogen (-196°C). The result showed that the percentage of motility after thawing in Andromed â + raffinose 0.4% (47.0 ± 2.4%), was significantly different (P 0.05) from that of Andromed â + raffinose 0.2% (46.0 ± 2.0%). The percentage of live sperm after thawing in control (52.2 ± 2.5%), was the lowest and significantly different (P 0.05) in the percentage of membrane integrity from all treatments. In conclusion, the addition of 0.2 and 0.4% raffinose into Andromed â extender could improve the percentage of motility and viability of post thawing spotted buffalo epididymal spermatozoa. Key Words : Epididymal Sperm, Cryopreservation, Raffinose, Spotted Buffalo

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5 mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 × g/5 min) just after sorting and stored 16–18 h at 17 °C (groups Cc and Hc) at 6 × 10 6 sperm/mL. The second aliquot was directly stored at 17 °C for 16–18 °C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 × 10 6 sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 × 10 6 sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16–18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower ( p < 0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer ( p < 0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.

Quality of canine epididymal spermatozoa during storage at 4C

The aim of this present study was to investigate the quality of canine epididymal spermatozoa during storage at 4°C. Spermatozoa was collected by flushing technique with physiological saline (NaCl 0.9% w/v) and diluted in modified Tris based extender containing 20% (v/v) of egg yolk for three days. The result showed that mean concentration of spermatozoa from cauda epididymal was 95.29.10 6 spz/ml. The percentage of progressive motility and membrane integrity of spermatozoa on time of collection was 70.71% and 72.85%, respectively. Quality of epididymal spermatozoa was decreased significantly (P<0,05) during storage at 4°C. The percentage of progressive motility during storage were 70.71% on day 0 (H-0, after diluted), 60.71% (H-1), 45.71% (H-2), and 33.57% (H-3). The percentage of membrane integrity during storage were 72.85, 68.88, 61.06 and 47.47% on H-0, H-1, H-2 and H-3, respectivelly. In conclusion, quality of canine epididymal spermatozoa was decreased during three days of storage at 4°C. Key Words : Epididymal Sperm, Storage, Canine