engleski

Comparison of some simmental sires for IVF:preliminary results

Simmental purebreed cattle is grown for meat and milk purpose in the farms of Croatia. Eighty percent of the herd is under selection programes and subjected to AI. The recent adoption of in vitro technologies with the aim to improve our selection possibilities put a new chalenge: test our sires for the in vitro fertilization. In this phase of the program we screened nine sires of proved field fertility where we studied the thawed sperm and the selected population of spermatozoides for in vitro fertilization and the results of the IVF. The sperm is routinelly frozen in a TRIS-Citrate-Egg yolk-Glycerol diluent, in 0.5 ml straws. The non return rates of those bulls ranges form 58 to 68 %. Four straws of the same day of production were thawed, at 39°C for 1 minute, for each of three repetitions per bull, each day were tested 4 bulls. Concentration was determined in a Thoma chamber. Motility was evaluated subjectively under phase-contrast microscop. The sperm membrane integrity were evaluated using the HOS Test. The status of the acrosome were evaluated using the Trypan blue-Giemsa stain under magnification x 400. Evaluations were made before and after sperm separation. The sperm separation and clean up for IVF were performed trough a discontinuous Percoll gradient centrifugation (45-90 % Percoll in TALP withouth Ca). The IVF was made in medium with 10 mg/mL Heparine. The final concentration of the sperm suspension was adjusted to 1x106 spermatozoa/ml in the mIVF with oocytes. Oocytes were recovered from abatoir derived Simmental ovaries. A total number of 1244 oocytes were fertilized. Cleaved embryos were cultured in vitro in SOFaaBSA medium untill the Day 9. On the Day 2 we registered the number of cleaved embryos, on Day 7 the total number of morulas (M) and blastocysts (Bl) and finally the number of hatched blastocysts (hBl) on Day 9. The statistical comparison between sires was done with ANOVA using the arcsin transformation of the percent values. The average parameters before and after Percoll separation show some differences: concentration of 67 vs 51x 106 (SD: 21 vs 18) spermatozoa/ml (P<0, 05), progressive motility of 63 vs 68 % (SD: 8 vs 8) NS, membrane integrity of 40 vs 53 % (SD: 9 vs 9) (P<0, 05), and intact acrosomes in 56 vs 63 % (SD: 18 vs 10) NS. The sperm was considered good enough for IVF for all the sires. The results of the IVF were quite variable between bulls and also within each sire. The average values were: 66 % (SD:18) cleaved on Day 2, 21 % (SD: 11) of M+Bl on Day 7 and 9 % (SD: 7) hBl on Day 9 of development. There are diferences between sires but high variabilities into the results of some sires difficult the statistical analysis. Seven sires shown high variability between repetitions: this is clearly visible in the percent of hatched Bl on Day 9: 4 % (SD: 8), 9 % (SD: 8), 5 % (SD: 3), 6 % (SD: 7), 15 % (SD: 10), 10 % (SD: 12), 11 % (SD: 8) while other bulls are more consistent 7 % (+/-2), 15 % (+/-3). We conclude that there are high variation between sires but also into the sires. Individual analysis of each bull should be done.

cattle; IVF; sperm evaluation

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