Eosin-nigrosin Staining Research Articles

Supplementation with seminal plasma and relationship of its components with the quality of frozen-thawed semen of donkeys (Equus asinus)

El objetivo fue evaluar la suplementación con plasma seminal y la relación existente entre algunos de sus componentes, con la calidad del semen congelado-descongelado de asnos (Equus asinus). El semen de cinco asnos criollos colombianos se colectó por el método de la vagina artificial. A partir de una fracción de cada eyaculado se separó el plasma seminal por centrifugación y se evaluó su contenido de vitamina C (VC), vitamina E (VE), proteínas totales (PT) y perfil lipídico (PL). La criopreservación del semen se realizó por congelación convencional en un diluyente suplementado con 20 % de plasma seminal. Se evaluó la movilidad, la integridad estructural de membrana (IEM), la morfología anormal (MA) y la integridad funcional de membrana (HOS) de los espermatozoides, mediante el sistema computarizado SCA®, el ensayo fluorescente SYBR14/IP, la tinción con eosina-nigrosina y la prueba hipoosmótica, respectivamente. Se ajustaron modelos mixtos y se realizó un análisis de correlación de Pearson entre los componentes del plasma seminal y la calidad espermática. Se encontró que un nivel alto de VC, VE, PT y ácido esteárico (C18:0) tuvo un efecto deletéreo sobre la calidad del semen criopreservado (P≤0.05). Mientras que un nivel bajo de PT y C18:0, y un nivel medio de VE y VC presentaron resultados post-descongelación superiores para la movilidad y la integridad de membrana (P≤0.05). Se hallaron coeficientes de correlación negativos de VC, VE, PT y C18:0 con diferentes parámetros de calidad seminal post-descongelación. Se concluye que la composición del plasma seminal suplementado para la congelación de semen de asno, influye en la calidad espermática post-descongelación.

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen.

Aim:The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters.Materials and Methods:A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test.Results:The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups.Conclusion:In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment.

The effect of antioxidant agents' addition and freezing method on quality parameters of frozen thawed ram semen.

The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2weeks. Equal number of good quality spermatozoa from each ejaculate (concentration ≥1×109spermatozoa/ml, motility ≥70%, motility score ≥3.5) were pooled. Three equal aliquots of the pooled sample were diluted using three different fractions of a milk-based and glycerol extender (control, quercetin-enriched, α-tocopherol-enriched). Three freezing methods were applied (slow and fast freezing rate in a programmable freezer, vapors of liquid nitrogen) in every aliquot. Sperm aliquots were tested before freezing, immediately after thawing and after 3h of incubation at 37°C. Sperm motility (%) was evaluated microscopically. The percentage of membrane and acrosome-intact spermatozoa (IL%) as well as the percentage of membrane-intact and acrosome-reacted spermatozoa (ARL%) were determined by eosin-nigrosin stain. Furthermore, the percentage of hypo-osmotic swelling (HOS) test-positive spermatozoa was estimated. The results revealed no beneficial effect of the antioxidant treatment on the parameters of post-thaw semen (P>0.05). However, the slow freezing rate method was more beneficial regarding motility, IL, ARL and HOS-positive spermatozoa compared to the other methods. In conclusion, the antioxidant agents used in this study failed to protect sperm against cryopreservation stress; however, the choice of the appropriate freezing method could contribute to the improvement of post-thaw ram sperm quality.

Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage

The purpose of the current experiment was to evaluate the protective effect of oleic acid on ram spermatozoa parameters during storage at refrigerator temperature. Ejaculates were collected from rams, pooled, diluted with extender and enriched with 0 (control), 0.125 (O 0.125), 0.25 (O 0.25), 0.5 (O 0.5) and 1 (O1.0)mM oleic acid at a final concentration of 500×106 spermatozoa/mL. Spermatozoa kinematics, viability and spermatozoa membrane integrity were evaluated by using computer-assisted sperm analysis (CASA), eosin–nigrosin staining and hypo-osmotic swelling (HOS) test, respectively. In addition, amounts of malondialdehyde (MDA), total antioxidant capacity (TAC), nitric oxide (NO) and superoxide dismutase (SOD) activities were assessed in the medium and spermatozoa at 0, 24, 48 and 72h of storage. Enrichment of semen with oleic acid resulted in a significant (P<0.05) increase in total motility, forward progressive motility and VCL (curvilinear velocity, μm/s) of O 0.5 and O1.0 groups at 48 and 72h of storage. In addition, viability and spermatozoa membrane functionality were greater in O 0.5 (81.81±2.29% and 72.10±2.63%) and O1.0 (82.32±2.36% and 74.53±1.90%) groups compared to the control group (73.28±1.60% and 59.34±2.12%) at 72h (P<0.05). The measurement of MDA levels in spermatozoa showed that, there were higher in control group (0.84±0.02 and 0.86±0.04μmol/g protein) in comparison with the O 0.125 (0.67±0.05 and 0.69±0.03μmol/g protein), O 0.25 (0.62±0.03 and 0.66±0.02μmol/g protein), O 0.5 (0.62±0.02 and 0.65±0.06μmol/g protein) and O1.0 (0.60±0.01 and 0.61±0.01μmol/g protein) groups at 48 and 72h of storage (P=0.002). Moreover, higher amounts of TAC were obtained in oleic acid treated groups compared to control group in spermatozoa and medium at 48 and 72h (P<0.05). Nitrosative status was lower and SOD activities were higher in spermatozoa of oleic acid treated groups than the control group at 48 and 72h (P<0.05). Our results indicated that oleic acid with the dose of 0.5 and 1mM would enhances ram semen quality during liquid storage at 48 and 72h.

Investigation of scrotal circumference, testicular dimensions and semen characteristics of the vulnerable Arabian oryx (Oryx leucoryx): an approach supports the future use of artificial insemination

Data regarding semen collection and characteristics in Arabian oryx are not available so far. Therefore, the current study aimed to evaluate scrotal circumference, testicular dimension and semen characteristics in four age groups of this species. Eleven male Arabian oryx bulls were divided into four groups according to age: group 1: 3 years old (n = 2); group 2: 4 years old (n = 2); group 3: 5 years old (n = 2) and group 4: 10 years old (n = 3). After immobilisation, scrotal circumference, testicular dimensions, volume and weight and gonadosomatic index (GSI) were estimated for each animal. Semen was collected from each male on a weekly basis for 3 weeks. Immediately after collection, semen was evaluated for volume, pH, osmolality total motility (TM), forward progressive motilities (PM) using a phase microscope. Furthermore, TM, PM, progressive velocity (PV), curvilinear velocity (VCL) and pathway velocity (VAP) have been evaluated using computer-assisted sperm analyser (CASA). Sperm concentration was evaluated using a haemocytometer, whereas sperm morphology was detected using phase contrast microscopy and staining methods by eosin-nigrosin and Spermac stains. Sperm viability was assessed using eosin-nigrosin stain under light microscopy and Sybr14/PI stain under fluorescence microscopy. The current findings indicated a positive correlation between body weight versus scrotal circumference, testicular weight, testicular volume and GSI. However, age did not have a positive association with percentage of normal sperm in Arabian oryx. Conclusively, the current findings provide a valuable data for future use of artificial insemination, which will be crucial for propagation and conservation of Arabian oryx.

Evaluating the spermicidal activity of an antimicrobial peptide from the Bufo kavirensis, MaximinBk: in vitro study

Abstract Objective The aim of this study was to evaluate the spermicidal activity of this peptide to introduce a new potent agent for prevention of sexually transmitted infections and unplanned pregnancies. Methods The purified MaximinBk (with amino acid sequence: ILGPVLGLVGRLAGGLIKRE) was diluted with Ham’s F10 solution in 100, 200, 400, 600, 800 and 900 μg/mL. One milliliter from peptide solution with different dosage was mixed with 200 μL prepared sperm solution in microtube. Sperm motility, viability and morphology were assessed at different time intervals (0.3, 5, 10, 15 min). Eosin–Nigrosin staining and Giemsa staining methods were applied for sperm viability and morphology detection, respectively. Results Total spermicidal activity was shown after addition of 900 μg/mL for 0.3 min without any morphological change in the sperm head, midpiece or tail. Also, Eosin–Nigrosin staining indicated MaximinBk can disturb membrane integrity of normal sperm that is in dose-dependent manner. Conclusion MaximinBk has spermicidal activity in addition to antimicrobial activities (especially on vaginal infections such as candidal vulvovaginitis). It seems this peptide might be a potential candidate in order to use in male contraception, although, this preliminary study needs more studies to elucidate final conclusion.

Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa.

Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions? DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions. DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown. Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples. Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay. DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction. N/A. Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications. The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function. National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

237 Diethylstilbestrol Activates Catsper and Disturbs Progesterone Actions in Human Sperm

Is the prototypical endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), able to evoke physiological changes in human spermatozoa and affects progesterone actions? DES promoted Ca2+ flux into human sperm by activating CatSper channel and suppressed progesterone-induced Ca2+ signaling, protein tyrosine phosphorylation and human sperm functions. Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a plate reader following sperm loading with Ca2+ indicator Fluo-4 AM and further whole-cell patch-clamp technique was performed to record CatSper currents. Sperm viability and motility parameters were assessed by eosin-nigrosin staining kit and a computer-assisted semen analysis (CASA) system, respectively. The ability of sperm to penetrate into viscous media was detected by 1% methylcellulose. Sperm acrosome reaction was measured using chlortetracycline (CTC) staining. The level of protein tyrosine phosphorylation was determine by western blot analysis. DES rapidly increased human sperm [Ca2+]i even at environmentally relevant concentrations (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and can be significantly inhibited by the CatSper blocker mibefradil, suggesting CatSper participate in this process. In fact, whole-cell patch-clamp recordings confirmed that DES could increase CatSper currents. DES did not affect sperm viability, motility, penetrate into viscous media, protein tyrosine phosphorylation and the acrosome reaction. However, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous medium and acrosome reaction.

43 CORRELATIONS OF METHODS OF SPERM ANALYSIS IN FRESH SEMEN OF SOUTH AFRICAN INDIGENOUS GOAT

This study investigated the correlations between methods of assessing sperm qualities; namely, sperm total motility (TM), sperm vitality, acrosome integrity, DNA integrity, and sperm membrane integrity. A total of 60 ejaculates from 6 bucks were collected and the spermatozoa evaluated. The overall mean percentages of sperm TM, sperm vitality (eosin-nigrosin and propidium iodide stains), sperm acrosome integrity (Spermac and SpermBlue® stains), sperm DNA integrity (acridine orange and halotech), and sperm membrane integrity (hypoosmotic swelling test and water test) were 94.7 ± 0.5, 81.0 ± 0.6. 79.6 ± 3.7, 79.7 ± 1.8, 78.6 ± 5.3, 75.7 ± 5.5, 74.3 ± 5.1, 73.1 ± 3.5, and 73.4 ± 3.6, respectively. There were significant correlations (P &lt; 0.05) between mean percentage live sperm evaluated with eosin-nigrosin stain and sperm TM (r = 0.813), between percentage intact acrosome assessed with SpermBlue® and sperm TM (r = 0.846), and between SpermBlue® and eosin-nigrosin (r = 0.965). There were highly significant correlations (P &lt; 0.01) between sperm membrane integrity evaluated with HOS test and sperm TM (r = 0.871), between percentage of intact sperm DNA assessed with halotech and SpermBlue® (r = 0.832), and between percentage of intact spermatozoa DNA assessed with acridine orange and percentage intact acrosome evaluated with spermac stain (r = 0.862). Under the conditions of this study, the correlated methods of sperm analysis proved suitable for analysis of goat spermatozoa and can serve as useful indicator of potential fertility for sperm. They could be used for accurate assessment of the individual sperm cell rather the population as a whole. Motility, eosin-nigrosin stain, SpermBlue®, halotech and acridine orange stain still remain practical and valuable tools for predicting sperm fertilizing ability.

24 RELATIONSHIP BETWEEN BOAR SPERM TRAITS AND FERTILITY RATE FOLLOWING ARTIFICIAL INSEMINATION UNDER SMALLHOLDER PRODUCTION SYSTEMS

The prediction of sperm fertility has a great economic importance to the pig breeding industry. The objective of the study was to determine the relationship between boar sperm quality and fertility following artificial insemination (AI) under smallholder production systems. A total of 18 ejaculates were collected from 3 breeding boars using a hand-gloved technique. Aliquots of diluted semen were assessed for sperm motility using a computer aided sperm analysis before AI. Sperm viability was evaluated using Synthetic Binding CD-14 (SYBR-14+)/propidium iodide (PI–), whereas sperm morphology was evaluated using Eosin Nigrosin staining. Fluorescent microscope was used at 100× magnification to count 200 sperm per slide. The semen was extended with Beltsville Thawing Solution and contained 3 × 109 sperm/dose. A total of 73 multiparous sows were inseminated twice. Fertility was measured by conception rate, farrowing rate, litter size and number of piglets born alive following AI. Sperm quality and fertility data were analysed using one-way ANOVA. Spearman’s rank correlation was used to determine the relationship between sperm quality and fertility traits. The mean values for total sperm motility ranged from 93.5 to 96.8%. Progressive and rapid sperm motility differed significantly (P &lt; 0.05) among the boars. However, no significant differences were found for sperm velocity traits. The mean values for morphologically normal sperm ranged from 47.8 to 60.9% and live sperm ranged from 71.8 to 77.2%, but did not differ significantly among the boars (P &gt; 0.05). Conception rate from different boars varied (P &lt; 0.05) from 63.6 to 93.3%. Of all fertility traits studied, conception rate was significantly related to total sperm motility rate (r = 0.34, P &lt; 0.0029), progressive motility (r = 0.29, P &lt; 0.0141) and rapid motility (r = 0.34, P &lt; 0.0032), although relatively low. There was a low positive relationship between morphologically normal sperm and fertility traits (P &gt; 0.05). In conclusion, total, progressive, and rapid sperm motility rate were the only sperm traits significantly related to conception rate. Conversely, litter size and number born alive were not correlated with sperm motility, viability, or morphology traits.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

Clouded leopards (Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n=9) were allocated to simple washing (control; resulting in 11.7%±1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9%±0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution (HOS, 60 mOsm fructose solution) containing 5mM dithiothreitol (DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 (TX) (a detergent) was added to the HOS/DTT-treated samples. After HOS/DTT incubation, the control samples and sperm-selected samples presented 73.4%±3.1% and 73.9%±2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8%±3.8% and 26.5%±3.4%; p=.1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6%±10.4% and 21.1%±7.9%; p=.06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation.

Heat stress has an effect on motility and metabolic activity of rabbit spermatozoa

In the warm months the function of the spermatozoa can be affected by the temperature of the reproductive tract of the female exposed to hyperthermic conditions. The aim of this study was to evaluate the impact of heat stress on sperm parameters in an in vitro model and to determine if there were seasonal effects on sperm heat tolerance. Sperm samples from 32 New Zealand White rabbits were collected in two seasons and incubated at scrotal (32.5°C), body (37°C) or hyperthermic (42°C) temperatures for 3h. Sperm viability and morphology were evaluated using nigrosin-eosin staining. Motility and metabolic activity parameters were determined using computer-assisted sperm analysis and the QBlue cell viability test, respectively. The incubation of spermatozoa at 42°C decreased (P<0.05) the mean values of total motility, curvilinear (VCL) and mean velocity (VAP) as well as the metabolic activity with respect to the incubation at 32.5°C and 37°C.No seasonal effects were observed except for the highest percentages of bent and coiled tails in the cold season, and the highest mean values of VCL, linear velocity and VAP in the warm season (P<0.01). The interaction between in vitro heat stress and season was significant for metabolic activity (P=0.02). Our results suggest that rabbit spermatozoa parameters are largely modified by a short exposure to hyperthermic conditions, in terms of metabolic activity and motility parameters. Thus, a short exposure of spermatozoa to an environment of 42°C in temperature for only 3h may compromise sperm functionality. Additionally, sperm metabolic activity is influenced by season.

Polymyxin B changes the plasma membrane integrity of cryopreserved bull semen

ObjectiveTo examine adding polymyxin B (PMB) if is effective and appropriate to bull extender, and decrease bacteria damage. MethodsPMB were added to extender (Andromed®) in different values (0, 50, 100, 500, 1000 μg/mL). The prepared samples were cryopreserved, thawed and analyzed for viability (by eosin–nigrosin staining) and plasma membrane function (by hypo osmotic solution test) in 3 different times (immediately, 1 h & 2 h later). ResultsThe result showed that both parameters significantly increased in 50 and 100 μg/mL treatments. Moreover, by passing time, plasma membrane function significantly increased. But, in high dosage (1000 μg/mL), the toxicity effect of PMB was seen, and in both parameters the differences were significantly lower than the control treatment (0 μg/mL). ConclusionOur study indicates that adding PMB to bull extender is efficient and appropriate, but, it must be mentioned that addictive amount will be due to extender composition, existence of other antibiotics and, importantly, the load of bacterial contamination in the semen.

Relationship between total protein concentration of seminal plasma and sperm characteristics of highly fertile, fertile and subfertile Barki ram semen collected by electroejaculation

The aim of this study was to determine the relationship between total protein concentration (TPC) of seminal plasma (SP) and semen characteristics in rams. A total of 105 ejaculates from 21 Barki rams, of known fertility (9 highly fertile, 9 fertile and 3 subfertile) were collected by electroejaculation (EE) and used in the present study. An aliquot (200μl) from each ejaculate was used for evaluation of semen characteristics (wave motion, motility, viability, sperm morphology, intact-acrosome, sperm concentration and total sperm count per ejaculate). The remaining volume was centrifuged to collect SP and TPC (g/dl) of SP was estimated with hand-held Refractometer; then SP proteins were separated on 15% (w/v) polyacrylamide gel. The results revealed that TPC of SP and almost all semen characteristics were significantly (P<0.05) lower in subfertile (SF) group than both highly fertile (HF) and fertile (F) groups but the longest (85.0±4.1s) stimulation time (ST) was recorded in SF group. Moreover, TPC of SP in HF and F groups showed highly significant (P<0.001) negative correlation with ST but showed highly significant positive correlation with all semen characteristics. However, in SF group neither ST nor semen characteristics showed any significant correlation with TPC of SP. Eosin-nigrosin (EN) stain, hypoosmotic swelling test (HOST) and glutaraldehyde fixation (GF) were capable to discriminate among HF, F and SF groups of rams. In total, fourteen protein bands with different molecular weights, ranging from 7 to 91kDa, were detected on polyacrylamide gel. Protein bands of 11, 13 and 22.5kDa were more prominent in HF group than F group but it was faint to absent in SF group. Moreover, bands of 91, 80 and 27kDa were prominent in F and SF groups than HF group. In conclusion, TPC and protein profile of SP were closely correlated with semen characteristics and might be useful tools to determine ram fertility or semen quality. Protein bands of 11, 13 and 22.5kDa were fertility-associated proteins in Barki rams SP. The effects of these fertility-associated proteins on sperm cell function required further investigation.

Effect of LDL Addition Into Selected Bull Sperm Diluters on Resistance of Spermatozoa Against Cold Shock

The aim of work was to determine the effect of LDL cholesterol addition into selected diluters on the resistance of spermatozoa against cold shock and on their short-term survivability during cold test. The hypothesis was that the addition of LDL cholesterol will positively affects sperm resistance to cold shock and ensures a higher survivability of spermatozoa during short-term cold survival test. Four bulls of different breeds and ages, from the same sire insemination center were used. A total of eight semen collections were processed. Each ejaculate was divided into 6 portions (3 controls and 3 samples). Three commercially produced diluters, AndroMed®, Bioxcell®, and Triladyl® were used, each in standard and LDL enriched variants. In the case of AndroMed® or Bioxcell®, 6% of LDL was simply added. In Triladyl®, 10% of LDL replaced the standard egg yolk component. Spermatozoa resistance to cold shock was evaluated by the percentage of live sperm using Eosin-Nigrosine staining. The results showed the influence of bull individuality as an important factor. It is possible to recommend Bioxcell® with addition of LDL cholesterol in 6% concentration, which survivability was 69.17% at the beginning of the test, and 52.94% after 2 hours of incubation.

Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit

The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

Effect of Akti-2 on sperm motility, capacitation and acrosome reaction in a mouse model

The aim of the present study was to investigate the effect of the Akt inhibitor, Akti-2, on the sperm motility and acrosome reaction in mice. Mature sperms from the adult mice, aged 8 weeks, were co-incubated with Akti-2 for ~30 min at 37°C in 5% CO2, and the sperm viability was assessed by eosin-nigrosin staining. The sperm total and progressive motility were analyzed by computer-aided sperm analysis. In addition, the acrosome reaction of sperms was detected by the acid phosphatase assay, Coomassie Brilliant Blue staining and fluorescein-isothiocyanate conjugated pisum sativum lectin staining, respectively. Compared with the control (dimethyl sulfoxide), Akti-2 had no effect on sperm viability, but it suppressed the total and progressive motility significantly. Furthermore, the capacitation-associated protein tyrosine phosphorylation and the acrosome reaction induced by calcium ionophore A23187 could be suppressed by Akti-2. These experiments confirmed that Akti-2 significantly impaired the sperm functions, including motility, capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity.

Sperm Quality Parameters and Reproductive Efficiency in Muscovy Duck (&lt;i&gt;Cairina moschata&lt;/i&gt;)

The in vitro sperm quality parameters (motility, M; viability, V; normal morphology, NM; plasma membrane integrity, PMI; mitochondrial function, MF) in Muscovy drakes (Cairina moschata) were evaluated by using microscopy and flow cytometry, the correlation among sperm quality parameters and results of artificial insemination was also assessed in present study. M, V and NM were detected by phase contrast microscopy assisted with eosinnigrosin staining, and PMI and MF were detected by using flow cytometry within appropriate fluorescence staining (SYBR-14/PI and R123/PI, respectively). Fertility (F), early embryonic mortality (EEM) and the survival embryo rate (SER) were assessed after the artificial insemination of Muscovy or Kaiya duck (Anas platyrhynchos) females. Sperm PMI and MF, the parameters detected by flow cytometry were positively correlated with sperm M, V, and NM, those were detected by phase contrast microscopy (P<0.05). Sperm V and PMI were negatively correlated with the percentage of early embryo mortality of Muscovy duck fertile eggs (P<0.05). The results of the present study showed the relationships among the AI results and the sperm quality parameters detected by microscopy as well as flow cytometry. In conclusion, flow cytometry assisted with microscopy can be an effective tool to evaluate in vitro sperm quality and may contribute to predict the reproductive performances of individual Muscovy drakes, which helps to improve duck production efficiency.

Efecto del suplemento de astaxantina sobre la calidad seminal en Moenkhausia sanctaefilomenae (Teleostei: Characidae)

This study was set out to: i) characterize the morphometry of spermatozoa of M. sanctaefilomenae ; and ii) evaluate the effect on the quality of semen by astaxanthin supplementation in the diet. The morphometry of the sperm was analyzed by optical microscopy (nigrosin-eosin staining). Semen quality of 360 fish was evaluated by spermograms (volume, total number, motility and viability). The length of the sperms was 16.83 ± 2.33 μm, the head diameter was 1.93 ± 0.21 μm without acrosome, the midpiece diameter was 0.91 ± 0.23 μm and the length of the flagellum was 13.18 ± 1.76 μm. In fish unexposed to astaxanthin (control group) the seminal volume and the total number of sperms was 2.14 ± 1.55 μL and 6.8x10 8 ± 29, respectively. The addition of astaxanthin in the diet significantly increased ( P 0.05). The results suggest that astaxanthin improves some parameters of the semen quality (volume and total number). This improvement may be attributable to the antioxidant properties ( i.e ., protection against oxidative stress in testicular cell structures) of astaxanthin.

Influence of selected factors on bovine spermatozoa cold shock resistance

The objectives of this study were to determine the effects of sire, extender, and addition of Low Density Lipoprotein (LDL) to extenders used on the percentage rate of spermatozoa survival after cold shock. Two groups of extenders were compared: without LDL addition (control variants) and LDL enriched (experimental variants). Three extenders were used: AndroMed®, Bioxcell®, and Triladyl®. Experimental variants included 4–8% LDL addition into the AndroMed® and Bioxcell® extenders, and 6–10% LDL addition into the Triladyl® extender. In total, 12 samples of fresh semen were collected from 4 bulls during a period of 8 weeks. Bovine spermatozoa cold shock resistance (1 ± 1 °C, 10 min) was evaluated by the percentage rate of live sperm using eosin-nigrosine staining immediately and after heat incubation (37 ± 1 °C, 120 min). The results showed the effect of sire as important and individual differences between selected sires in their sperm resistance against cold shock were confirmed. AndroMed® and Bioxcell® were found to be providing better protection of bull semen to cold shock compared to Triladyl® due to lower decline of live sperm proportion. Our results detected a positive effect of LDL addition on sperm resistance against cold shock, especially on lower decrease of live sperm percentage rate after 120 min of the heat test (P &lt; 0.05). Further studies are needed to assess the optimal concentration of LDL in various kinds of extenders as well to state ideal time and temperature conditions for ensuring LDL reaction with sperm.