Effects of ovum pick-up frequency and FSH stimulation: a retrospective study on seven years of beef cattle in vitro embryo production.

The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non-stimulated Ovum Pick-up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH-LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day -8). The dominant follicle was ablated by ultrasound-guided follicle puncture on day -6. On day -3 and day -2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm), i.e. a total dose of 160 mug FSH and 40 mug LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14-day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set-up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2-week periods are considered (four non-stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non-stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better-quality, oocytes.

Characterisation of low, medium and high responders following FSH stimulation prior to ultrasound-guided transvaginal oocyte retrieval in cows.

In human IVF, the concept of 'low responders' is well known and generally defined as women with poor-response to gonadotrophin stimulation in a previous induction cycle. The objective of this retrospective study is to describe and characterise the concepts of 'low-, medium-, and high-response' and 'low, medium, and high responders' in bovine-assisted reproduction by analysing the OPU-IVF results obtained following 665 gonadotrophin-stimulated sessions conducted in 112 animals, nearly all of which were previously unsuccessful in traditional multiple ovulation and embryo transfer (MOET) programs. They were submitted to OPU and IVP between 1999 and 2003. In reference to these 665 OPU sessions, categories of response were defined based on the overall mean+/-S.D. follicles aspirated and COC obtained i.e., for follicles 14.7+/-9.8 and for COCs 11.7+/-8.1. So arbitrary cut-off values to define the categories of sessions were for follicles 5 and 25, and for COC 4 and 20. The three categories for follicles punctured in one session were therefore follicle low-response (FLR)or=25 follicles and for COCs recovered in one session COC low-response (CLR)or=20 COC. In addition, four categories of animals were also defined: (1) a low responder animal (LRA) had at least one OPU session in which FLR and CLR were observed (genuine low-response, see Section ); these animals did not have any high-response sessions, (2) a medium responder animal (MeRA) had only medium-responses, (3) a high responder animal (HRA) had at least one OPU session in which FHR and CHR were observed; these animals did not have any low-response sessions, and (4) mixed responder animals (MiRA) had both low and high-responses. Finally, we distinguished biological (animals) and technical (recovery rate and ultrasound resolution) causes of response differences. In 'low, high, medium and mixed reponders,' different results were obtained (p<0.05): mean follicle numbers (8.8+/-4.8a, 22.4+/-10.5c, 13.2+/-5.2b,15.1+/-10.2d), COC numbers (6.3+/-3.9a, 18.5+/-8.2c, 10.4+/-4b, 12.0+/-8.3d), embryo numbers (1.8+/-2.1a, 5.6+/-4.9c, 2.5+/-2.7b, 3.5+/-3.8d) and also for recovery rate (72%a, 83%b,79%, 79%) and percentage embryo development (29%, 30%a, 24%b, 29%). In conclusion, the results of this study demonstrate that variability in OPU results has technical (ultrasound resolution and recovery rate) as well as biological (animal) aspects. Selection of animals with extreme (high or low) follicle and COC production results allows us to distinguish three populations: 'low, medium, and high responders' to FSH stimulation.

Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol.

The aim of this experiment was to apply an ovarian superstimulation protocol prior to ovum pick up (OPU), tailored to the individual donor response, to evaluate its advantages and disadvantages in terms of follicle numbers and diameters, the numbers of retrieved oocytes and day 7 cultured blastocysts. Ten adult non-lactating dairy cows were superstimulated with pFSH and subjected to ovum pick up-in vitro fertilisation (OPU-IVF) 6 times at 2-week intervals. On day 0 of each 2-week period, all follicles >8mm were ablated and an ear implant (Crestar, Intervet, Belgium) was inserted. On day 2, 48 h after follicle ablation the animals were administered six equal doses of pFSH, divided into morning and evening doses for 3 days. On day 7, 48 h following the last pFSH injection, follicle diameters were measured by ultrasound and all follicles were subjected to OPU. All cumulus-oocyte complexes (COC), regardless of their quality, were subjected to in vitro maturation-in vitro fertilisation-in vitro culture (IVM-IVF-IVC). The total dose of pFSH prior to the first OPU session was 300 microg per animal. During the following OPU sessions, the total pFSH dose was either kept unchanged, increased or reduced (+/-50 microg), according to the percentage of follicles of more than 11 mm in diameter, present in the previous session of that particular donor. The mean number of punctured follicles per session was 11.9 +/- 7.7 (mean +/- S.D.), with 16% of follicles exceeding 11 mm. These follicles yielded a mean of 5.6 +/- 4.1 cumulus oocyte complexes (COC), 32% of which had >/=3 layers of cumulus cells (quality 1 and 2). The recovery rate was 47%. Finally, all COC were subjected to IVM-IVF-IVC, which resulted in a mean of 2.0 +/- 2.3 blastocysts on day 7 postinsemination. The subtle changes in pFSH dose influenced the sizes but not the numbers of follicles, the latter parameter was influenced by the individual donor and the OPU session.

Increase of plasma eCG binding rate after administration of repeated high dose of eCG to cows.

Equine chorionic gonadotrophin (eCG) is still used to promote follicular growth in cattle and, more recently with an increased frequency of administration, in ovum pick-up protocols. The aim of this experiment was to verify the possible effect of high frequency of administration on the immune response to eCG. The profiles of eCG binding rate, in the blood of two groups (A, B) of 4 primiparous cross breed beef cows (3-3.5 years old) submitted weekly for 5 to 10 weeks to repeated high doses (1000-2000 IU) of equine chorionic gonadotrophin, are presented in this paper. A sensitive radiometric method was used to detect antibodies in plasma. The profiles clearly indicated a marked increase of eCG binding rate after 3 to 5 injections of the exogenous hormone to the females. The statistical analysis of the results established that treatments induced a significant increase (P < 0.01) in binding rates after 6 and 3 injections in group A and B respectively. These binding rates remained elevated for at least 1 week following the last injection and decreased afterwards. The values of plasma binding rates following repeated eCG administration differed significantly between groups (0.90+/-1.04 and 1.04+/-0.11 for groups A and B before treatment versus 11.77+/-0.92, 6.70+/-0.85 for groups A and B after treatment, P < 0.01) and from one cow to another (P < 0.01) with some cows presenting no significant immune response while others were more reactive against the hormone (at least 3 injections).

Development of vitrified matured cattle oocytes after thawing and culture in vitro.

Bovine oocytes were partly denuded either at the beginning (t0) or six hours (t6) after the beginning of maturation and vitrified by the open pulled straw method at the end of the maturation process. After warming and fertilisation, their development in vitro and in vivo was assessed. The rates of production of blastocysts achieved in vitro were 3.4 per cent for the t0 group and 0.9 per cent for the t6 group compared with 40.4 per cent for the control oocytes. After transfer at the blastocyst stage pregnancies have been established in the three groups. Some of these pregnancies originated from vitrified oocytes which were further vitrified at the blastocyst stage before being transferred into synchronised recipients.

Embryo production by ovum pick up in unstimulated calves before and after puberty.

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.

Nuclear transplantation using bovine primordial germ cells from male fetuses.

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.