The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C) or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively). On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.
The aim of this study was to determine the minimum effective intrafollicular doses of luteinizing hormone and human chorionic gonadotropin in order to induce ovulation in cycling dairy heifers that have not yet been adequately established. Application of 10, 5, 1, 0.5, 0.1, 0.01 and 0.001 µg luteinizing hormone as well as 10, 1, 0.1, 0.01 and 0.001 international units (IU) of human chorionic gonadotropin in dominant follicles was performed on day 7 of the oestrous cycle. Control animals were given luteinizing hormone (12.5 mg and 25 mg) or human chorionic gonadotropin (2000 IU) intravenously. Accessory corpus luteum on day 14 of the oestrous cycle was considered as an evidence of ovulation. Ovulation was observed in 2 out of 3 heifers in each treatment group (n = 3) after administration of 10-0.1 µg luteinizing hormone (except for 0.5 µg -ovulation in 3 of 3 heifers), in all heifers after administration of 10-0.01 IU human chorionic gonadotropin as well as in all control heifers. Administration of 0.01 µg and 0.001 µg luteinizing hormone as well as of 0.001 IU human chorionic gonadotropin did not result in ovulation. Higher progesterone concentration on day 14 vs. day 7 of the oestrous cycle was found after all treatments. Nevertheless, the differences were significant (P < 0.05) only after intrafollicular treatments with 5, 1 and 0.001 µg luteinizing hormone as well as 10, 1 and 0.01 IU human chorionic gonadotropin. In conclusion, minimum efficient doses for intrafollicular treatment of the dominant follicles in cycling heifers capable of inducing ovulation were 0.1 µg of luteinizing hormone and 0.01 IU of human chorionic gonadotropin. This is the first study describing the intrafollicular luteinizing hormone administration in cycling dairy heifers. Intraovarian injection, ovum pick-up, ultrasound-guided transvaginal ovarian puncture, accessory corpus luteumUltrasound-guided transvaginal intrafollicular injection of human chorionic gonadotropin (hCG) in cattle was first reported as a useful research tool by Kot et al. in 1995. Since then, intrafollicular injection of different substances such as phosphate-buffered saline or insulin-like growth factor-I (Bergfelt et al. 1998;Ginther et al. 2004;Shahiduzzaman et al. 2010) as well as intrafollicular insemination (Lopez-Gatius and Hunter 2011) have been described. In our former study, intrafollicular treatment (IFT) with luteinizing hormone (LH) injected in the dominant follicle in heifers previously treated by deslorelin implants was reported. Ovulation was proven after the intrafollicular treatment using various doses of LH . However, minimum effective doses of LH for IFT in comparison with IFT using hCG have not yet been adequately established.The aim of this study was to determine the minimum efficient doses of LH and hCG for intrafollicular treatment to induce ovulation in cycling dairy heifers. Materials and Methods Intrafollicular treatment equipmentIntrafollicular treatment was performed using a newly developed double channel instrument enabling asp...
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