Transcriptomic analysis of in vivo and in vitro produced bovine embryos revealed a developmental change in cullin 1 expression during maternal-to-embryonic transition.

Pre-implantation embryos derived by in vitro fertilization differ in their developmental potential from embryos obtained in vivo. In order to characterize changes in gene expression profiles caused by in vitro culture environment, we employed microarray constructed from bovine oocyte and preimplantation embryo-specific cDNAs (BlueChip, Université Laval, Québec). The analysis revealed changes in the level of 134 transcripts between in vitro derived (cultured in COOK BVC/BVB media) and in vivo derived 4-cell stage embryos and 97 transcripts were differentially expressed between 8-cell stage in vitro and in vivo embryos. The expression profiles of 7 selected transcripts (BUB3, CUL1, FBL, NOLC1, PCAF, GABPA and CNOT4) were studied in detail. We have identified a switch from Cullin 1-like transcript variant 1 to Cullin 1 transcript variant 3 (UniGene IDs BT.36789 and BT.6490, respectively) expressions around the time of bovine major gene activation (8-cell stage). New fibrillarin protein was detected by immunofluorescence already in early 8-cell stage and this detection correlated with increased level of fibrillarin mRNA. The qRT-PCR analysis revealed significant differences in the level of BUB3, NOLC1, PCAF, GABPA and CNOT4 gene transcripts between in vivo derived (IVD) and in vitro produced (IVP) embryos in late 8-cell stage. The combination of these genes represents a suitable tool for addressing questions concerning normal IVD embryo development and can be potentially useful as a marker of embryo quality in future attempts to optimize in vitro culture conditions.

Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows.

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

[Intracytoplasmic sperm injection--a possible therapy for male subfertility]. / Intracytoplazmatická injekce spermie (ICSI)--mozná lécba muzské subfertility.

The authors describe a new method of assisted fertilization, i.e. intercytoplasmic injection of spermatozoa. They submit an account of their technical equipment and the initial experience with this method on the degenerated human oocyte as well as on the blastocyst of cattle. Before this method can be introduced into clinical practice, it must be approved by the ethical commission.

[Descent of ova and embryos in cows superovulated with serum gonadotropin]. / Sestup vajícek a embryí u krav superovulovaných sérovým gonadotropinem.

The descent and localization of eggs and embryos in individual segments of the reproductive tract of superovulated cows were studied in this work. For the induction of superovulation, serum gonadotropin (PMSG, Ivanovice in Haná) at a dose of 2,500-3,500 I.U. was used, in combination with 0.5 mg of Cloprostenol (Oestrophan, Spofa), administered 48 hours after gonadotropin treatment. The start of superovulation fell on days 9 to 12 of the sexual cycle and was conditioned by the presence of the corpus luteum (CL). After the onset of the heat, 2-3 inseminations were carried out using fresh semen. Donor cows were slaughtered 3, 4, 5, 6, and 7 days after the second insemination and isolated reproductive organs (Fig. 1) were divided into five segments (two on oviducts and three on uterine horns) by the applied ligature. In laboratory conditions superovulation response was determined accurately, the volume of ovaries was assessed according to water displacement and the segments of oviducts and uterus were rinsed with TCM 199 or PBS supplemented with FCS. 3, 4, 5, 6, and 7 days after insemination (Tab. I). 18.1 (+/- 3.55), 12.4 (+/- 0.91), 19.2 (+/- 2.86), 20 and 23 (+/- 2.44) CL on average were recorded, which corresponded to the ovulation of 64, 50, 56, 71 and 72 percent of stimulated follicles (Fig. 2). Within 3 to 7 days after insemination nearly triple enlargement of ovaries was also observed (Tab. I, Fig. 3). During the lavage of individual segments of the tubular reproductive tract, 38 per cent of eggs and embryos were detected in the uterus as early as 3 days after insemination (Tab. II). Unfertilized eggs and degenerated embryos were found in the 2nd and 3rd uterine segment, embryos at the stage of 8-16 blastomeres were localized in the 1st and 2nd segment of the uterus. Four days after insemination (Tab. III), about 64 per cent of eggs and embryos at the stage up to 16 blastomeres were found in the uterus, but embryos up to 32 blastomeres were still flushed out of the oviduct. On day 5 after insemination, 92 per cent of eggs and embryos were released into the uterus, being localized mostly in the cranial and medial part of the uterus (Tab. IV). 7.5 per cent of recovered eggs and embryos at the stage of early or compacted morulae were still detected in the oviducts.(ABSTRACT TRUNCATED AT 400 WORDS)

[The embryo yield in relation to the method of processing the irrigation fluid in superovulated donor cows]. / Výteznost embryí ve vztahu ke zpracování výplasku superovulovaných krav-dárcu.

Total 240 flushings of superovulated donor cows were examined after double sedimentation. Altogether 2,810 ova and embryos, i.e. average of 11.7 per donor, including 9.0 (76.97%) transferable embryos, were obtained after the first sedimentation. The sedimentation was carried out separately for the respective uterine horns. The second sedimentation, which was carried out in the flushing from both uterine horns simultaneously, yielded ova and embryos in 82.9% (199/240) of the cases. Total 679 ova and embryos were found, i.e. 3.41 per donor, including 2.61 (76.58%) transferable embryos per donor. The increase of the yield of total ova and embryos, and transferable embryos, was 2.82 and 2.16 (24:16 and 24.04%) respectively after the second sedimentation. Altogether 3.489 total ova and embryos were obtained from 240 donors. The average embryo yield was 14.53 per donor with 76.89% (11.17) transferable embryos. The embryo yield/corpora lutea ratio was at the number of corpora lutea 12.04 on the average 120.68%. It is concluded that the second sedimentation of the flushing provides for the release of a part of the embryos for the mucous and cell aggregations, which density is usually lower. The effect of the second sedimentation is considerable, because as much as 24% of the total number of transferable embryos can be saved. The total efficiency of superovulation may be considerably decreased if no attention is paid to the above mentioned facts.

[Use of synchronized heifer-recipients in view of the effectiveness of embryo transfer in cattle]. / Vyuzitelnost synchronizovaných jalovic-príjemkyn ve vztahu k efektivite transferu embryí u skotu.

A possibility was investigated of using heifers prepared for embryo reception by i. m. double application of Oestrophan (Spofa) luteolytic preparation. After a clinical examination of ovaries and corpus luteum, out of the total number of 2894 animals synchronized for nonsurgical ipsilateral transfer in the D7 stage only 2038 (70.4%) recipients were used. Almost 30% (856) heifers were discarded from the role of recipients, mostly due to the functional disorders of the cyclic activity of ovaries manifesting themselves as luteal cysts (22.5%), cysts of corpus luteum (16.5%), small or young corpora lutea (12.7%), postovulation states (4.5%), and also as a high frequency of the follicular activity without the presence of corpus luteum (37.9%). The occurrence of follicular cysts (1.4%) and nonfunctional ovaries (2.8%) was considerably lower. The high number of discarded recipients which results, in the complex of causes, from qualitative nutritional disorders, husbandry and zoo-hygienic shortcomings and stress-inducing factors in the course of recipient selection and preparation, diminishes the transfer efficiency and considerably increases the claims for the numbers of recipient prepared for ovulation. In the conditions of our workplace where 10.6 to 12.8 transferable embryos are recorded from one successful superovulation and their survival is 65 to 68.6%, it will be necessary, at the present level of recipient discarding, to prepare 13.8 to 16.6 heifers per donor to make full use of the superovulation effect.

[Initial experience with cryopreservation of human embryos]. / První zkusenosti s kryokonzervací lidských embryí.

We present our first experience with human embryo cryopreservation. In our hands the best results were obtained with cryopreservation of pronuclear-staged embryos using propanediol as cryoprotectant. In this group 3 embryos out of 4 continued their development after thawing. The advantages of embryo cryopreservation in IVF programs are discussed.

[Increasing the superovulatory effect in heifers by trans-zonal bisection of embryos in practice]. / Moznost zvysování superovulacního efektu u skotu transzonálním delením embryí v praxi.

Early and late blastocysts (D7) of excellent quality were bisected by the transzonal method. The developing semiembryos without zonal protection were transferred to synchronised recipients--heifers. After bilateral transfer of the identical pair (always one semiembryo into one uterine cornu) 79.7% of the heifers were impregnated. The total gravidity was 58.8%. After ipsilateral transfer (the halves transferred individually) gravidity was confirmed in 48.2% of the cases. The survival efficiency of the halves with respect to the number of bisected embryos was 117.6% in bilateral transfer and it decreased to 93.3% after ipsilateral transfer. Identical pregnancies of twins were recorded in 36.8% of the bilateral transfers and in ipsilateral transfers only 20% of the identical pairs survived. We demonstrated the high recuperative and regenerative ability of the bovine 7 day old blastocyst by means of transzonal bisection. This was dependent on the selection of the appropriate embryo, careful manipulation aseptic technique, biologically compatible cultivation medium and rapid transfer. It is possible to attain satisfactory semiembryo survival levels in transplantation work and by means of this a significant increase of the superovulation effect.

[Use of a rapid method of cryopreservation of bovine embryos for non-surgical transfer in transplantation practice]. / Vyuzití rychlého zpusobu kryokonzervace embryí skotu pri nekravavém prenosu v transplantacní praxi.

The embryos were frozen and thawed in Cassou minipaillette by a rapid method. Embryos with cryoprotective agent (glycerol, 1.5 M) were placed directly into the freezing medium at the temperature of -6 to -7 degrees C, frozen after seedling at the temperature decrease by 0.3 to 0.5 degrees C per minute to the temperature of -32 degrees C and then transferred directly into liquid nitrogen. They were thawed in a bath warm 20 to 37 degrees C. After thawed the cryoprotective agent was evacuated in 1.1 M sucrose. The best-quality embryos were selected for freezing. Out of these 366 thawed so far, with average survival of 74.31%. The total of the 268 thawed embryos were transferred ipsilaterally, by a non-surgical method, to 190 synchronised heifers, out of which 105 (55.26%) got in calf. Rapid freezing method based on 1.5 M of glycerol and thawing at the presence of 1.1 M sucrose proved effective and suitable for practice, as not only sufficient reviviscence of embryos and their survival in womb are guaranteed, but also a substantial shortening of the freezing as well as thawing process.

[Bloodless transfer of early bovine embryos in relation to the biological properties of the materials used]. / Nekrvavý prenos raných embryí u skotu ve vztahu k biologickým vlastnostem pouzívaných materiálu.

Attention is drawn to the need of the cytotoxic testing of instruments used in the process of the transfer of early embryos. It was demonstrated by spermiotoxic test that some instruments were very toxic to bull sperm and may, analogically, threaten the life of embryos. The new catheters Foley Barum, produced by the Optimit Corporation and developed in recent time, are highly compatible with bull sperm: a slight reduction of sperm motility (by 5.7%) occurred only after 90 minutes of incubation. Teflon embryonation capillaries had a similar behaviour. The introduction of biologically compatible materials in the process of embryo transfer contributed to a substantial improvement of embryo survival: 63.01% of animals (46) got in calf after 68 bloodless transfers.