Summary. Blastocysts, obtained from cows on Day 10–11 after oestrus, were cultured for 20 h. Most (81·3%) blastocysts grew in culture and about 50% took up glucose. There was no morphological difference between the blastocysts which did or did not take up glucose but development in vivo was better for blastocysts which had taken up glucose (69·2%) than for those which did not (14·2%).
J.-P. Renard, A. Philippon and Y. Menezo
J.-E. Fléchon and J.-P. Renard
Summary. The blastocysts were recovered from cows 7–10 days after oestrus and cultured. The zona pellucida has a spongy fibrous structure. Hatching begins, about 24 h after culture, through a relatively small hole out of which the blastocyst appears to escape by its own activity. Later the zona becomes split and the two edges of the slit surround part of the blastocyst. The emerging cells were large or small blastomeres which were generally covered by a dense mass of microvilli.
J.-P. Renard, Bui-Xuan-Nguyen and V. Garnier
Summary. The effect of rapid freezing and thawing on the survival of 2-cell rabbit embryos was examined. When embryos in 2·2 m-propanediol were directly plunged from room temperature to liquid nitrogen some of them survived after thawing (8%) but only if they had been pretreated by exposure to an impermeable solute, sucrose, that makes the blastomeres shrink osmotically before cooling. High survival (77–88%) in vitro was obtained when pretreated embryos were first held at – 30°C for 30–240 min before immersion into liquid nitrogen. Transfer of such frozen—thawed embryos gave a survival rate to live young similar to that obtained with controls (26% and 32% respectively). DMSO was less effective than propanediol; only 2 out of 38 sucrose-pretreated frozen—thawed embryos developed in vitro.
The present work shows that a combination of partial dehydration of blastomeres at room temperature with their permeation by a cryoprotective agent offers a simple method for successful rapid freezing and thawing of rabbit embryos.
M Saint-Dizier, JP Renard and S Chastant-Maillard
In contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation. Moreover, the bitch is receptive to mating while immature oocytes are present in the oviducts. The aims of this study were to examine the influence of fertilization in immature oocytes on the resumption of meiosis, and the modification of both male and female chromatin in fertilized oocytes. Canine cumulus-oocyte complexes collected from routine ovariectomies were cultured in medium 199 with 20% fetal calf serum for 24 h, incubated in the same medium with fresh semen for 24 h, washed, cultured for a further 24 h and fixed. Control oocytes were cultured in the same medium but without spermatozoa for 24, 48 or 72 h. After fixation, chromatin was stained with propidium iodide and examined using laser scanning confocal microscopy. The data indicate that sperm penetration can occur in immature canine oocytes and that it induces resumption of meiosis. After 72 h of culture, the percentage of oocytes at the germinal vesicle stage was significantly lower in fertilized oocytes (40% versus 60.3% for control oocytes; P < 0.05) and the percentage of oocytes beyond metaphase I was significantly greater in fertilized oocytes (28.3% metaphase I and II, and two pronuclei versus 10.2% metaphase I and II for control oocytes; P < 0.01). Observation and measurement of the area of chromatin in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage.
M Tamassia, Y Heyman, Y Lavergne, C Richard, V Gelin, JP Renard and S Chastant-Maillard
There have been few studies on a possible maternal influence on in vitro embryo production in cows. The objective of this study was to evaluate the maternal influence on oocyte production and in vitro blastocyst formation rate using repeated ovum pick-up and in vitro fertilization. Six contemporary cows raised on the same farm and with varied genetic origins were submitted to 42 weeks of ovum pick-up organized into four series. Collected oocytes were fertilized in vitro with spermatozoa from a different bull for each series. In total, 1933 oocytes were recovered from 3936 follicles with a recovery rate of 57.2% and a mean oocyte collection of 4.6+/-0.2 (mean+/-SEM) per animal per session. Animals were ranked according to their oocyte production. The best oocyte donor was the same female in all four series. No relationship was identified between oocyte production and blastocyst production rate (r=-0.08). The mean blastocyst rate was 28.8% with significant variation among animals. The best and the worst blastocyst producers were always the same animals independent of the semen used. The results of the present study support the hypothesis that in cattle, the oocyte donor influences the production of blastocysts. Furthermore, they demonstrate that oocyte and embryo production are independent factors. Further studies are necessary to identify the maternal or oocyte factors responsible for such differences.
S Chastant-Maillard, H Quinton, J Lauffenburger, N Cordonnier-Lefort, C Richard, J Marchal, P Mormede and JP Renard
The purpose of this study was to evaluate the impact of repeated follicular puncture used in the ovum pick-up technique on the welfare of cows. The evaluation relies on the physiological measurement of stress, milk production criteria, immune status, and the histological examination of ovaries. Two groups of five Holstein cows were submitted to epidural anaesthesia and genital palpation with insertion of an intravaginal ultrasound probe for transvaginal puncture (the puncture was not performed in the control group). Animals were manipulated twice a week for 8 weeks (16 manipulation sessions). The blood cortisol concentrations increased after each session; however, the concentrations were the same in both the control and the punctured groups. Two adrenocorticotrophic hormone challenge tests, performed before the first session and after the last session, showed an unchanged adrenal sensitivity through repeated puncture sessions. The transvaginal puncture did not affect milk production, or blood and milk somatic cell counts. Ovariectomies were performed on another group of four Holstein cows at various intervals (0 to 30 days) after five similar puncture sessions. Histological examination of the ovaries 4 days after puncture revealed blood-filled follicles and haemorrhagic foci in ovarian stroma, but the examination 30 days after the last puncture session demonstrated very limited, if any, fibrosis. On the basis of the criteria chosen for this study, repeated transvaginal follicular puncture on its own does not impact adversely on the welfare of cows.