engleski

Simmental sperm separation protocols, evaluation for IVF

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 A.I.. The recent adoption of in vitro technologies with aim to improve our selection possibilities put a new chalenge: test our sires for the in vitro fertilization. In the first phase of this program we tested three protocols for sperm separation and clean-up for in vitro fertilization. The sperm is routinelly frozen in the Center for Reproduction and Animals Breeding of Croatia in a TRIS-Citrate-Egg yolk-Glycerol diluent, in 0.5 ml straws. Only straws with more than 50% post-thaw motility were stored for A.I. For this evaluation one sire was selected. The non return rate of this bull is 66.6 % over 2, 910 inseminations. Four straws were thawed for each of five repetitions and the motility, concentration, membrane integrity and acrosome status were evaluated before and after sperm separation. Straws were thawed at 39°C for 1 minute, and the concentration was determined in a Thoma chamber. Motility was evaluated subjectively by a trained evaluator 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 and the total number of spermatozoa with intact acrosome were counted under 400X magnification . The sperm separation and clean up for IVF were performed with three different protocols. a) Dilution and Washing of the thawed sperm in Tyrode medium supplemented with Pyruvate and BSA and without Calcium (Medium without Calcium: M-Ca), two washings with 10 and 5 ml of medium respectively, centrifugated at 200 x g for 5 min, the pellet was resuspended in medium for fertilization (mIVF: Tyrode supplemented with BSA, Penicillamine, Epinefrine, Heparine, Pyruvate and Caffeine). b) Swim up protocol: in M-Ca (1 ml, one hour at 39°C), the upper fraction of 1 ml was collected and washed twice with 3 ml of M-Ca and resuspended with mIVF. c) Discontinuous Percoll gradient centrifugation (45-90 % Percoll in M-Ca), centrifugated at 700 x g for 15 min, washed in M-Ca, and resuspended in mIVF. The final concentration of the sperm suspension was adjusted to 1x106 spermatozoa/ml in the mIVF with oocytes. An average of 40 in vitro matured oocytes were used for each of five repetitions. The oocytes were fertilized with the separated frozen/thawed bull semen. Cleaved embryos were cultured in vitro in SOFaaBSA medium till the Day 10. On the 2nd day after IVF we registered the number of cleaved embryos, the total number of morulas (M) and blastocysts (Bl) on day 7 and finally the number of hatched blastocysts (hBl) on day 10. The statistical comparison between protocols was done with ANOVA (GraphPad, Instat, V2.00) using the arcsin transformation of the percent values. The initial parameters of the thawed sperm were: 105 x 106 (&plusmn ; ; ; 27) spermatozoa/ml and 70 % (&plusmn ; ; ; 7.1) progressive motility, 51 % (&plusmn ; ; ; 19.8) membrane integrity and 12.5 % (&plusmn ; ; ; 2.12) of spermatozoa with intact acrosome. Table 1. Comparison of three sperm separation protocols: final sperm quantity and quality and IVP results. SeparationProtocol Motility%(&plusmn ; ; ; S.D.) Concentration x106 sp./ml (&plusmn ; ; ; S.D.) Membrane Integrity% (&plusmn ; ; ; S.D.) IntactAcrosome% (&plusmn ; ; ; S.D.) CleavageDay 2% (&plusmn ; ; ; S.D.) M + Bl Day 7% (&plusmn ; ; ; S.D.) hBl Day 10% (&plusmn ; ; ; S.D.) Washing 62 (7.6) 35.4 (10.4)a 60.5 (0.7) 48 (2.8) 76.6 (12.2) 22.4 (8.2) 11.8 (6.5) Swim up 65 (10.0) 11.0 (2.7)b 55 (4.2) 14 (5.7) 73.6 (16.6) 24.8 (17.9) 11.7 (10.4) Percoll 69 (7.4) 70.0 (16.5)c 55.5 (12) 22.5 (14.9) 67.5 (13.8) 20.9 (8.8) 10.8 (7.6) Values in the same column with different superscripts differ significantly (P<0.05). Although the studied parameters shown differences in the final concentrations and slight differences in the percent of intact acrosome recovered after sperm separation protocols, the sperm used for in vitro fertilizations shown the same results in terms of Cleavage, percent of Morulas and Blastocysts on Day 7 and hatched Blastocysts on Day 10 for all three protocols. The final concentration and quality parameters of the separated sperm not seem to be related with the results of the IVP, at least in the range obtained in our experiments. So, we concluded that all three protocols gave suitable sperm for IVF and can be used for in vitro production under our conditions.

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