Percentage Of Total Motility Research Articles

Stress preconditioning of rooster semen before cryopreservation improves fertility potential of thawed sperm

Avian semen cryopreservation is not as successful as that seen in mammals. This failure is mostly attributed to unique physiological characteristics of poultry semen that make it susceptible to cryo-damages. Utilization of sublethal oxidative stress for preconditioning of sperm, as an innovative approach, improves the cryo-survival of sperm in certain mammalian species. The purpose of this study was to investigate the effects of preconditioning of rooster semen with sublethal oxidative stress [very low concentrations of nitric oxide (NO)] before cryopreservation on the quality and fertility potential of thawed sperm. Semen samples were collected from 20 roosters, twice a wk, and different concentrations of NO [0 (NO-0), 0.01 (NO-0.01), 0.1 (NO-0.1), 1 (NO-1), 10 (NO-10), and 100 μM (NO-100)] were used to investigate the effects of controlled induction of sublethal stress before semen cryopreservation on the thawed sperm performance. A significantly higher (P < 0.05) percentage of total motility was observed in semen treated with NO-1 compared to NO-0, NO-0.01, NO-0.1, NO-10, and NO-100. NO-1 and NO-100 produced the highest and lowest percentages of progressive motility, which were significantly different from that of the other groups (P < 0.05). A significantly higher (P < 0.05) percentage of sperm mitochondria activity was observed in semen exposed to NO-0, NO-0.01, NO-0.1, and NO-1. Moreover, the lowest (P < 0.05) concentration of malondialdehyde (MDA) was measured in samples treated with NO-1 in comparison to the other groups. Abnormal morphology, acrosome integrity, and velocity parameters [velocity average path (VAP) and linearity (LIN)] of sperm were not significantly (P > 0.05) affected by different concentrations of NO. Sperm exposed to NO-1 produced the highest percentage of viable spermatozoa (Annexin-/PI-), which was significantly different from the other samples. Finally, rate of fertility after artificial insemination was significantly higher (P < 0.05) following treatment with NO-1 compared to NO-0 and NO-0.1. Application of 1μM NO as a sublethal oxidative stress before cryopreservation of sperm efficiently increased numerous quality indices of thawed sperm as well as its fertility potential.

Geographical differences in semen characteristics of 13 892 infertile men

ObjectiveTo assess the relationship between geographical differences and all semen parameters, across 13,892 infertile men of 84 diverse nationalities, recruited at a specialised tertiary hospital that represents the main healthcare provider in Qatar. Male infertility is an important and global public health problem. Despite this, there is a significant scarcity of epidemiological male infertility and semen analysis research in the Middle East and North Africa (MENA) region, as well as geographical comparisons with other parts of the world. Patients and methodsRetrospective study of semen findings of 13 892 infertile men assessed at the Male Infertility Unit at Hamad Medical Corporation, in Qatar between January 2012 and August 2015. Based on country of origin, patients were categorised into those from the MENA region (n = 8799) and non-MENA patients (n = 5093). The two groups were compared across demographic features and semen characteristics: age, sperm volume, sperm total motility, sperm progressive motility (PMot), abnormal sperm forms (ABF), and sperm DNA fragmentation (SDF). ResultsThe whole sample’s mean (SD) age was 35.7 (0.7) years, sperm concentration was 32.3 (0.25) × 106 sperm/mL, total motility was 45.4 (0.2)%, sperm PMot was 25.1 (0.2)%, and ABF was 79.9 (0.2)%. Overall, 841 patients had azoospermia (6.05%), 3231 had oligospermia (23.3%), 4239 had asthenospermia (30.5%) and 6772 had teratospermia (48.7%). SDF (1050 patients) was abnormal in 333 patients (31.7%). MENA patients were significantly younger than their non-MENA counterparts and had a greater semen volume. Non-MENA patients had significantly higher sperm counts, total motility and PMot, and lower ABF. SDF showed no statistical difference between the two groups. MENA patients had significantly higher prevalence of oligospermia, asthenospermia, and teratospermia; and lower prevalence of normal sperm concentration, normal motility, and normal morphology. Throughout the 4 years of the study, MENA patients constantly had significantly lower sperm counts; generally lower sperm total motility percentage and generally lower quality sperm morphology. We compared patients by age (≤40 and >40 years): in the patients aged ≤40 years, the same results as for the overall study were reproduced; in the >40-years group, the same results were reproduced with the exception of morphology, which was not significantly different between the MENA and non-MENA patients. ConclusionSemen quality is generally lower in male infertility patients from the MENA region compared to non-MENA regions.

The effect of in-vitro myoinositol supplementation of human sperm on the outcome of cryopreservation

Objective: To investigate the effects of in vitro Myo- inositol supplementation of cryopreserved human semen on the post-thaw sperm quality. Design: A randomized controlled clinical trial (RCT). Materials and Methods: The study included 41 fresh semen samples obtained from 41 infertile men. Following routine semen analysis and estimation of sperm motion kinetics by CASA, each sample was divided into 2 identical aliquots. One aliquot (Myo- aliquot) was cryopreserved after Myo-inositol supplementation, while the other aliquot was cryopreserved as it is. Semen analysis and sperm motion kinetics were re – estimated and sperm cryosurvival rate was calculated for both aliquots after thawing. Results: Among patients with abnormal semen samples, Myo aliquot showed a highly significant increase in the following parameters in comparison with control aliquot: Percentage of progressive motility (P< 0.00001), percentage of total motility (P=0.00001), cryosurvival rate (P< 0.00001) and PR recovery rate (P< 0.0001). All sperm motion kinetics were also higher in Myo aliquot than control aliquot among this group, but failed to reach a statistical significant difference. Conclusion: in vitro myoinositol supplementation of human sperm prior to cryopreservation resulted in a highly significant increase in cryosurvival rate among abnormal semen samples, and an increase in cryosurvival rate among normozoopermic samples that failed to reach a statistical significant difference. These findings may pave the way to include myoinositol in the manufacture of human sperm cryopreservation media.

Effect of modifiable lifestyle factors and antioxidant treatment on semen parameters of men with severe oligoasthenoteratozoospermia.

Severe oligoasthenoteratozoospermia (OAT) refers to impaired count, motility and abnormal sperm morphology of infertile men associated with high chromosomal abnormalities. The objective of the present study was to define a management protocol for severe OAT cases and discover new routes to improve their basic semen parameters. We have applied a therapeutic treatment protocol in a cohort of 210 infertile men diagnosed with extreme severe idiopathic OAT. This therapeutic treatment based on modifying the lifestyle factors combined with antioxidant treatment for 6months in severe OAT to study its effect on basic semen parameter. Basic semen parameters were assessed before and after applying the therapeutic treatment strategy. Sperm concentration, percentage of total motility and progressive motility were significantly increased after applying the therapeutic treatment (p=.006, p=.001 and p=.001 respectively). On the other hand, abnormal sperm morphology was significantly reduced after therapy (p<.01). In conclusion, the present results suggested that antioxidative supplement in combination with modifying the lifestyle factors in a cumulative treatment period significantly improves the basic semen parameters.

Porcine sperm vitrification II: Spheres method.

Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n=3; r=4) and raw (n=5; r=2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5×106 spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30s at 37°C) and an ultrarapid method (7s at 75°C, followed by 30s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6-carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.

Effect of dietary fish oil supplementation on ram semen freeze ability and fertility using soybean lecithin– and egg yolk–based extenders

Ram semen cryopreservation is not efficient for artificial insemination in commercial herds. Beneficial effects of dietary fish oil have been evaluated for cryopreservation of ram semen in soybean lecithin (SL) and egg yolk (EY)–based extenders. A factorial study (two diets × two extenders) was used to analyze the effects of two diets supplemented with fish oil (n-3 fatty acid) or palm oil (saturated fatty acids; [SFAs]) to freeze ram semen in two extenders containing SL or EY. Motility characteristics, membrane integrity, abnormal morphology, mitochondria activity, acrosome integrity, apoptotic status, and fertilizing ability were assessed after freeze-thawing. Although diet had significant (P ≤ 0.05) effects on the quality parameters of frozen-thawed sperm, effects of extenders on these traits were not significant (P > 0.05). The higher significant (P ≤ 0.05) percentage of total motility and progressive motility were observed in n-3/SL (44.83 ± 1.56 and 28.33 ± 1.4) and n-3/EY (43.33 ± 1.56 and 28.50 ± 1.4) than SFA/SL (32.16 ± 1.56 and 14.00 ± 1.4) and SFA/EY (31.66 ± 1.56 and 12.66 ± 1.4) groups. Moreover, n-3/SL and n-3/EY produced the higher significant (P ≤ 0.05) percentage of membrane integrity of sperm (39.83 ± 1.4 and 37.33 ± 1.4) than SFA/SL and SFA/EY (29.83 ± 1.4 and 28.5 ± 1.4). For viability results, the higher significant percentage of live sperm was observed in n-3/SL and n-3/EY (43.16 ± 1.38 and 45.66 ± 1.38) than SFA/SL and SFA/EY (28.66 ± 1.38 and 27.5 ± 1.38). For fertility trials, n-3–based diets (n-3/SL and n-3/EY) improved significantly (P ≤ 0.05) pregnancy rate (44% and 46%), parturition rate (42% and 42%), and lambing rate (46% and 44%) compared with the SFA-based diets (SFA/SL and SFA/EY). No interaction effects have been found between diets and extenders (P > 0.05). It seems that dietary fish oil can improve the semen performance after freezing–thawing process and artificial insemination aside from type of extenders.

Effect of sperm concentration on characteristics and fertilization capacity of rooster sperm frozen in the presence of the antioxidants catalase and vitamin E

The objective of this study conducted was to determine the influence of different levels of sperm concentration, including catalase (CAT) and vitamin E (VitE) in rooster semen extender on postthawed quality and fertility of rooster semen. Semen was collected twice a week from six roosters (Arian) and diluted according to experimental treatments consisting of sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) without antioxidant supplementation as control (Con) groups (Con200, Con400, and Con600, respectively), sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplemented with 5-μg/mL VitE (VitE200, VitE400, and VitE600, respectively) and different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplementation with 100 IU/mL CAT (CAT200, CAT400, and CAT600, respectively). After thawing; sperm motility, membrane integrity, and mitochondrial function were assessed. Fertility and hatchability rates were determined by using 100 artificially inseminated hens. The percentage of total motility (TM) and activity of mitochondria decreased (P < 0.05) as the sperm concentration increased in control groups. So, the lowest percentage of the TM and activity of mitochondria were observed in the Con600 as compared with other treatment groups. Extenders containing 100 IU/mL CAT and 5-μg/mL VitE resulted in higher (P < 0.05) TM, progressive motility, membrane integrity, and activity of mitochondria compared with control groups. Adding VitE and CAT in different sperm concentrations, the percentage of TM, membrane integrity, and activity of mitochondria decreased (P < 0.05) as the sperm concentration decreased. The highest (P < 0.05) membrane integrity, TM, and progressive motility were recorded at VitE400 and CAT400. Including VitE and CAT in rooster extender with different level sperm concentrations had no effect (P > 0.05) on fertility and hatchability rates. In conclusion, although adding VitE and CAT in extender with different levels of sperm concentration improved postthawed quality of rooster semen, but adding VitE and CAT in the extender have no effect on fertility rate.

Are the optimum levels of the catalase and vitamin E in rooster semen extender after freezing-thawing influenced by sperm concentration?

To date, there has no report to evaluate the interaction effects of antioxidant and sperm concentration in rooster semen cryopreservation. This study was aimed to investigate the effects of vitamin E (VitE) and catalase (CAT) at different sperm concentrations on the rooster post-thawed sperm quality. Semen samples were collected twice a week from ten roosters (ROS 308) and diluted according to experimental treatments. The treatments consist of different sperm concentrations (200, 400 and 600 × 106 sperm/mL) with supplementation VitE (5 μg/mL; VitE200, VitE400, and VitE600, respectively) or CAT (100 IU/mL; CAT200, CAT400, CAT600, respectively) and without antioxidants [Control (Con); Con200, Con400, Con600, respectively]. After thawing, motion characteristics were assessed using a CASA system. Plasma membrane integrity and malondialdehyde (MDA) level were evaluated with Hypoosmotic swelling test (HOST) and Thiobarbituric acid (TBA), respectively. The higher percentage of total motility, progressive motility, viability and membrane integrity were obtained in VitE400 (81.16 ± 1.21, 18.44 ± 1.19, 85.47 ± 1.07, 86.91 ± 1.16, respectively) and CAT400 (79.38 ± 1.21, 17.19 ± 1.19, 83.42 ± 1.07, 85.73 ± 1.16, respectively) compared to control groups. Moreover, the lowest percentage of MDA was measured in VitE400, VitE600 and CAT400 rather than other groups (1.489, 1.500, 1.510 ± 0.06, respectively). In conclusion, the results of the present study demonstrate that VitE (5 μg/mL) and CAT (100 IU/mL) independently at sperm concentration, 400 million sperm/mL could beneficial effect for preservation of rooster semen during cryopreservation.

Coenzyme Q10 and α-Tocopherol Prevent the Lipid Peroxidation of Cooled Equine Semen.

Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with the following concentrations: α-TOH: 2 mm; CoQ10: 40 μg/ml; and CoQ10 + α-TOH: 40 μg/ml + 2 mm for control (C) without the addition of antioxidants and for vehicle control (EtOH) with 100 μl ethanol. The CoQ10 group had a higher percentage of total motility (69.1 ± 16.2%) compared to control (62.1 ± 16.2%) and EtOH (58.1 ± 18.6%). CoQ10 + α-TOH and α-TOH groups were most effective in preventing LPO compared to controls (1765.9 ± 695.9, 1890.8 ± 749.5, 2506.2 ± 769.4 ng malondialdehyde/10(8) sptz, respectively). In conclusion, CoQ10 and α-TOH were effective during the cooling process of equine semen at 5°C for 72 h, providing increased levels of total motility, as well as lower LPO.

Role of amino acids as additives on sperm motility, plasma membrane integrity and lipid peroxidation levels at pre-freeze and post-thawed ram semen

The possibility of including amino acids for cryopreservation of ram semen to improve the quality of frozen semen was explored in this study in sheep model. 24 samples were collected in triplicate from 8 rams of 2–3 year old Bannur cross bred rams maintained at the Institute Experimental Livestock Unit. Semen was diluted in tris-egg yolk glycerol diluent and made into 7 aliquots as follows: aliquot 1 served as control, “l-alanine” was added at 100 and 135mM in the aliquots 2 and 3, “l-glutamine” was added at 20 and 25mM in the aliquots 4 and 5 and “l-proline” was added at 25 and 50mM in the aliquots 6 and 7, respectively. Diluted semen was filled in 0.25ml French straws and frozen in LN2. Inclusion of “l-proline” and “l-glutamine” in the diluent increased the percent live sperm (P<0.001), total motility (P<0.05) and maintained higher functional membrane and acrosomal integrity (P<0.001) by decreasing lipid peroxidation (P<0.001) compared to the control group. In contrast, “l-alanine” decreased the percentage of total motility, fast progressive spermatozoa and increased (P<0.01) the percentage of immotile spermatozoa. It can be concluded that 20mM “l-glutamine” and 25mM “l-proline” can be used as semen additive to freeze ram semen as they prevented cryoinjuries to sperm and improved the pre-freeze and post-thaw semen characteristics.

The sperm motility pattern in ecotoxicological tests. The CRYO-Ecotest as a case study

Changes in environmental stressors inevitably lead to an increasing need for innovative and more flexible monitoring tools. The aim of this work has been the characterization of the motility pattern of the cryopreserved sea bream semen after exposure to a dumpsite leachate sample, for the identification of the best representative parameters to be used as endpoints in an ecotoxicological bioassay.Sperm motility has been evaluated either by visual and by computer-assisted analysis; parameters concerning motility on activation and those describing it in the times after activation (duration parameters) have been assessed, discerning them in terms of sensitivity, reliability and methodology of assessment by means of multivariate analyses. The EC50 values of the evaluated endpoints ranged between 2.3 and 4.5ml/L, except for the total motile percentage (aTM, 7.0ml/L), which proved to be the less sensitive among all the tested parameters. According to the multivariate analyses, a difference in sensitivity among “activation” endpoints in respect of “duration” ones can be inferred; on the contrary, endpoints seem to be equally informative either describing total motile sperm or the rapid sub-population, as well as the assessment methodology seems to be not discriminating. In conclusion, the CRYO-Ecotest is a multi-endpoint bioassay that can be considered a promising innovative ecotoxicological tool, characterized by a high plasticity, as its endpoints can be easy tailored each time according to the different needs of the environmental quality assessment programs.

Effect of amino acids on sperm motility, velocity parameters, plasma membrane integrity and lipid peroxidation levels at cooled and post–thawed ram epididymal semen

This study is focused to look into the possibility of including amino acids during cryopreservation to improve the quality of frozen semen in sheep. Cauda epididymal semen was collected from 2-3 year-old Bannur crossbred rams slaughtered at the local abattoir. The semen samples were diluted in tris egg yolk glycerol diluent along with different additives and made into 7 aliquots. Aliquot 1 served as control, L-alanine was added @ 100 and 135mM in the aliquot 2 and 3, L-glutamine was added @ 20 and 25mM in the aliquot 4 and 5 and L-proline was added @25 and 50mM in the aliquot 6 and 7, respectively. Diluted semen was filled in 0.25 ml French straws, sealed manually and equilibrated at 4oC for 2 h, cooled in LN2 vapour before being plunged into LN2 till further evaluation. Inclusion of L-proline and L-glutamine in the diluent significantly increased the percent live sperm, total motility, lipid peroxidation and maintained higher functional membrane and acrosomal integrity than the control group. In contrast, L-alanine decreased the percentage of total motility, fast progressive spermatozoa and increased the percentage of immotile spermatozoa. It can be concluded that 20mM L-glutamine and 25mM L-proline can be used as semen additive to freeze ram epididymal semen as they prevented cryo injuries to sperm and improved the pre-freeze and post-thaw semen characteristics.

Crioconservación de semen y calidad espermática en sabaleta Brycon henni (Pisces: Characidae)

La crioconservación de semen es una técnica utilizada tanto en humanos como en animales, permitiendo la congelación del material genético que será utilizado en la reproducción. El objetivo de esta investigación fue evaluar el efecto de tres crioprotectores sobre los parámetros de movilidad y vitalidad en el semen post descongelado de la sabaleta Brycon henni (Pisces: Characidae). Tres crioprotectores: dimetilformamida (DMF), dimetilsulfoxido (DMSO) y etilenglicol (EG), fueron utilizados independientemente para la crioconservación seminal, a una concentración de 5%, el semen fue empacado en pajillas de 0.5 ml. Diecinueve machos sexualmente maduros y 116 pajillas fueron analizadas en el laboratorio de Biotecnología Animal del Politécnico Colombiano Jaime Isaza Cadavid. En la evaluación del semen fresco y post descongelado se utilizó el Sistema de Análisis de Clase (SCA®), para los parámetros de movilidad total (MT), movilidad progresiva (MP), velocidad curvilínea (VCL), velocidad rectilínea (VSL) y velocidad promedio (VAP), entre otros. La vitalidad espermática post descongelación se evaluó mediante microscopia de fluorescencia utilizando la combinación de sondas SYBR/IP. Para el análisis estadístico se ajustaron modelos lineales generalizados (GLM) y las medias se compararon por la prueba de Tukey. Los porcentajes de MT fueron 96.1±3.3% (semen fresco), 52.7±9.2% (DMF), 59.7±9.3% (DMSO) y 62.1±11.2% (EG).Mientras que para la MP fueron de 73.3±14.2 (semen fresco), 11.7±6.7% (DMF), 13.8±6.8% (DMSO) y 15.6±7.8% (EG). No hubo diferencias significativas entre DMSO y EG, pero fueron superiores a DMF (P≤0.05), para ambas variables. VCL, VSL y VAP fueron estadísticamente superiores para EG (P≤0.05). DMSO presentó valores mayores de vitalidad espermática (82.0±1.9%), cuando fue comparado con DMF (73.5±5.9%) y EG (69.2±6.7%) (P≤0.05). Se concluye que DMSO y EG presentan porcentajes superiores de MT y MP que DMF, mientras DMSO produce mejores resultados de vitalidad, cuando se evalúa el semen post descongelado de sabaleta Brycon henni.

Effect of ethanol induced mild stress on post-thawed bull sperm quality

This study was performed to investigate the effect of sub-lethal exposure of bull semen to ethanol on the post-thaw spermatozoa quality. Semen samples (n=24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled. Pooled samples were divided into 4 equal parts and each part was frozen after being diluted with Optidyl® extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9) and 0.15 (O-E15) % (v/v) absolute ethanol. Sperm motility and velocity, plasma membrane integrity and functionality, mitochondrial activity, malondialdehyde concentration, and apoptosis status were evaluated after thawing. A higher percentage of total motility was observed in the O-E9 group as compared to the O-E0, O-E3 and O-E15 groups (p<0.05). Also, plasma membrane integrity was higher (p<0.05) in the O-E9 group compared to the O-E3, and O-E15 groups. However, the difference between the O-E9 and O-E0 groups was not significant (p>0.05). In terms of the proportion of sperm abnormality and plasma membrane functionality no differences (p>0.05) were observed between the groups. Our results revealed that malondialdehyde level was lower in ethanol treated (O-E3, O-E9 and O-E15) groups compared to the O-E0 group (p<0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in the O-E9 and O-E15 groups compared to the O-E0 and O-E3 groups (p<0.05). The O-E3 and O-E9 groups resulted in the highest and lowest percentage of apoptotic spermatozoa, respectively (p<0.05). The results of this study demonstrate that supplementation of semen extender with sub-lethal concentration of ethanol influences post-thawed bull sperm quality in a dose dependent manner.

51 STRESS PRECONDITIONING OF SPERMATOZOA WITH SUBLETHAL CONCENTRATIONS OF ETHANOL IMPROVE POST-THAW SPERM QUALITY IN BULL

A variety of controlled mild stressors have been applied for activation of temporary response in oocytes, embryos, and somatic cells. So far, several stressors have been used to induce mild stress, including that of hydrostatic pressure, osmotic stress, mechanical stress, and oxidative challenges. Based on these evidences, we hypothesised that the ethanol in sublethal concentration would be capable of generating mild stress that may ultimately leads to an adaptive response in spermatozoa. To evaluate this hypothesis, semen samples (n = 24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled for each replicate. Pooled samples were divided into 5 equal parts and each part diluted with tris-glycerol-based (Optidyl®) extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9), and 0.15 (O-E15) % (vol/vol) ethanol and frozen. After thawing, sperm motility and velocity parameters (sperm class analysis), apoptosis status (Phospatidylserin Translocation Detection commercial kit), plasma membrane integrity (eosin-Nigrosin staining), malondialdehyde concentration (thiobarbituric acid reaction), and mitochondrial activity (rhodamine-123 and propidium iodide) were evaluated. The data were analysed using Proc Mixed of SAS 9.1 (version 9.1; SAS Institute Inc., 2002, Cary, NC, USA). Tukey's test was used to compare least squares means. As a result, the O-E9 group showed higher (85.2%) percentage of total motility compared with O-E0 (73.6%), O-E3 (51.9%), and O-E15 (67.5%) groups (P &lt; 0.05). A highest (P &lt; 0.05) percentage of live spermatozoa were observed in the O-E9 (62.9%) group as compared with O-E0 (49.4%), O-E3 (50.3%), and O-E15 (49.6%) groups, and also the proportion of apoptotic spermatozoa in the O-E9 (10.6%) group tended to be lowest as compared with those of O-E0 (15.6%), O-E3 (17.2%), O-E15 (14.1%) groups (P &gt; 0.05). The plasma membrane integrity was higher (P &lt; 0.05) in O-E9 (90.8%) compared with O-E3 (75%) and O-E15 (77.2%) groups; however, the difference was not significant when the O-E9 group was compared with the O-E0 group (83.2%; P &gt; 0.05). Obtained results revealed that malondialdehyde level was lower in O-E3 (1.03%), O-E9 (0.63%), and O-E15 (0.89%) groups compared with the O-E0 (1.94%) group (P &lt; 0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in O-E9 (57.7%) and O-E15 (57.5%) groups compared with O-E0 (49.1%) and O-E3 (38.2%) groups (P &lt; 0.05). These results strongly suggest that supplementation of Optidyl® extender with sublethal concentration of ethanol influences post-thawed bull sperm quality in a dose-dependent manner. However, further studies are needed to empirically determine the effect of supplementation on fertilization and pregnancy outcome.

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.

FREEZABILITY AND DNA INTEGRITY OF DROMEDARY CAMEL SPERMATOZOA IN SEMEN COLLECTED BY ARTIFICIAL VAGINA AND ELECTRO-EJACULATOR

Two methods of semen collection from dromedary camel bulls, including electro-ejaculation (EE) versus conventional artificial vagina (AV) were compared in term of their effects on reaction time, physical semen characteristics, sperm biometry, alkaline comet assay of spermatozoa, sperm freezability and concentration of testosterone, some minerals and biochemicals as well as activity of some enzymes in blood serum of camel bulls. Results showed that using EE significantly increased reaction time, total sperm output per ejaculate, head length and tail width of spermatozoa, total percentage of sperm head showing comet and serum asprtate transaminase (AST) activity, while significantly decreased sperm cell concentration, serum cholesterol, magnesium, zinc, inorganic phosphorus and testosterone concentrations. However, ejaculate volume, percentages of total motility, forward motility, dead, abnormality and acrosome damage of spermatozoa, head width, tail length, and total length of spermatozoa, grades of comet assay, sperm freezability, concentration of total proteins and their fraction, serum Na, Ca and K concentrations and activity of alanine transaminase (ALT) and alkaline phosphatase (ALP) were not affected by collection method. Electro-ejaculation is considered as a suitable and repeatable technique for semen collection from dromedary camel bulls when semen collection by conventional artificial vagina is impossible.

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24h at 17°C) and after freezing and thawing (at 30 and 150min post-thawing in semen samples kept in a water bath at 37°C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P<0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk–based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate–conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration.

Effects of age and sexual maturation on expression of glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase histochemical activity in stallion reproductive tissue

6.1 mM cholesterol-loaded cyclodextrin was added after dilution. Semen of both groups was cooled-stored at 5 C for 24 and 48 hours. Semen samples were evaluated before and after cooling by CASA for total motility (TM), progressive motility (PM), rapid sperm (RAP) and plasma membrane integrity (PMI) by epifluorescence microscopy after staining with carboxyfluorescein and propidium iodide. All parameters were analyzed by ANOVA, followed by unpaired T-test. Cholesterol-treated samples showed higher percentages of TM, PM, RAP and PMI at both 24 and 48 hours (P < 0.001) than control samples (Table 1).