Molecular forms of selected antioxidant enzymes in dog semen – electrophoretical identification

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

Acrosin system of dog spermatozoa and reproductive tract secretions

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

Semen characteristics and selected biochemical markers of canine seminal plasma in various seasons of the year

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

Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa

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

Effect of dialysis of dog semen on sperm characteristics and some biochemical components of seminal plasma

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

Effects of the platelet-activating factor (PAF) supplementation on ATP content of cryopreserved bull spermatozoa (AI)

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

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

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

Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine (<i>Canis lupus familiaris</i>) and stallion (<i>Equus caballus</i>)

Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content of carbonyl groups in the SP was measured on the basis of the reactions with DNPH. The following reagent variants were used to dissolve protein precipitates: Variant 1 (V1) – 6M Guanidine solution and Variant 2 (V2) – 0.1M NaOH solution. It has been shown that to obtain reliable results for the measurement of protein carbonylated groups in the dog and horse SP, both 6M Guanidine and 0.1M NaOH may be used. A correlation was also found between the number of carbonyl groups and the total protein content in the canine (V1: r = -0.724; V2: r = -0.847) and stallion (V1: r = -0.336; V2: r = -0.334) SP. Additionally, the study showed a higher content (p≤0.05) of protein carbonyl groups in the stallion SP in the non-breeding season compared to the breeding season. The method based on the reaction with DNPH, due to its simplicity and cost effectiveness, appears to be suitable for large-scale application in the determination of the SP proteins oxidative damage in dog and horse semen

Effects of storage in different semen extenders on the pre - freezing and post - thawing quality of boar spermatozoa

The aim of this study was to investigate the effects of storage of semen in different commercial extenders on the pre-freezing and post-thawing quality of boar spermatozoa. Semen was diluted in BTS, Androhep (AH) and Gedil (GD), stored for 24 h at 17°C, and then frozen in accordance with the cryopreservation protocol. Analyses of the quality of spermatozoa included: motility, normal apical ridge (NAR) acrosome, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), measurements of ATP content and activity of superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Prior to the freezing process, no significant effect of the extender on the sperm quality parameters was noted. After thawing the spermatozoa it was demonstrated that the type of extender used influenced PMI, MMP, ATP content and activity of GPx. In the AH extender the percentage of spermatozoa with PMI and ATP content in spermatozoa was significantly higher (P<0.05) as compared to the BTS or GD extenders. In addition, semen stored in the AH was characterised by a statistically higher (P<0.05) percentage of spermatozoa with MMP and increased activity of GPx as compared with the BTS. The results obtained indicate that for the cryopreservation process, boar spermatozoa stored for 24 hours in liquid state can be used. However, the type of extender used prior to freezing may have a significant effect on the post- thawing quality of the spermatozoa. The AH extender better secured the quality of thawed boar spermatozoa as compared with the BTS or GD

Semen quality assessments and their significance in reproductive technology

Semen quality assessment methods are very important in predicting the fertilizing ability of persevered spermatozoa and to improve animal reproductive technology. This review discusses some of the current laboratory methods used for semen quality assessments, with references to their relevance in the evaluation of male fertility and semen preservation technologies. Semen quality assessment methods include sperm motility evaluations, analyzed with the computer-assisted semen analysis (CASA) system, and plasma membrane integrity evaluations using fluorescent stains, such as Hoechst 33258 (H33258), SYBR-14, propidium iodide (PI), ethidium homodimer (EthD) and 6-carboxyfluorescein diacetate (CFDA), and biochemical tests, such as the measurement of malondialdehyde (MDA) level. This review addresses the significance of specific fluorochromes and ATP measurements for the evaluation of the sperm mitochondrial status. Laboratory methods used for the evaluation of chromatin status, DNA integrity, and apoptotic changes in spermatozoa have been discussed. Special emphasis has been focused on the application of proteomic techniques, such as two-dimensional (2-D) gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/MS), for the identification of the properties and functions of seminal plasma proteins in order to define their role in the fertilization-related processes