Agnieszka Partyka

The effect of cysteine and superoxide dismutase on the quality of post-thawed chicken sperm.

The study was conducted to determine the influence of N-acetyl-l-cysteine (NAC) and superoxide dismutase (SOD) on chicken sperm motility, plasma membrane and acrosome integrity, mitochondrial activity, lipid peroxidation (LPO) and apoptotic changes after freezing-thawing process. Semen samples from fifteen Greenlegged Partridge roosters were pooled, diluted with EK extender without antioxidants (control), or supplemented with 5 mM NAC, or 200 U/mL SOD. Samples were subjected to cryopreservation. After thawing, sperm parameters evaluated by using CASA system and flow cytometry were assessed. The extender supplemented with NAC and SOD caused the increase (P < 0.01) in sperm motility and provided the higher percentage of rapid sperm (P < 0.01) compared to control. The addition of NAC increased the progressive motility of cells (P < 0.01). In NAC and SOD groups post-thaw plasma membrane integrity was higher (P < 0.05) and less spermatozoa showed apoptotic changes (P < 0.01, P < 0.05). Post-thaw percentage of sperm with high mitochondrial activity was the greatest (P < 0.05) with NAC addition. The SOD supplementation only reduced (P < 0.05) the percentage of sperm with LPO, following the cryopreservation. These results indicate that the addition of NAC (5 mM) and SOD (200 U/mL) is beneficial for the function of frozen-thawed chicken spermatozoa. The antioxidants prevented the reduction in motility, viability and mitochondrial membrane potential, as well as protected from apoptotic changes in sperm. Lipid peroxidation in sperm plasma membrane was decreased by SOD supplementation. Therefore, these antioxidants can be recommended as an additional component of chicken freezing extender.

Lipid peroxidation and antioxidant enzymes activity in avian semen

The present study compared the antioxidant system and lipid peroxidation in semen of two avian species: chicken and goose. The experiment was conducted on Greenleg Partridge roosters and White Koluda(®) ganders, each represented by 10 mature males. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma. In gander spermatozoa, the amount of MDA was 10 times greater (P<0.01) than in rooster spermatozoa. Each of the investigated antioxidant enzymes had greater (P<0.01) activity in goose than chicken sperm. Catalase activity was detected in seminal plasma and spermatozoa from both studied species for the first time. In seminal plasma, the activity of GPx was two times greater (P<0.01) in the White Koluda(®) than in chickens, whereas SOD activity was less (P<0.01) than in chickens. This is the first study describing the presence of CAT in avian semen and the occurrence of indicator of lipid peroxidation (LPO) in geese. Data from the present study clearly show the species-specific differences in the activity of antioxidant defense and LPO. The greater amount of lipid peroxidation and greater activity of antioxidant enzymes in goose semen might suggest that spermatozoa were under greater oxidative stress and the enzymes were not utilized for the protection of functionally and structurally impaired cells. In turn, in fresh chicken semen a lesser activity of antioxidant enzymes accompanied with a lesser lipid peroxidation amount and good semen quality could indicate that fowl spermatozoa were under oxidative stress, but the enzymes were employed to protect and maintain sperm quality.

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen.

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.

Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10(6)/mL, 5 × 10(6)/mL, and 1 × 10(6)/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10(6)/mL dilution rate, spermatozoa diluted to 1 × 10(6) sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-β-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl spermatozoa.

Methods of Assessment of Cryopreserved Semen

Despite the significant progress, the post-thaw viability and fertility of the cryopreserved sperm are still reduced, as a consequence of accumulated cellular injuries that arise throughout the cryopreservation process. Many laboratory tests have already been carried out to verify these detrimental effects and their origin. Their is needed to well understand the whole process of cryopreservation and its influence on sperm function. As a consequence, it would lead to a subsequent improvement of sperm viability by means of reformulated protocols and approaches helping to minimize the detrimental effect of cryopreservation.

The Effect of L-Carnitine, Hypotaurine, and Taurine Supplementation on the Quality of Cryopreserved Chicken Semen

The objective of this study was to investigate the effect of L-carnitine (LC), hypotaurine (HT), and taurine (T) on the quality of frozen-thawed chicken semen. Pooled semen samples were divided into seven aliquots (control, 1 mM LC, 5 mM LC, 1 mM HT, 10 mM HT, 1 mM T, and 10 mM T) and subjected to cryopreservation. Postthaw sperm motility was determined by IVOS system and sperm characteristics were assessed with fluorochromes and flow cytometry. The highest sperm motility and the highest percentage of viable sperm were in the HT1 group (P < 0.01 and P < 0.05) following cryopreservation. After thawing, we observed a higher percentage of sperm without apoptosis and membrane reorganization changes in the LC1 and T1 group when compared to the control (P < 0.05). There was a higher percentage of live sperm without lipid peroxidation (LPO) in all treatments (P < 0.01; P < 0.05), when compared to the control group. The percentage of sperm with high mitochondrial potential significantly increased with LC1, T1, and T10 (P < 0.05). Supplementation of the diluent with LC1, LC5, and T1 significantly (P < 0.05) reduced DNA susceptibility to fragmentation, compared to the control and HT1 groups. These results indicate that the addition of examined antioxidants improves the quality of cryopreserved chicken semen.

Effects of N-acetyl-L-cysteine and catalase on the viability and motility of chicken sperm during liquid storage

The purpose of the current study was to investigate the effects of N-acetyl-L-cysteine (NAC) and catalase (CAT) on chicken sperm parameters during liquid storage for up to 48 h at 5 °C. Supplementation of EK extender with NAC (15 mM) increased sperm motility after 24h. After 48 h, an increase in sperm viability with NAC (5, 15 mM) and CAT (100, 300 U/mL) was observed, but only treatment with 15 mM NAC improved sperm progressive motility.

Cyclodextrins or cholesterol-loaded-cyclodextrins? A better choice for improved cryosurvival of chicken spermatozoa

This study was designed to test if treating chicken sperm with i) the cyclodextrins 2-hydroxypropyl-β-cyclodextrin (HBCD) and methyl-β-cyclodextrin (MBCD) alone improve fresh, liquid-stored and cryopreserved semen quality, or ii) cholesterol-loaded cyclodextrins (CLCs): 2-hydroxypropyl-β-cyclodextrin loaded with cholesterol (HCLC) and methyl-β-cyclodextrin loaded with cholesterol (MCLC) enhance chicken semen quality for application to assisted reproductive technologies. Three consecutive experiments were performed with different concentrations of additives: Exp. 1: 1, 2, 4 mg of HBCD and MBCD in fresh and liquid stored semen; Exp. 2: 1, 2, 4 mg of HCLC and MCLC in fresh and liquid stored semen; and Exp. 3: 1, 2 mg of HBCD, HCLC, 1 mg MBCD, MCLC in cryopreserved and post-thaw storage semen. Sperm motility parameters were assessed by CASA system and comprehensive sperm characteristics were evaluated by flow cytometry. Supplementation with 4 mg HBCD, MBCD, HCLC and MCLC resulted in the lowest motility and functional parameters of fresh and stored spermatozoa for 24 h at 5 °C. After cryopreservation, spermatozoa stored with CLCs showed significantly lower progressive motility and velocity of movement, and exhibited the lowest percentage of cells with intact plasma membranes and acrosomes, mitochondrial activity and cells without apoptosis. These results indicated that CLCs did not improve chicken sperm viability after cryopreservation. However, spermatozoa treated with 2 mg HBCD showed higher proportion of motile sperm (28%; P < 0.01) together with higher proportion of sperm cells with high mitochondrial potential (25%; P < 0.05) compared to the control (18%; 21%, respectively) after 3 h of post-thaw storage. CLC, especially with methyl-β-cyclodextrin, appeared to be detrimental to chicken spermatozoa, causing apoptosis, impairment of mitochondrial potential, and damaged acrosomes and sperm plasma membranes.

The usefulness of Real Time Morphology software in semen assessment of teratozoospermic boars

The aim of the present study was to compare two different techniques of sperm cell morphology evaluation in teratozoospermic boars: computer assisted semen morphology analysis and conventional assessment of stained semen smears. The semen samples were collected manually from 30 boars with reduced semen quality. In all samples the percentage of morphologically normal spermatozoa was below 70%. Computer assisted semen morphology assessment was performed using the Real Time Morphology (RTM) software (IVOS ver. 12.2, Hamilton Thorne Bioscience). The assessment was made by phase contrast optics, with the magnification of 20 x 3.8 and without staining. Conventional morphology assessment was performed by bright field microscopy with 1,000 x magnification after staining with Giemsa. At least 200 spermatozoa were evaluated per slide in both methods. The Bland-Altman plot indicated a general agreement between both methods of sperm morphology evaluation. The plots revealed the widest limits of agreement (mean ± 1.96 SD) for the percentage of midpiece anomalies (from −16 to 13.2), and the narrowest for the percentage of looped tail (from −1.49 to 1.09). The Bland Altman plot indicates general agreement between RTM and Giemsa staining in the percentage of major and minor defects. However, it was not possible to evaluate acrosomes using RTM. Otherwise, RTM proved to be a valuable tool in sperm morphology assessment, with accuracy equal to typical conventional methods.