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The aim of the study was the electrophoretical identification of molecular forms of selected antioxidant enzymes in dog semen. Ejaculates to be studied were chosen from five dogs, aged from two to eight years. Polyacrylamide gel electrophoresis was carried out under non-denaturing conditions and then gels were stained for the activity of the following enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Sperm homogenates and all fractions (pre-spermatic, spermatic and post-spermatic) of dog ejaculate demonstrated one protein band with SOD activity characterized by low electrophoretic mobility. Based on the confirmed sensitivity to H₂O₂, it can be assumed that the detected SOD is an enzyme containing ions of Zn²⁺ and Cu²⁺ (Cu,Zn SOD). In sperm homogenates one protein band with GPx activity was characterized by high electrophoretic mobility, whereas in the spermatic and post-spermatic fractions of dog ejaculate three protein bands with different (low, medium and high) electrophoretic mobility were identified. CAT molecular forms were not found in either sperm homogenates or in the analyzed fractions of ejaculate.
The aim of this study was to investigate the effect of dog semen dialysis on sperm characteristics and some biochemical components of seminal plasma. Whole ejaculates were dialyzed against Triscitrate-fructose extender for a 5 h period at room temperature (using semi-permeable cellulose tubing of 12-14 kDa molecular weight cut-off). It has been demonstrated that the long-term dialysis of dog semen causes a significant decrease in sperm quality parameters and disrupts the biochemical properties of seminal plasma. This procedure requires further improvement.
The resazurin reduction test (RRT) was subjected to spectrophotometric analysis to evaluate the quality of canine semen. Twenty four samples of canine semen were analysed. The absorption peaks for resazurin and resorufin were determined at 615 and 580 nm, respectively. The RRT ratio (RRTₚₗₐₛₘₐ-the ratio for samples containing spermatozoa, RRTpiasma-the ratio for samples containing seminal plasma) was calculated by dividing the absorbance at 580 nm by the absorbance at 615 nm. Spearman's correlation test was used to determine the significance of correlations between the analysed sperm parameters and the results of the resazurin reduction assay. The RRT ratio was highly correlated with sperm motility (r=0.68, P<0.01), progressive sperm motility (r=0.61, P<0.01), the subpopulation of cells with rapid velocity (r=0.72, P<0.01), and the subpopulation of cells with medium velocity (r= -0.54, P<0.05). A negative correlation was observed between the reducing capacity of seminal plasma vs. sperm with plasma membrane integrity (r= -0.60, P<0.01) and sperm with normal morphology (r= -0.58, P<0.01). The RRT test can be used as an additional tool for evaluation of the quality of canine semen.
The aim of this study was to determine the activity of proacrosin and acrosin in spermatozoa originating from the sperm-rich fractions (SRF) and whole ejaculates (WE) of dog semen. In addition, experiments were conducted to determine the activity of antitrypsin inhibitors in the fluids of different ejaculate fractions and whole seminal plasma. Ejaculates were collected from five dogs of mixed breed and one Beagle dog (aged from 2 to 9 years). In the SRF, it was confirmed that the activity of the free acrosin form was predominant (acrosin / proacrosin; 2.38 ± 0.22 / 1.05 ± 0.08 mIU / 106 spermatozoa). On the other hand, spermatozoa originating from the WE exhibited significantly higher (p<0.05) proacrosin activity (proacrosin / acrosin; 2.19 ± 0.19 /1.30 ± 0.11 mIU / 106 spermatozoa). Furthermore, acrosin inhibitor activity was lower in the fluids of the pre-sperm fraction (0.09 ± 0.006 IU / cm3), whereas it was higher in the fluids of the post-sperm fraction (0.11 ± 0.007 IU / cm3). Using PAGE analysis, the antitrypsin activity of the enzyme was represented by the presence of one electrophoretic band in the fluids of the pre-sperm and post-sperm fractions and whole seminal plasma. Furthermore, two electrophoretic bands were detected in the fluids of the SRF. The findings of this study indicate that specific proteinase inhibitors present in the individual ejaculate fractions of dog semen may act by stabilizing the sperm acrosin system.
The aim of this study was to evaluate the influence of season on selected qualitative semen characteristics and biochemical markers of canine seminal plasma. Whole ejaculates were collected from 5 crossbred dogs aged 2-8 years. The study covered a period of one year divided into four seasons: spring (March, April, May), summer (June, July, August), autumn (September, October, November) and winter (December, January, February). Semen samples were subjected to macroscopic and microscopic analyses to determine semen volume, total sperm counts and sperm morphology parameters. The study also involved the determination of sperm motility parameters (CASA system), sperm plasma membrane integrity (SPMI, fluorescent staining SYBR-14/PI), sperm mitochondrial membrane potential (MMP, fluorescent staining JC-1/PI) and the ATP content of sperm cells. Total protein content (TPC) and the activity of alkaline phosphatase (AP) and acid phosphatase (AcP) were determined in biochemical analyses of seminal plasma. No significant differences in ejaculate volume, SMPI or ATP content of sperm cells were observed between seasons. The highest total sperm counts were reported in ejaculates acquired in summer and autumn. The lowest MMP values were determined in summer ejaculates. No significant differences in sperm motility (MOT) were observed throughout the experiment, but ejaculates collected in autumn and winter were characterized by the highest progressive motility (PMOT). AP activity and TPC were not significantly affected by season. However, AcP activity levels were significantly lower in autumn than in the remaining seasons. Seasonal variations in the analyzed macroscopic and microscopic parameters of ejaculates and biochemical markers of seminal plasma did not exert a clear negative effect on the quality of canine semen.
The aim of the study was to characterize the enzymatic and non-enzymatic components comprising the antioxidant system in spermatozoa and individual fractions of dog ejaculate. Ejaculates were collected from six dogs of mixed-breeds. Total protein content, activity of antioxidant enzymes and the content of low-molecular antioxidants, such as L-glutathione (GSH), L-ergothioneine (ERG), L-ascorbic acid and total SH-group, were analyzed in the ejaculated spermatozoa and seminal plasma of the pre-spermatic, spermatic and post-spermatic fractions. The total antioxidant status (TAS) and antiperoxidant activity of the seminal plasma were also determined. The enzymatic antioxidant system of canine spermatozoa is mainly represented by superoxide dismutase (SOD) activity and, to a lesser extent, by glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity. Catalase activity was not detected either in the spermatozoa or in the different ejaculate fractions. GSH and ERG were detected in each fraction. Furthermore, a high level of L-ascorbic acid was observed in fractions of the ejaculate. Proteins and low-molecular weight antioxidants could influence the total antioxidant status and antiperoxidant activity of the seminal plasma. Increased suppressive activity against lipid peroxidation was shown only in the pre-spermatic and post-spermatic fractions. The different fractions of dog ejaculate, which are the main source of enzymatic antioxidants and low-molecular antioxidants, play an important role in protecting spermatozoa against reactive oxygen species.
The aim of this study was to determine the effect of two variants of Tris-citric acid-fructose (TCF) extender containing whole hen egg yolk (TCF-HEY) and lyophilized lipoprotein fractions extracted from ostrich egg yolk (TCF-LPFo) on selected biological properties of cryopreserved sperm cells. Post-thaw percentage of motile sperm (MOT) was significantly higher (P<0.05) for TCF-HEY extender (66.3 ± 3.2%) than for TCF-LPFo extender (52.4 ± 3.4%). Moreover, there was no significant difference in the percentage of sperm with progressive motility (PMOT). Both diluents effectively preserved sperm plasma membrane integrity and mitochondrial function. However, it was observed that cryopreservation impaired the functionality of antioxidant sperm enzymes. The above was manifested by reduced SOD activity, in particular in samples preserved in the TCF-HEY extender, as well as decreased GPx activity. Both diluents inhibited the rate of lipid peroxidation in sperm plasma membrane during freezing-thawing. Our results suggest that LPFo is a satisfactory alternative to hen egg yolk in the extender used for canine sperm cryopreservation.
A limited field trial was performed to evaluate the fertilizing capacity of boar spermatozoa frozen in an extender supplemented with lipoprotein fractions isolated from ostrich egg yolk (LPFo). Boar semen, diluted in an extender containing lactose with lyophilized lipoprotein fractions, glycerol and Orvus Es Paste (lactose-LPFo-G), was frozen using a controlled programmable freezer. Sperm characteristics, such as motility, plasma membrane and acrosome integrity, and mitochondrial function were monitored. Post-cervical artificial inseminations (post-CAIs) in multiparous sows (Polish Large White) were performed using the Soft & Quick® catheter/cannula set. Sows were inseminated 2 to 3 times within one oestrus. Possible returns of sows to oestrus were determined from 21 to 30 days after post-CAIs. In this field trial, sows inseminated with 2 x 109 motile frozen-thawed spermatozoa resulted in pregnancy and farrowing rates of 75%, respectively. The average piglets born live was 10.5 ± 0.4 (mean ± SEM). The data of this study showed that post-CAI of boar semen frozen in LPFo-containing extender has the potential to provide acceptable fertility results. Further investigations are needed to elucidate the cause of variations in pregnancy/farrowing rate associated with frozen-thawed boar semen.
The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 × 10⁻³M, 1 × 10⁻⁴M, 1 × 10⁻⁵M and 1 × 10-6M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 × 10⁻³ M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 × 10⁻³ M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 × 10⁻³ M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.
The aim of this study was to investigate the effect of PAF supplementation in semen extender on ATP content in cryopreserved bull spermatozoa used for artificial insemination at different time intervals. Cryopreserved semen was treated with different concentrations of PAF: 1x10⁻⁵M, 1x10⁻⁶M, 1x10⁻⁷M, 1x10⁻⁸M and 1x10⁻⁹M at 37°C. In the present work we showed that content of ATP in cryopreserved semen supplemented with 1x10⁻⁹M PAF was statistically significantly higher at 90 and 120 minutes of incubation in comparison to the control group (p≤0.05). Present study indicates the potential influence of PAF on ATP content in male spermatozoa via it’s protective role towards mitochondria metabolic activity.
Oxidative stress has a significant impact on the quality of the mammalian semen. An excessive amount of reactive oxygen species (ROS) present in the environment of the spermatozoa, which rises as a result of prooxidative-antioxidative balance disorders, leads inter alia to a decrease of semen motility, changes in the conformation of the spermatozoa membrane, acrosome damage, decrease of mitochondrial activity, and appearance of the lipid peroxidation, which can become a cause of reduced fertility or infertility. For this reason the estimation of the influence of oxidative stress on semen can turn out to be a very useful tool in diagnostics of problems with fertility. Nowadays, in order to evaluate the intensity of the oxidative stress it is possible to use the direct method and indirect method that evaluate both the amount of ROS in the semen and the increase of changes in the spermatozoa caused by this compound. A precise analysis of the changes, resulting from prooxidative-antioxidative balance disorders with use of a suitable method of analysis is a base for accurate diagnosis.
The effect of reactive oxygen species (ROS) on spermatozoa can be twofold. Depending on their concentration, moment of activity, and exposure time, ROS can be an essential element in modulating many physiological processes or a cause of serious damage to the gametes. ROS of the greatest importance to the quality of semen are superoxide anion O₂⁻˙ and hydrogen peroxide – H₂O₂. Trace amounts of these compounds are essential for such processes as sperm maturation, capacitation, hyperactivation, acrosome reaction, and the fusion of the sperm with the oocyte. Their excess results in the development of oxidative stress, which significantly reduces the quality of sperm through the peroxidation of lipids, proteins, and other elements of cellular structure. The antioxidative system of semen limits the negative effects of oxidative stress on sperm. The proper function of the reproductive system and successful fertilization depend on maintaining a delicate balance between the amount of ROS and the activity of the antioxidant system.
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