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 glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates

This study was performed to gain insight about how fructose and glucose modulate dog spermatozoa motility in the absence of other motility-modulating factors. Incubation of dog spermatozoa from fresh ejaculates in a basal medium without sugars for 60 min at 37 degrees C induced a progressive decrease in the percentage of motile spermatozoa and in some mean motility parameters, such as mean velocity (VAP), linear coefficient (LIN) and dance (DNC), and an increase in the mean frequency of head displacement (BCF). This indicates a progressive loss of linearity and an increase in oscillatory movement. Addition of 10 mM fructose prevented these effects. Incubation in a basal medium with 10 mM glucose for 60 min at 37 degrees C provoked a fast and intense decrease of LIN and a slight increase of DNC, inducing a less linear and more oscillatory mean movement. Neither fructose nor glucose modified the percentage of motile spermatozoa. The response to both sugars was dose-dependent, with differences appearing at concentrations as low as 1 mM. An analysis of the spermatozoa subpopulation placed above the 95th percentile of the whole population and a factorial analysis of the data indicated that the changes in the mean values of the motility parameters were mainly due to a specific motile subpopulation that had a strong reaction to the two sugars. Our results indicate that fructose, at concentrations from 1 to 10 mM, induced a more linear and less oscillatory motility pattern than glucose. Moreover, from our results we suggest the presence of motile dog sperm subpopulations with an increased sensitivity to fructose and glucose.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

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.     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

Mechanism for procaine-mediated hyperactivated motility in guinea pig spermatozoa

Hyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine-treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca²⁺ did not inhibit procaine-induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca²⁺ for sperm to express hyperactivated motility. © 1994 Wiley-Liss, Inc.     

Chemically modified cellulose acetate membrane for biosensor applications

The chemical modification of cellulose acetate by acylation of the C-2 position produces a membrane that is more hydrophobic and biocompatible. The efficacy of this membrane in biosensor applications is compared with the unmodified cellulose acetate membrane.     

Determination of labeled adenine by means of adsorption on cellulose nitrate filters: a sensitive method for estimation of nucleosidase activity.

Cellulose nitrate filters strongly adsorb adenine up to 4.4 nmoles/cm² of the filter with a constant 90% efficiency. Based on this phenomenon a sensitive and fast method of estimation of labeled adenine in amounts over three orders of magnitude was developed. This method has been successfully applied to estimate the activity of minute amounts of a nucleosidase system, catalyzing the decomposition of ATP to adenine. Other purines and pyrimidines are also adsorbed by cellulose nitrate filters in the following order: Adenine > cytosine ? thymine > uracil > guanine. The affinity of corresponding nucleosides as well as nucleotides toward cellulose nitrate filters is greatly reduced. The kinetics of adsorption and desorption of [¹⁴C]adenine was studied with regard to temperature and time of adsorption, additives, source of the filters and their pore size. The mechanism of adsorption is discussed.     

Influence of temperature on the motility of human spermatozoa]

The mean swimming speed of a suspension of human sperms has been estimated by applying a kinetic theory and using an image analysing computer. This technique allowed the study of the evolution of motility in terms of temperature: the global swimming speed significantly increases from 22 degree C to 31 degrees C, and then remains rather constant. However the percentage increases are very varying according to samples.     

Effect of quercetin on the motility of cryopreserved canine spermatozoa

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 μg/ml and 10 μg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 μg/ml of quercetin in 0.1% DMSO was improved by approximately 10–20% at 30–180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 10⁸ spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.