Eosin-nigrosin Staining Research Articles

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurement of spermatozoal membrane status using computer-assisted spermatozoal quantification (CASQ) and eosin-nigrosin (EN) staining with manual counting after CFDA/PI staining. Analysis was performed on both fresh and thawed cryopreserved canine semen. Membrane-disrupted spermatozoa (MDS) were counted using CASQ (n=311) in an untreated sample and a completely membrane-disrupted sample, and the percentage of membrane-intact spermatozoa (MIS) calculated: (Total count-Untreated sample count)÷Total count×100. Spermatozoa were stained with a one-step EN stain (n=501), and then, at least 100 spermatozoa were manually examined under ×1,000 magnification and classified as MDS (stained with eosin) or MIS (non-stained). Spermatozoa from the same samples were also stained with CFDA/PI, and then, at least 200 spermatozoa were manually examined under ×1,000 magnification and classified as MIS (completely stained by CFDA) or MDS. The percentage of progressively motile spermatozoa (PMS) was determined by both computer-assisted semen analysis (CASA) and subjective methodologies, and the data were subsequently analysed to measure the agreement between the CASQ and EN methods with the CFDA/PI technique using Bland-Altman methodology. Pearson's correlation was measured between the MIS and PMS percentage samples and correlation coefficients compared. The mean MIS percentage was lower for CASQ and higher for EN than in CFDA/PI for all comparisons. The agreement of MIS percentage between CASQ and CFDA/PI was -20.2% to 32.0%, and between EN and CFDA/PI was -32.9% to 14.9%. In all methods, the MIS and PMS percentages were correlated (p<.001). Measurement of CFDA/PI appeared to be the most reliable and accurate method of determining MIS percentage in dogs. Further investigation is required to determine whether the CASQ technique can be improved. Eosin-nigrosin staining also appeared to be unreliable at MIS <80% and overestimated the MIS percentage.

A non-invasive method for semen collection and evaluation in small and median size lizards.

The aim of this study was to develop a non-invasive procedure to stimulate ejaculation in median and small lizards for semen collection. After semen collection, we applied a battery of tests to assess the motility (wave and progressive), viability (eosin-nigrosin stain), morphology (normal/abnormal), sperm concentration and ejaculate number, seminal volume and colour. We obtained this fluid from all males of the four species of sceloporine lizards (n=30) and one species of Mexican horned lizards (n=7). We found that semen from all males had a liquid-like consistency and a milky-white appearance. Ejaculate volume and sperm concentration varied among the males and the species studied. We also observed that although the mobility, viability and normal sperm morphology showed a wide variability, their average value in each species was high. We conclude that "genital papilla pressure" was an efficient and non-invasive semen obtaining method for small- and medium-sized lizards that does not imply damage to donors and could be used in other lizard species.

Famotidine does not affect human sperm fertility characteristics (motility, viability and DNA integrity) in vitro.

Famotidine, a histamine-2 receptor antagonist, is commonly used to relieve the acid-related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10mM concentrations in vitro. Forty-nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin-nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10mM had insignificant effect on human sperm motility (progressive, p=.9594; and total, p=.8420), sperm viability (p=.6471), and content of DNA breaks in sperm (p>.05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.

Characteristics of the post-thawed Balinese bull semen extended in three different extenders and equilibration times

The objectives of the present study were to compare and determine the best post-thawed characteristics of balinese bull sperm cryopreserved in three different extenders; animal based (Tris-clarified egg yolk (Tris-cEY)), and non-animal based extenders (Bioxcell® (lecithin based) and Optixcell® (liposome based)) in combination with three different equilibration times (30 minutes, 2 hours, 4hours). Thirty six ejaculates were collected from six Balinese bulls and frozen in three extenders (Tris-cEY, Bioxcell® and Optixcell®) after equilibration in three different times (30 minutes, 2hours and 4hours). Computer-assisted sperm analysis (CASA), hypo-osmotic swelling test (HOST) and eosin nigrosin staining were used in the post-thawed semen analysis. There was a significant interaction between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P&lt;0.05) for all the evaluated parameters independent of extender type. Overall, semen extended in Tris-cEY, Bioxcell® and Optixcell® were similarly better when equilibrated at 4 hours (P&gt;0.05). Moreover, post-thawed semen which were extended in Optixcell® for 2 hours equilibration showed a better motility compared with the other extenders (P&lt;0.05). In conclusion, two hours equilibration of semen with Optixcell® is sufficient for semen freezing. Four hours equilibration has the best sperm survival, independent of the extender type.

Correlation between sperm motility and sperm chromatin/DNA damage before and after cryopreservation and the effect of folic acid and nicotinic acid on post-thaw sperm quality in normozoospermic men.

Cryopreservation exposes sperm to physical and chemical stresses causing cell damages and impairs sperm functions. The aim of this study was to evaluate the association between motility and sperm chromatin/DNA damage before and after cryopreservation and investigate the effects of folic acid and nicotinic acid on post-thaw sperm quality. Thirty semen samples were obtained from 30 normozoospermic men, aged between 25 and 45years old. Each sample were divided into five aliquots to form the following groups: fresh, cryopreserved with sperm-freeze only (control), with nicotinic acid (10mM), with folic acid (50nM), and with a combination of folic acid (50nM) + nicotinic acid (10mM). Sperm viability and motility in each group were assessed by eosin-nigrosine staining and computer-aided sperm analysis respectively. Sperm chromatin quality was studied by aniline blue, toluidine blue, acridine orange staining methods and sperm chromatin dispersion test. Cryopreservation led to a significant reduction in sperm quality in comparison to fresh sample groups (p < 0.05). Sperm chromatin damage was negatively correlated with the percentage of progressively motile cells. Supplementation of the cryopreservation medium with folic acid or nicotinic acid induced a significant improvement in sperm parameters and chromatin quality, compared to control groups (p < 0.05). Meanwhile, the combination of folic acid + nicotinic acid showed a significant protective effect in post thaw sperm. In conclusion, cryopreservation generated oxidative stress, inducingsperm cryodamage, reducing progressive motility and sperm quality, as an indicator of significant chromatin/DNA damage. Folic acid and nicotinic acid exhibited a potential cryoprotective effect by enhancing sperm quality.

A point of confusion for embryologists in the identification of viable spermatozoa by the eosin-nigrosin test.

A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients’ semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Effect of butylated hydroxytoluene on quality of pre—frozen and frozen buffalo semen

Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 °C for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 °C and then frozen to -196 °C in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 ×) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.

Evaluation of Acrosomal Integrity and Viability in Bull Spermatozoa: Comparison of Cytochemical and Fluorescent Techniques

This experiment was aimed to compare two well known methods for assessment of acrosomal integrity and another two methods for assessment of viability in bull spermatozoa during cryopreservation. For this purpose, ejaculates were collected from four Hariana bulls using artificial vagina at biweekly interval. The semen samples which possesses more than 70% progressive motility and above 500 million/ml spermatozoa concentration was subsequently subjected to processing for liquid nitrogen (LN2) vapour freezing. Semen samples were extended in Tris-egg yolk-glycerol extender and further subjected to two staining methods, cytochemical by Giemsa staining and fluorescent method by fluorescein isothiocyanate - pisum sativum agglutinin (FITC-PSA) at two stages i.e. pre freeze and post thaw. In the same way to assess the viability eosin-nigrosin staining is compared with fluorescent method i.e. Annexin-V/PI method. Results showed that fluorescent staining were more sensitive as compare to cytochemical method for spermatozoa of Hariana bulls.

Influence of idebenone on ram semen quality stored at 4°C.

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris-based extender supplemented with 0, 1, 2, 4 and 8µM idebenone. Computer-assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin-nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72hr. Total and forward progressive motility were greater in 1, 2 and 4µM idebenone treated groups compared to control at 24, 48 and 72hr time points (p<0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p<0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id-treated groups compared to control one at 24 and 48hr of storage (p<0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4µM group compared to the control at 72hr (p<0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4µM idebenone treated groups in comparison with the control at 72hr (p<0.05). Results of the current study indicated that ram semen supplementation with Id at 4µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.

Effect of Vitamins and Amino Acids as Cryoprotectant Solution Supplementation for the Cryopreservation of Tambaqui Semen (Colossoma macropomum)

Background: The addition of antioxidant substances to a cryoprotective solution can increase its protective capacity, shielding spermatozoa from the oxidative stress caused by the seminal cryopreservation process. However, there is no record of a seminal cryopreservation protocol of tambaqui (Colossoma macropomum) using antioxidants as a supplement to the cryoprotective solution. Thus, the objective of this study was to evaluate the effects of adding vitamin C, vitamin E, cysteine, and/or taurine to the seminal cryopreservation of tambaqui.Materials, Methods &amp; Results: Pools of semen (n = 10) were diluted in cryoprotective solutions supplemented with: vitamin C (T1), vitamin E (T2), vitamin C + vitamin E (T3), cysteine (T4), taurine (T5), and taurine + cysteine (T6). The control treatment (T7) was not supplemented. Diluted semen was loaded in 0.5 mL straws, frozen in a dry-shipper, stored in a cryogenic cylinder, and then thawed in a water bath (45ºC for eight seconds). The quality of fresh and cryopreserved semen was evaluated by measuring total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity, and straightness using a computerized system of sperm analysis. Sperm membrane integrity parameters were analyzed using eosin-nigrosin staining, sperm morphology was assessed using pink bengal staining, and motility duration was measured by a digital timer. Data were analyzed using the statistical program SAS (2002) and the results were expressed as means ± standard error of the mean. The results showed that, in general, there was no significant increase in seminal quality when antioxidants were added to the cryoprotective solution. The T5 treatment promoted an increase (P &lt; 0.05) in progressive motility when compared to T1 (6.33 ± 1.14% and 2.98 ± 0.88%, respectively). However, it did not differ significantly (P &gt; 0.05) from the other treatments. Treatments T2 and T5 presented the highest values of VCL (34.74 ± 2.58 and 33.60 ± 1.81 μm.s-1, respectively). These were higher (P &lt; 0.05) than T1 (26.31 ± 1.64 μm.s-1) but not different (P &gt; 0.05) from the T7 control (30.87 ± 1.49 μm.s-1). The VSL and VAP results showed that T1 presented the lowest velocity (9.89 ± 1.75 and 15.06 ± 1.92 μm.s-1, respectively) compared to the other treatments (P &lt; 0.05) that did not differ from each other. Combining the two vitamins (T3) or the two amino acids (T6) was not advantageous in relation to the use of only one of these antioxidants.Discussion: The present study reports, for the first time, results of the addition of antioxidants to the tambaqui seminal freezing medium. The addition of taurine and vitamin E, although not significantly different from the control treatment, resulted in a tendency to increase sperm kinetics. This effect may be due to the action of taurine as a regulator of Ca2+ transporters, which is necessary to trigger sperm activation, and to the ability of vitamin E to scavenge reactive oxygen species produced during lipid peroxidation. On the other hand, the reduced sperm quality observed when vitamin C was used may have been related to the toxicity caused by a high concentration of this vitamin. In addition, once the safe dose of antioxidants has been exceeded, the normal physiological functions of reactive oxygen species can be inhibited. Thus, it is concluded that the use of vitamin E and taurine promotes promising results of curvilinear velocity after thawing of sperm. Therefore, these treatments are recommended, as well as more tests to determine their optimal concentrations.

Effect of taurine on turkey (Meleagris gallopavo) spermatozoa viability and motility

The effect of taurine on the turkey spermatozoa motility and viability during the in vitro incubation was assessed. Experimental samples were prepared by diluting the raw semen in nine different concentrations of taurine – from 10 mg/ml to 0.078125 mg/ml. The motility parameters were evaluated by the CASA system (Computer Assisted Semen Analyser) using the program Sperm Vision&lt;sup&gt;®&lt;/sup&gt; and for spermatozoa viability assessment the eosin-nigrosin staining was performed. Selected parameters were evaluated at six time periods: 0, 1, 2, 3, 4, and 5 h at 5°C and 41°C. At 5°C, a significantly lower percentage of motility and progressive motility was detected only in the samples with the highest concentration of taurine (10 mg/ml) at time 0 and 1. After 2 h of incubation a significant preventive effect of taurine on spermatozoa parameters was observed. The tendency of the taurine effect on motility parameters was different during the in vitro incubation at 41°C. Significantly lower values of motility parameters were detected in all experimental samples in comparison to the control after 5 h. The analysed concentrations of taurine did not significantly affect viability of turkey spermatozoa during all time periods. A higher percentage of dead spermatozoa were observed at 41°C (4.87–9.90%) if compared to 5°C (2.12–4.88%). The results indicated that the addition of taurine (from 2.5 to 7.5 mg/ml) to turkey spermatozoa positively affected the monitored spermatozoa parameters incubated at 5°C.

Age effect on post freezing sperm viability of Bali cattle (Bos javanicus)

Post freezing sperm viability is one of factors which determine artificial insemination success. In the other side, bull’s or sire age influences the semen quality through sperm membrane constituent. It is known that freezing process change the sperm membrane during the processing stage. This research aims to know the effect of sire age on post freezing sperm viability of Bali cattle. The samples were collected in Singosari Artificial Insemination Centre, Malang, East Java, Indonesia on September - November 2016. Eight Bali cattle (4 and 7 y.o, 4 heads in each group) were used as semen source. Semen was collected using artificial vagina, 10 times spanning for 5 weeks (2 times per week, interval 3 and 4 days) in a row. The samples were then evaluated at fresh, chill and frozen stage. Fresh semen was diluted in Tris-citrate-egg yolk 20% (v/v) followed with chilling and freezing. Semen qualities were observed as sperm % motility (MOT), % live sperm using eosin-nigrosine staining (EOS) and % sperm membrane integrity using hypoosmotic swelling test (HOS). Variable comparisons between age groups were done using t-test. On the average, 4 y.o bulls showed higher semen quality at fresh, chill and frozen compared to 7 y.o in MOT (68.00±6.39 vs 65.9±7.62; 56.40±3.71 vs 54.33±5.83; 44.25±3.52 vs 40.40±7.06), EOS (72.08±6.63 vs 71.82±7.38; 57.81±3.83 vs 57.41±6.32; 53.16 ±8.41 vs 46.49±9.13) and HOS (60.85±13.91 vs 54.84±13.43; 53.16 ±8.41 vs 46.49±9.13; 44.6±9.39 vs 33.8±10.70) respectively. Statistical analysis results showed that age was significantly (P<0.05) affecting HOS at chill stage and MOT and HOS at frozen. In conclusion, younger Bali cattle (4 y.o) have more viable post freezing sperm compared to the older ones (7 y.o).

The test about blood serum capabilities in maintaining the quality of bull spermatozoa during storage in cep diluent at refrigerator temperature

The storage of spermatozoa requires a protective material from cold shock events and the presence of free radicals.In CEP diluent contain BSA, that was used as spermatozoa protection. This study aim was to examine the ability of cow blood serum in replacing BSA as spermatozoa protective in CEP diluent. Fresh semen from Limousin bull was diluted with CEP diluent + BSA as control, in the treatment group were CEP without BSA, but replaced with 3%, 5%, and 7% serum from fresh blood. Spermatozoa quality tests included motility and viability. The motility of spermatozoa was observed by two people using a light microscope with 200 X magnification at temperature of 37°C. The method of viability observation was eosin nigrosin staining, and observed under a light microscope with 400 X magnification. The results showed that the replacement of cow blood serum with various concentrations gave different effects on the quality of spermatozoa. The best motility and viability of the treatment group was at serum concentrations of 5% after eight days storage and was not significantly different from the controls. The conclusion in this study was cow blood serum can replace BSA in CEP diluents.

Caffeine strongly improves motility parameters of turkey spermatozoa with no effect on cell viability.

The purpose of this study was to evaluate the impact of caffeine on turkey spermatozoa during in vitro incubation. Experimental samples were prepared by diluting the raw semen with nine different concentrations of caffeine - from 0.078125 mg/mL to 10 mg/mL. The individual motility parameters were evaluated by the Computer Assisted Semen Analyser (CASA) system, and the viability of spermatozoa was evaluated using eosin-nigrosin staining. Selected parameters were recorded at six time periods: 0, 1, 2, 3, 4 and 5 h at 5 °C and 41 °C. A significantly higher motility and progressive motility of spermatozoa (P < 0.01 and P < 0.001, respectively) was detected in the samples containing caffeine ranging from 0.15625 to 7.5 mg/mL as compared to the control sample at 5 °C. At an incubation temperature of 41 °C the positive effect of caffeine on motility parameters was observed only at the beginning of incubation (at times 0 and 1). The tested caffeine concentrations showed no significant effect on the viability of turkey spermatozoa at any time period of incubation. A higher percentage of dead spermatozoa was observed for incubation at 41 °C (from 5.96% to 11.1%) in comparison to 5 °C (from 1.62% to 5.79%). The results suggest that caffeine can be used as a suitable component of turkey semen extenders and has the potential to improve fertility.

Morphological defects of sperm and their association with motility, fertility, and hatchability in four Korean native chicken breeds.

ObjectiveThis study was conducted to compare morphological defects, viability, motility (MOT), fertility (F), and hatchability (H) in four Korean native chicken breeds (KNCBs), and to evaluate whether defective segments of spermatozoa are associated with MOT, F, and H.MethodsFour KNCBs, including Korean Ogye (KO), Hwangbong (HB), Hyunin Black (HH), and Hoengseong Yakdak (HY) were used. White Leghorn (WL) was used as a control. Nine cocks from each breed were randomly assigned into three groups. Semen was collected by abdominal massage method. Eosin-nigrosin staining method was used to identify live-dead spermatozoa. Different segments and specific morphological defects of spermatozoa were identified using 4′, 6-diamidino-2-phenylidole and MitoTracker Red CMXRos. F and H rates were evaluated following artificial insemination (AI).ResultsKO had the highest MOT rate compared to HY. Viable normal sperm rates of KO and HH were high and comparable with WL. HY spermatozoa had the highest viable abnormal sperm (VAS) or morphological defect rate followed by HB. Likewise, HB spermatozoa had the highest dead sperm (dead) rate compared to KO, HY, and WL. Bent, coiled, detached, broken, and knotted were common identified specific morphological defects for all breeds. Most morphological defects were at the head and tail in all breeds. VAS showed strong negative correlation with MOT (r = −0.697) and F (r = −0.609). Similarly, defective tail was negatively correlated with MOT (r = −0.587), F (r = −0.797), and H (r = −0.448). The F and H rates of KO and WL were comparable.ConclusionThese data indicate that most identified specific morphological defects are at the head and tail. VAS and defective tail were associated with poor motility, F, and H. KNCBs showed more morphological defects than WL. Finally, these results will facilitate successful AI and semen cryopreservation.

130 In Vitro Production of Hybrid Desert Bighorn×Domestic Sheep Embryos Using Frozen–Thawed Epididymal Semen from a Hunter-Harvested Ram

Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 106 cells mL−1. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 106 sperm mL−1) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 106 sperm mL−1). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.

Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm.

The purpose of this study is to investigatewhether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation. Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation. No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation. ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

Relationship between semen quality and meat quality traits in Belgian Piétrain boars

Abstract The main objective of this study was to assess the semen quality of Pietrain boars originating from Belgian AI centers and to correlate these results with their meat quality traits. Freshly diluted semen doses from 140 boars originating from 10 artificial insemination (AI) centers were used and stored for five days at 17 °C. Motility was assessed daily using a computer assisted semen analyzer (Hamilton-Thorne), while morphology and concentration were assessed on the day of semen collection (Day 0) by eosin-nigrosin staining and the Burker counting chamber, respectively. These data were correlated with the lean meat percentage, loin eye depth and backfat thickness using linear mixed models taking into account the clustering of boars within each AI center and the daily measurements for each semen dose. The mean values (± SD) on Day 0 were: motility 79.7 ± 8.2%, live sperm 91.5 ± 4.3%, live normal sperm 83.6 ± 7.4%, and concentration 29.0 ± 10.6 (×10 6 sperm/mL). The average five-day motility across all AI centers was 77.7 ± 8.9%. None of the assessed semen quality traits were associated with lean meat percentage. Motility and progressive motility on Day 0 were positively associated with backfat thickness ( P

Effect of brain-derived neurotrophic factor (BDNF) on sperm quality of normozoospermic men

The neurotrophin family of proteins and their receptors act as important proliferative and pro-survival factors in differentiation of nerve cells and are thought to play key roles in the development of reproductive tissues and normal function of spermatozoa. The objective of the present study was to evaluate the effect of Brain-Derived Neurotrophic Factor (BDNF) on the sperm viability and motility, lipid peroxidation (LPO), mitochondrial activity and concentration of leptin, nitric oxide (NO) and insulin in normozoospermic men. Semen samples from 20 normozoospermic men were divided into three groups: (i) control, (ii) BDNF and (iii) BDNF + K252a. BDNF and K252a were added in the dose of 0.133 and 0.1 nM, respectively. Viability was assessed by eosin–nigrosin staining technique, and motility was observed by microscopy. NO concentration and mitochondrial activity were measured with flow cytometry, and LPO was analyzed using enzyme-linked immunosorbent assay (ELISA) kits. Results showed that exogenous BDNF at 0.133 nM could significantly (p < 0.05) influence viability, motility, NO concentration, mitochondrial activity and LPO content. Secretions of insulin and leptin by human sperm were increased in cells exposed to the exogenous BDNF, whereas viability, mitochondrial activity and insulin and leptin secretions were decreased in cells exposed to the K252.