No abstract

Replacement of animal-origin components in extenders used for bull semen freezing is of high importance for individuals involved in cattle breeding. The experiment was designed to compare efficacy of 5 different semen extenders in cryopreservation of bull semen: sodium citrate-based extender containing egg yolk (CT), commercially available Bioxcell� (IMV Technologies, L'Aigle, France), and 3 custom-made homogenized plant lipidsbased, egg yolk-free extenders (Y-1, Y-2, and Lipo) . The objective was to determine whether homogenization procedures of lipids improve the quality of the extender. Lipid homogenates of custom-made extenders were prepared in Tris buffer using a high pressure homogenizer (Nira Saovi, Parma, Italy). Ten (Y-1) or 5 (Y-2) homogenization cycles were applied and then 8% glycerol was added. Lipid liposomes were produced by simultanous high pressure homogenization of lipids and glycerol supplementation (Lipo). Semen was collected from young bulls of 3 different breeds (Simmental, Polish Red, and Holstein; 1 ejaculate/bull). Each ejaculate with at least 70% motility was split into 5 parts and processed further by a standard freezing protocol: semen was diluted at 35�C with each of the 5 extenders to a concentration of 100 � 106 spermatozoa per mL, cooled to 4�C over 5 h, aspirated into 0.25-mL plastic straws, frozen in liquid nitrogen vapor to –140�C, and then plunged into LN2. Straws were thawed in a water bath at 37�C for 20 s. Sperm motility was estimated microscopically immediately after thawing and after 5 h of storage at 22�C. Immediately after thawing, flow cytometry and SYBR-14/PI staining were used for examination of sperm membrane integrity (live/dead assay). A total of 20 000 spermatozoa of each sample were counted. Student's t-test was used to estimate statistical differences between experimental groups. The mean sperm motility after thawing ranged from 45.6% (SD = 13.7) for CT (egg yolk extender) to 57.8% (SD = 7.1) for Lipo. A significant difference (P < 0.05) was observed betweenY-1 (50.0%, SD = 9.7) and Lipo and Bioxcell (56.1%, SD = 8.6). After 5 h of storage at 22�C, the mean motility for all tested bulls ranged from 25.0% (SD = 7.1) for CT to 42.2% (SD = 7.5) for Lipo. Significant differences were observed between Lipo (P < 0.01), Y-2 (P < 0.05) and CT, and between Y-1 and Lipo (P < 0.01). Mean percentage of 'live' spermatozoa with intact membrane after freezing/thawing was 51.85% (SD = 11.49) for Y-1, 45.72% (SD = 9.36) for Y-2, 47.57% (SD = 7.93) for Lipo, 45.47% (SD = 8.35) for Bioxcell, and 49.06 (SD = 11.59) for CT. No significant differences were observed except forY-1 and Bioxcell extenders (P < 0.05). It can be concluded that both methods of lipid/glycerol homogenization can be successfully applied in the preparation of bull semen extender. In addition, extensive lipid homogenization (10 cycles) produced more transparent extender that in turn improved visualization of sperm. Custom-made plant origin lipids homogenization may provide a valuable alternative for the preparation of extenders that more closely match the membrane composition of bull sperm cells and thus contribute to development of an efficient extender free of animal-origin components for bull semen freezing.

ATP level, cell motility and viability, oxygen uptake, pyruvate kinase activity, and ultra-weak photon emission (UPE) induced by red-ox Fe(2+)-ascorbate cycling system were studied in fresh, in previously equilibrated in a glycerol diluent, and in cryopreserved bull spermatozoa, exposed to thermal stress by incubation of the cells at 44 degrees C. A sharp drop in motility and viability of fresh spermatozoa and even more so, of equilibrated and cryopreserved cells was accompanied by accumulation of ATP. When cell movement was totally inhibited, ATP utilization was decreased, while chemical energy continued to be produced by cell pyruvate kinase, one of the key glycolytic enzymes, which in spermatozoa is very active (6500 IU/g protein) and insensitive to feed-back inhibition by excess of ATP and L-cysteine. Accumulation of ATP during incubation at 44 degrees C in 0.9% NaCl was accompanied by rapid decrease in oxygen consumption by fresh spermatozoa and an increase in Fe(2+)-ascorbate induced UPE, followed by a sharp decrease in ATP level observed at the end of induced UPE measurement. The increase in photon emission due to lipid peroxidation was highly correlated with the increase in cell ATP level caused by thermal stress.

The aim of the study was to improve cryopreservation of young bulls’ semen. Bioxcell and Tris extenders were modified by adding α-linoleic acid (ALA) or linoleic acid albumin (LAA). Non-modified Bioxcell extender was used as control. Semen diluted in modified, glycerol-free Tris extender was subjected to a longer equilibration (26-28 hours at +4°C). Thirty-one ejaculates collected from 12 bulls aged 12-17 months were used in this research. After collection, fresh semen was divided into 7 portions, and each portion was diluted in one of experimental diluents, as well as in the control diluent. After equilibration, semen was frozen in a freezer. Subsequently, the semen was thawed and its motility was assessed (approximately and by CSA) along with cell membrane integrity (SYBR-14 and PI). The highest motility was observed in Bioxcell diluent supplemented with α-linoleic acid (ALA), and it was significantly higher (p ≤ 0.5) than in Bioxcell diluent (control). No significant differences in cell membrane integrity were observed between experimental groups that were subjected to a 2.5 hour equilibration. The extension of the equilibration time to 26-28 hours caused a significant (p ≤ 0.01) decrease in both the motility and the cell membrane integrity of spermatozoa compared to semen frozen in Bioxcell diluent (control) as well as to all other experimental diluents. In the case of semen that did not meet qualitative standards (motility below 50%) when frozen in the control diluent, significantly better results (p ≤ 0.01 and p ≤ 0.05) in terms of motility (assessed approximately and by SCA) were observed when the semen was diluted in Bioxcell diluent supplemented with α-linoleic acid (ALA) as well as in Bioxcell diluent supplemented with linoleic acid albumin (LAA) (p ≤ 0.05). No significant differences in cell membrane integrity were observed. The extension of equilibration time caused a significant (p ≤ 0.01) decrease in both the motility and the cell membrane integrity of spermatozoa. The study confirmed the possibility of improving cryopreservation of young bulls’ semen by modification of freezing diluents.

The objective of the study was to determine the effect of high hydrostatic pressure (HHP) on quality of cryopreserved semen of young bulls. Semen for this study was collected from 8 bulls aged between 13 and 18 months at monthly intervals, from June to September. After collection, semen was diluted in a commercial Bioxcell® extender (one part at 1:1 and a second part to give a sperm concentration of 20 million/0.2 mL), filled into straws and treated with HHP at 30 MPa for 90 min. After HHP treatment, pre-diluted semen (1:1) was diluted to a sperm concentration 20 million/0.2 mL and filled into straws. In addition, part of the semen diluted to a concentration of 20 million/0.2 mL was not treated with HHP (control). All of it was held at +4°C and frozen in a freezer after 2.5-h equilibration. Semen was thawed in a water bath at 38°C and subjected to estimation of the percentage of motile sperm both subjectively and using a computer-assisted semen analyzer and cytometric assessment of sperm cell membrane integrity. Subjective motility and fast progressive motility were significantly higher with pre-diluted (1:1) and HHP treated semen compared to control (P<0.05). No significant differences were observed in percentage of membrane-intact spermatozoa between control and experimental groups. Additionally, the influence of HHP on the sperm of individual bulls was assessed. In bull number 2, the HHP treatment after semen pre-dilution significantly improved progressive motility from 54.1 to 63.4 percent (P <0.05). In bull number 4, the HHP treatment after semen pre-dilution significantly improved subjective motility, rapid motility and progressive motility by 12.5, 16.8 and 16.3 percent, respectively (P<0.05). No effect was seen for 6 bulls. It is concluded that for some bulls, the application of HHP before semen freezing may improve the cryopreservation outcome. However, this requires further research in this area, also to determine the fertilizing capacity of bull semen exposed to high hydrostatic pressure.