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

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

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

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

Endogenous carbon monoxide (CO) as a modulating factor of molecular biological clock in the hypothalamic structures involved in light transmission in pig and wild boar hybrid during long and short day season

Mature males of a wild boar-pig crossbreed, during the long and short day season, were used for the study which demonstrates that the chemical light carrier CO regulates the expression of biological clock genes in the hypothalamus via humoral pathways. Autologous blood with experimentally elevated concentrations of endogenous CO (using lamps with white light-emitting diodes) was infused into the ophthalmic venous sinus via the right dorsal nasal vein. Molecular biology methods: qPCR and Western Blot were used to determine the expression of genes and biological clock proteins. The results showed that elevated endogenous CO levels, through blood irradiation, induces changes in genes expression involved in the functioning of the main biological clock located in suprachiasmatic nuclei. Changes in the expression of the transcription factors Bmal1, Clock and Npas2 have a similar pattern in both structures, where a very large decrease in gene expression was shown after exposure to elevated endogenous CO levels. The changes in the gene expression of PER 1-2, CRY 1-2, and REV-ERB α-β and ROR β are not the same for both POA and DH hypothalamic structures, indicating that both structures respond differently to the humoral signal received. The results indicate that CO is a chemical light molecule whose production in an organism depends on the amount of light. An adequate amount of light is an essential factor for the proper functioning of the main biological clock

Seasonal-dependent variations in metabolic status of spermatozoa and antioxidant enzyme activity in the reproductive tract fluids of wild boar/domestic pig hybrids

This study investigated seasonal changes in the metabolic performance of spermatozoa and activity of the antioxidant enzymes in the seminal plasma of three wild boar/domestic pigs (aged 1.5 to 2.5 years) and the activity of the antioxidant enzymes in fluids of the cauda epididymidis and vesicular glands from 16 wild boar/domestic pig hybrids (aged 1 to 3 years). Parameters of the sperm metabolic activity, such as total motility, mitochondrial functions, and measurements of oxygen uptake, ATP content and L-lactate production, were analyzed during the spring-summer and autumn-winter periods. Besides these sperm metabolic parameters, the sperm membrane integrity was also assessed. Total protein content and activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were measured in the reproductive tract fluids. There were no marked significant differences (P > 0.05) between the seasonal periods in terms of sperm motility, mitochondrial function and oxygen uptake; however, spermatozoa collected during the autumn-winter period exhibited higher (P < 0.05) ATP content and L-lactate production than those harvested during the spring-summer period. It was found that the vesicular gland fluid exhibited a higher level of SOD activity during the spring-summer period compared with the autumn-winter period. Furthermore, CAT activity in the seminal plasma and vesicular gland fluid was greater during the autumn-winter. Total protein content was significantly higher in the vesicular gland fluid, whereas the cauda epididymidal fluid exhibited greater SOD and GPx activities, irrespective of the seasonal period. The findings of this study confirmed seasonal-related differences in the metabolic performance of spermatozoa and activity of antioxidant enzymes of the reproductive tract of the boar/domestic pig hybrids

Effect of boar ejaculate fraction, extender type and time of storage on quality of spermatozoa

The aim of this study was to investigate the effect the sperm-rich fraction (F1) and the post-F1 fraction (F2) on the quality of boar spermatozoa stored in a liquid state. Ejaculates were collected from three Polish Landrace boars. Each ejaculate fraction was diluted with BTS short-term extender and Safe-Cell Plus (SCP) long-term extender and stored for seven days (D1-D7) at 17°C. Analyses included sperm motility parameters, normal apical ridge (NAR) acrosomes and plasma membrane integrity (PMI). Prior to the dilution of fractions, marked changes (p<0.05) were noted between F1 and F2 in progressive motility (PMOT), velocity average pathway (VAP) and velocity straight line (VCL). After the ejaculate was diluted, the type of fraction and type of extender significantly affected (p<0.05) PMOT, being markedly higher (p<0.05) for F1 extended in BTS. No marked changes (p<0.05) were observed between F1 and F2 extended in SCP for any of the analyzed sperm quality parameters during seven days of storage. Significantly higher (p<0.05) values of sperm quality parameters were noted in F1 compared with F2 for BTS on D7 of storage. The results of the four-way ANOVA analysis indicate that boar, fraction of ejaculate, extender type and day of storage had significant effects on the quality of boar stored spermatozoa. The F1 was characterised by higher quality of spermatozoa during storage in comparison with F2 in the short-term extender. Using the long-term extender containing the proteins allowed for a better application of F2, which could be important for the pig industry

The Quality and Fertilizing Potential of Red Deer (<i>Cervus elaphus</i> L.) Epididymal Spermatozoa Stored in a Liquid State

The aim of this study was to assess the quality and fertilizing potential of red deer epididymal spermatozoa stored in a liquid state for up to 11 days (D11). In Experiment 1, sperm quality was determined. In Experiment 2, the efficiency of in vitro fertilization (IVF) and artificial insemination (AI) of stored sperm were evaluated. An analysis of sperm quality on D5 of storage revealed a decrease (p < 0.05) in motility and morphology, and a higher proportion of apoptotic spermatozoa. On D1, D7 and D10, the total motility of sperm for IVF and AI was determined to be 82.6%, 71.0% and 64.8%, respectively. The results of IVF and AI demonstrated that the fertilizing potential of spermatozoa differs between days of storage. The percentage of blastocysts was higher when oocytes were fertilized on D1 (17.4 %) compared to D7 (8.5%) and D10 sperm (10.5%). Differences were noted in the pregnancy rates of inseminated hinds. The insemination with D1, D7 and D10 sperm led to live births (33% from D7 and D10). The results indicate that the quality of red deer epididymal spermatozoa remains satisfactory during ten days of storage in a liquid state, and that these spermatozoa maintain their fertility potential

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