The effect of temperature and pH on the motility and viability of ostrich sperm

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.     

The effect of ZnO nanoparticles on rabbit spermatozoa motility and viability parameters in vitro

Zinc plays a very important role in various biological activities of the body. Multifaceted role of zinc is also known in testes development, spermatogenesis, capacitation and has effect on spermatozoa motility. On the other hand, the growing industry of nanotechnology has created reasonable interest of the risk assessment for nanoparticles. The aim of this study was to evaluate in vitro effect of zinc oxide (ZnO) nanoparticles on rabbit spermatozoa. Fresh semen was collected from sexually mature New Zealand rabbits. Experimental groups were prepared by diluting semen with ZnO nanoparticles in seven different concentrations (6–391 mg/mL). The experimental groups were compared with control group. Semen was assessed using computer assisted semen analysis (CASA) at intervals of 0, 1, 2 and 3 h of incubation. The mitochondrial toxicity assay (MTT) assay was used to determine cell viability. The results of monitored motility parameters in experimental groups showed a decreasing trend during whole experiment. Significant decrease (P < 0.001) of motility and progressive motility was observed after 3 h of incubation in samples cultured with higher ZnO nanoparticles in comparison to the control group. After 3 h of incubation, viability of rabbit spermatozoa showed slightly increased values in group with the lowest concentration of ZnO nanoparticles, but in other groups viability showed non-significant decrease compared to control. Similar tendency was detected for spermatozoa membrane integrity. These original data show the negative dose–dependent effect of ZnO nanoparticles on spermatozoa motility and viability parameters.     

The degree of resistance to freezing-thawing is related to specific changes in the structures of motile sperm subpopulations and mitochondrial activity in boar spermatozoa

The main aim of this work was to analyze the possible relationship between the structures of motile-sperm subpopulations and boar (Sus scrofa domesticus) sperm resistance to freezability. For this purpose, 45 boar ejaculates were subjected to a standard freezing-thawing protocol, and afterwards they were classified into three groups, in accordance with their resistance to freezing-thawing. Our analysis yielded four separate motile-sperm subpopulations in all of the studied ejaculates, both in fresh samples and after freezing-thawing. Furthermore, whereas curvilinear velocity (VCL), mean velocity (VAP), and dance (DNC) of sperm from Subpopulation 1 underwent significant increases after freezing-thawing in samples with a good response to freezing-thawing, the same parameters of Subpopulation 1 either did not undergo significant variations (VCL and DNC) or even showed a decrease (VAP) (from 20.4 ± 0.4 μm/sec in fresh samples to 15.2 ± 2.2 μm/sec after freezing-thawing) in samples with the poorest response. Similarly, the behavior of other motility parameters in each subpopulation was also very different in the worst samples when comparing them with those with a good or average response to cryopreservation. Additionally, the DNC of all four subpopulations was in all cases lower in samples with the poorest characteristics of freezability. This was not the only difference, and significant changes in parameters such as the VCL of Subpopulations 2 and 4, linearity coefficient (LIN) of Subpopulations 1, 2, and 3, and wobble coefficient (WOB) of Subpopulations 2 and 3 were also observed in samples with different response to freezing-thawing. Meanwhile, the determination of mitochondrial activity and mitochondrial-linked reactive oxygen species formation indicated that the samples with the poorest freezability characteristics were also those with the lowest mitochondrial activity. We conclude that boar ejaculate resistance to cryopreservation seems to be related to the specific, initial motile-sperm subpopulation structure. In turn, this structure would be closely related to the specific, overall mitochondrial activity, which would be a very important indicator of sperm function. Furthermore, and as a practical conclusion, an in-depth analysis of motile sperm subpopulation structure together with functional tests could improve the design of predictive strategies for the freezability of boar sperm.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.     

Factors in seminal plasma of bulls that affect the viability and motility of spermatozoa

Bound sialic acids on rat spermatozoa were assayed by oxidation with 1 mM-NaIO4 at 0 degree C, liberating C-9 as formaldehyde which was further quantitated using 3-methyl-2-benzothiazolinone. The mean +/- s.d. (n = 20) content of bound sialic acids of spermatozoa from the caput and cauda epididymidis was 50.9 +/- 8.0 and 25.2 +/- 3.8 nmol/10(8) spermatozoa respectively. About 85% of the former and 75% of the latter could be extracted by 1% Triton X-100 and 2 mM-dithiothreitol. About 70% of the former and 20% of the latter were released by neuraminidase from Vibrio cholerae. About 40% of the former and 30% of the latter were sensitive to trypsin. During sperm maturation, the decrease in the total bound sialic acids was due to the decrease in the neuraminidase-sensitive but not the neuraminidase-resistant sialic acids.     

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.     

Subzonal microinjection of mouse spermatozoa: insufficient sperm motility might induce phagocytosis

Acrosome-reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty-one percent of the microinjected oocytes reached a morphologically apparent two-cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.     

The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa

The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Single layer colloid centrifugation technique improves motility,viability and chromatin integrity of ram spermatozoa after thawing

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC – selected samples in comparison to control (not SLC – selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC – selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC – selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC – selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.     

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16°?C for 24?h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240?×?10⁶ spermatozoa/mL using animal protein–free commercial BIOXcell^® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell^® extender supplemented with buserelin acetate and stored 0, 8 and 24?h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell^® extender did positively affect the total motility (was higher in the observed samples with the addition of 2?µg/mL and 4?µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p?<?0.05) over the control group at the 0?h and 8?h of incubation following the supplementation of 2, 4 and 8?μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p?<?0.05) in 2?µg/mL and 4?µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4?µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4?µg/mL to the extender to improve the quality of bovine semen.     

Effects of semen extender and semen processing on motility and viability of frozen-thawed dog spermatozoa

The aims of the present study were, to assess the effects of semen centrifugation, two different diluents and two different freezing methods on post-thaw semen quality in canine semen, and to elucidate the interdependence of these parameters. For this purpose, the sperm-rich fractions of ejaculates from 12 healthy male beagles were divided into four aliquots. Two aliquots were centrifuged and resuspended with two TRIS-egg yolk based extenders: with Uppsala and Gill extender (Gill). The diluents differed in the concentration of glycerol and in the admixture of Equex STM paste (Nova Chemical Sales Inc., Scituate, MA, USA). Diluted semen was frozen either in a styrofoam box or with a computerized freezing machine and an optimized freezing curve (IceCube 1,810; Sy-Lab, Purkersdorf, A). The change in temperature inside the straws was measured during the freezing procedure. Thawed semen samples were assessed for motility and viability (SYBR-14/PI) using the computer assisted sperm analyzer SpermVision (Minitüb, G) and a modified triple staining technique (flow cytometry). Deep freezing in the machine resulted in better motility and viability than in the box. The combination centrifugation-Uppsala extender-machine was superior to all other combinations, which was most evident after storage at +5 degrees C for 7 h (motility: 53.1%, viability: 64.9%). Post-thaw longevity and progressive motility were significantly improved by the use of the here introduced freezing curve. This was shown to be partly caused by less pronounced fluctuations of temperature inside the straws when compared to box-freezing.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Preventive effect of Desferal on sperm motility and morphology

Transition metal ions, mainly iron, are involved in the generation of highly reactive hydroxyl radicals, which are the most powerful inducers of oxidative damage to all biomolecules. The lipids in sperm membranes are highly susceptible to oxidation. Sperm lipid peroxidation (LPO) leads to decrease of motility and reduction of likelihood for sperm‐oocyte fusion. The excess radical production may affect also the spermatozoa morphology. The aim of the present study was to investigate the effect of Desferal on the LPO, motility, and morphology of boar sperm subjected to oxidative stress. After collection, the ejaculates were equally separated and diluted in a commercial semen extender (experiment 1) or in physiological saline (experiment 2). The ejaculates of the 2 experiments were divided into aliquots, which were incubated with one of the following agents: FeSO₄ (0.1mM), H₂O₂ (0.5mM), or FeSO₄ + H₂O₂ (Fenton system), in the presence or absence of Desferal. The application of Desferal in the incubation medium had a protective effect against FeSO₄ + H₂O₂‐induced sperm damage, namely, decrease of LPO; decrease the quantity of immotile spermatozoa and decrease the number of morphological abnormalities, regardless of the used medium. In experiment 2, the presence of FeSO₄ in the incubation medium induced LPO in the same range as the combination FeSO₄ + H₂O₂, in which the effect was reduced by Desferal. Thus, the supplement of Desferal to media used for sperm storage and processing could be a useful tool for diminishing oxidative injury and improving the quality of the semen.