Uncentrifuged Control Research Articles

Comparison of sperm selection techniques in donkeys: motile subpopulations from a practical point of view.

The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post-thaw semen characteristics.

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen-thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species-specific colloid, Bovicoll. After centrifugation for 20min at 300g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l'Aigle, France); the SLC-selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC-treated samples had a higher percentage of live, superoxide-positive spermatozoa than uncentrifuged samples (27.9±5.1% versus 21.7±6.7%; p=.03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9±8.2% versus 40.5±15.1%; p=.03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non-treated samples (42.0±8.5% versus 55.9±14.4%; p=.04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide-positive spermatozoa and live, hydrogen peroxide-negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC-selected spermatozoa compared to unselected samples.

Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without Single Layer Centrifugation

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from “normal” ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem.

Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation

There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidium and dichlorodihydrofluorescein diacetate. In addition, the effects of a new method of selecting good quality spermatozoa by colloid centrifugation, known as Single Layer Centrifugation (SLC), on ROS-content were investigated. Superoxide production by stallion spermatozoa was found to be higher at the start than at the end of the breeding season in Sweden (22±16% versus 9±6%, P<0.05), whereas sperm motility was lower (total motility 80±9% versus 90±6%, P<0.01; progressive motility 55±12% versus 60±8%, P<0.05, at the beginning and end of the breeding season respectively). The mean values of the other parameters of sperm quality measured did not differ with time within the breeding season although differences did occur for individual stallions. SLC was found to select motile spermatozoa that contained less superoxide (16±14% versus 23±18%, P<0.01) and less peroxide (0.3±0.8 versus 1±2%, P<0.01) than uncentrifuged controls, although they were capable of producing ROS when stimulated with menadione. This reduced peroxide production may contribute to the enhanced sperm survival (retention of motility) seen in the SLC samples during storage.

The impact of cushioned centrifugation protocols on semen quality of stallions

The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/mL. Sperm recovery rate among centrifugation treatment groups was compared. Motion characteristics, plasma membrane intactness (SMI), and DNA quality (COMPαt) of sperm were compared among treatment groups and uncentrifuged controls immediately following centrifugation (Time 0 h) and following 24 h of cooled storage (Time 24 h). Centrifugation treatment did not affect sperm recovery rate (P > 0.05). At Time 0 h, no differences in experimental end points were detected between cushion-fluid volumes tested (P > 0.05). Values for percent total sperm motility, percent progressive sperm motility, and track straightness were similar between sperm-number treatments subjected to centrifugation (P > 0.05). At Time 24 h, values for all experimental endpoints were similar between centrifugation treatments for cushion volume per tube, and between centrifugation treatments for sperm number per tube (P > 0.05). Centrifugation treatments and control treatments were similar for five of six variables tested (P > 0.05). Sperm storage concentrations of 25 × 106 and 250 × 106/mL yielded similar values for percent total sperm motility, percent progressive sperm motility, percent SMI, and percent COMPαt (P > 0.05). A storage concentration of 250 × 106 sperm/ml yielded higher values for curvilinear velocity, and lower values for straightness, than all other groups (P < 0.05). In conclusion, centrifugation with as little as 1 ml of cushion fluid and a sperm number of up to 3 × 109 sperm in 50-ml conical-bottom centrifuge tubes had no detrimental effect on initial or cool-stored sperm quality. Additionally, storage of centrifuged sperm at a concentration of 250 × 106/mL with 20% seminal plasma (v/v) did not have a detrimental effect on percentages of motile or progressively motile sperm, or sperm DNA quality.

Single layer centrifugation with androcoll-p can be scaled-up to process larger volumes of boar semen.

The objective of this study was to scale-up the procedure for Single Layer Centrifugation (SLC) through AndrocollTM-P, as a preliminary step towords processing the whole ejaculate. The first experiment compared Single Layer Centrifugation using 4.5 mL and 15 mL extended ejaculate (SLC-4.5 and SLC-15, resp.), assessing sperm quality by objective motility analysis, morphology, viability, and the production of reactive oxygen species (ROS). In the second experiment, SLC-4.5 was compared to Single Layer Centrifugation with 25 mL extended ejaculate (SLC-25) using motility analysis and morphology. In both experiments, normal morphology and linear motility were significantly higher in the SLC-selected samples than in the uncentrifuged controls (P < .001), whereas total motility and membrane integrity were unchanged. Although ROS production was higher in the SLC-selected samples than in the controls (P < .01), this might have been due to the presence of antioxidants in seminal plasma in the latter. In conclusion, there was no difference in sperm quality between SLC-4.5 and SLC-15 samples, or between SLC-4.5 and SLC-25 samples, indicating that the SLC method can be scaled-up successfully.

Selection of Boar Spermatozoa Using Centrifugation on a Glycidoxypropyltrimethoxysilane-Coated Silica Colloid

Use of sperm separation methods such as density gradient centrifugation for selecting the best spermatozoa for animal breeding is constrained by the problem of dealing with the large volumes of ejaculate produced by the males of some species, such as boars. The purpose of this study was to compare density gradient centrifugation (DGC) with centrifugation on a single layer of colloid (SLC) for the preparation of ejaculated boar spermatozoa using Androcoll(TM)-P. There was no difference between the two techniques in terms of sperm motility or duration of motility after selection, and sperm motility was retained for at least 24 h longer in the centrifuged sperm preparations than in controls (uncentrifuged aliquots). Sperm motility was significantly better (P<0.001) in the centrifuged sperm preparations (means +/- sd: SLC 79.6 +/- 8.1% and DGC 74.2 +/- 12.0%) than in the uncentrifuged controls (62.9 +/- 12.7%). The mean yield of motile spermatozoa for SLC was 67.5 +/- 25.6%, and for DGC it was 59.6% +/- 22.3% (not significant, ns). Sperm survival was significantly increased by colloid centrifugation (control 3.1 +/- 0.3 days, SLC 5.5 +/- 0.79 days, DGC 5.75 +/- 0.62 days; P<0.001 for uncentrifuged versus centrifuged; SLC vs. DGC, ns). Moreover, boar spermatozoa could be stored for 24 h before centrifugation without any detrimental effect on sperm motility or duration of motility. In a further experiment, larger volumes of ejaculate were processed easily on a modified SLC, indicating that this method may be practical for processing large volumes of boar ejaculates.

Effect of centrifugation on the removal of extracellular polymers and physical properties of activated sludge

Activated sludge is a flocculated suspension of living and dead microorganisms enmeshed together in a polymer matrix along with some organic and inorganic materials. Extracellular polymers, main components of which are the polysaccharides, proteins, and nucleic acids, act as a protective barrier to the living microbial community, and give the floc its integrity and rigidity. These polymers have been found to affect the dewatering and settling characteristics of activated sludge. Even though they were studied considerably in the past there is not a complete understanding of the relationship between the amount of polymer and changes in the physical properties of sludge. The purpose of this research is to observe the effects of centrifugal force on the extraction of the extracellular polymers from an activated sludge and to correlate the amount of polymer extracted to the subsequent physical properties of the sludge. Centrifugal forces of 2000, 5000, 8000, 11,000, 14,000, and 16,500 G were tested for their effects on the amount of extracellular polymer collected, as well as the final properties of sludge like viscosity, filterability, compactability and supernatant turbidity. Results were evaluated in comparison to those of an uncentrifuged control waste activated sludge. Removal of extracellular polymers seems to affect the viscosity of activated sludge. Filterability, as measured by the capillary suction time instrument, was affected by the extraction of polymers to a great degree. Results of the independent tests correlated well with each other and with the amount of polymers extracted. Results of this study have important implications on sludge pumping and dewatering.

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Density gradient centrifugation of hemopoietic colony‐forming cells

AbstractBuoyant density distributions of hemopoietic colony‐forming units (CFU) from normal mouse marrow were determined by equilibrium density gradient centrifugation in bovine serum albumin (BSA) gradients. The distributions were compared with those obtained for the total population of nucleated cells from normal mouse marrow. The buoyant density distribution for CFU was found to differ from the density distribution for the total nucleated cell population, and the portion of the total cell population with densities much less than the mean value was found to contain up to a 30‐fold greater proportion of CFU than an uncentrifuged control. These results provide a preliminary approach to the purification and characterization of normal hemopoietic colony‐forming stem cells.