Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
Poster Abstracts 175<br />
P447<br />
The effect of plant lipid exten<strong>de</strong>r component on quality of<br />
frozen ram semen<br />
Palasz, A 1 , Bochenek, M 2 *, Gogol, P 2 , Kareta, W 2 , Smorag, Z 2<br />
1INIA, Dpto Reproduccion Animal, Crta. Coruña, km 5, 9-28040 Madrid,<br />
Spa<strong>in</strong>; 2 Dept. Biotechnology of Animal <strong>Reproduction</strong>, NRIAP, 32-083 Balice,<br />
Poland<br />
Experiment was <strong>de</strong>signed to test different concentrations and different<br />
homogenization protocols of soybean lipids liposomes for the use as a<br />
milk replacement <strong>in</strong> ram semen freez<strong>in</strong>g exten<strong>de</strong>r. Lipids were<br />
homogenized by high pressure homogenization (800 bars) and then<br />
5% (Y1 exten<strong>de</strong>r) or 10% (Y2 exten<strong>de</strong>r) liposomes were mixed with<br />
8% glycerol. As additional group, lipids and glycerol were<br />
homogenized together (LIPO exten<strong>de</strong>r). All 3 exten<strong>de</strong>rs (Y1, Y2,<br />
LIPO) were prepared <strong>in</strong> Tris buffer conta<strong>in</strong><strong>in</strong>g citric acid and fructose.<br />
As control group milk/egg yolk (MY) exten<strong>de</strong>r was used. The semen<br />
was collected from 3 rams with artificial vag<strong>in</strong>a. Post thaw motility<br />
and survival time (total of 11 ejaculates), lipid peroxidation (total of 7<br />
ejaculates) and sperm membrane <strong>in</strong>tegrity (total of 3 ejaculates) were<br />
exam<strong>in</strong>ed <strong>in</strong> 4 exten<strong>de</strong>rs tested. Immediately after collection each<br />
ejaculate was divi<strong>de</strong>d <strong>in</strong>to 4 parts, diluted with Y1, Y2, LIPO and MY<br />
exten<strong>de</strong>rs and processed accord<strong>in</strong>g schedule. Post thaw motility and<br />
survival time: Mean sperm motility exam<strong>in</strong>ed immediately after<br />
thaw<strong>in</strong>g for Y1, Y2, LIPO and MY exten<strong>de</strong>rs was 51.4%, 46, 8%,<br />
50.6% and 44.4% respectively. Mean survival time of spermatozoa<br />
kept at 42°C was 200, 220, 255 and 135 m<strong>in</strong>utes for Y1, Y2, LIPO<br />
and MY exten<strong>de</strong>rs respectively. Lipid peroxidation was monitored by<br />
chemilum<strong>in</strong>escence method. Iron <strong>in</strong>duced lum<strong>in</strong>escence of<br />
frozen/thawed sperm cells was assessed us<strong>in</strong>g a lum<strong>in</strong>ometer. It was<br />
shown that Y1, Y2 and LIPO semen exten<strong>de</strong>rs had significantly lower<br />
peroxidation level than MY exten<strong>de</strong>r. The values of Integral<br />
parameter for Y1, Y2, LIPO, MY exten<strong>de</strong>rs was 4,57; 5,49; 4,31;<br />
28,70 respectively. Sperm membrane <strong>in</strong>tegrity: After thaw<strong>in</strong>g semen<br />
sample was diluted with PBS to 20mln/ml concentration and kept for<br />
1h at room temperature. Sperm membrane exam<strong>in</strong>ation (“live/<strong>de</strong>ad”)<br />
was performed by double sta<strong>in</strong><strong>in</strong>g with SYBR-14/propidium iodi<strong>de</strong><br />
fluorochromes and analysis by flow cytometry. Data of 20 000<br />
spermatozoa were collected for each sample. The percentage of<br />
membrane <strong>in</strong>tact (“live”) spermatozoa was taken for statistical<br />
analysis. The mean percentage of live spermatozoa for Y1, Y2, lipo<br />
and milk exten<strong>de</strong>rs were 10.79%, 10.61%, 10.82% and 4.71%<br />
respectively. Statistically significant differences was found between<br />
milk and Y1, Y2, LIPO (test t, P