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B. Fischer

Summary. The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocyts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions.

Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed.

No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P > 0·05) and was generally lower than in chronologically aged asynchronously transferred embryos (P < 0·05 for Day-3 and P < 0·001 or P > 0·05 for Day-4 embryos).

Keywords: uterine transfer; asynchrony; blastocyst; rabbit preimplantation development; in-vitro culture

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B. Fischer

Summary. Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine.

In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P < 0·001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.

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B. Fischer and A. Schumacher

Summary. The correlation between strain fecundity and (i) development and (ii) rate of aneuploidy was studied in rabbit preimplantation embryos obtained from 2 strains of different fecundity. Embryos were investigated at Days 3–6 (preimplantation development) or Days 2, 4 and 6 post coitum (aneuploidy). Embryonic size and cell proliferation varied on the days of investigation, but with no consistent tendency in favour of one strain. The incidence of aneuploidy did not differ significantly between embryos from the 2 strains (P>0·05). The multifactorially determined criterion of prolificacy was not selectively correlated with overall differences in embryonic preimplantation growth and rate of aneuploidy.

Keywords: preimplantation development; aneuploidy; fecundity; strain differences; rabbit

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B. Gosch and K. Fischer

Summary. Four adult male fallow deer were investigated for 1–4 consecutive years to study the relationships between annual changes in testis volume, sperm quality and antler status. Testicular volume started to increase in July/August, peaked just before the rut, declined until December to 50% of maximum, persisted at this level up to February/March and reached minimal volume after antler casting in late April. There was no apparent age effect on the seasonality of testis size fluctuations. Velvet shedding and antler casting occurred at about 80% and 25%, respectively, of maximal testis volume.

Spermatozoa had the same general appearance as those of related ruminants. Viable spermatozoa appeared between August and early May which corresponds almost exactly to the time when fallow deer are in hard antler. From September to March sperm quality would fulfil artificial insemination standards for cattle semen. In June and the first half of July 14 out of 15 ejaculates were devoid of any sperm cells. There were no indications of a secondary seasonal peak in values monitored.

Keywords: fallow deer; antler cycle; seasonality; electroejaculation

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A. Schumacher and B. Fischer

Summary. During in-vitro culture rabbit early cleavage stages (Day 1 p.c.) and compacted morulae (Day 3 p.c.) were exposed to visible light or to room temperature (23°C) for various lengths of time (0·5–24 h). The light source used resembled closely routine laboratory lighting. Controls were cultured simultaneously for 24 h under standard conditions (37°C, darkness). Development was assessed by incorporation of tritiated thymidine as an indicator of cell proliferation.

In comparison to non-exposed controls cell proliferation of Day-1 embryos was more impaired by light than by room temperature whereas in Day-3 embryos thymidine incorporation was more reduced following exposure to room temperature than to light. No statistically significant decrease in thymidine incorporation was detectable up to 1 h (light) and 8 h (room temperature) in Day-1 embryos. Morulae tolerated room temperature and visible light for up to 3 h and 8 h, respectively. Split-dose exposure (e.g. 4 × 1 h) to visible light or room temperature revealed no statistically significant differences compared with one long en-bloc exposure (e.g. 1 × 4 h). These results demonstrate a stage-dependent susceptibility of preimplantation embryos to physical environmental factors. The major risk, indicated by the shortest tolerance times, was provoked by visible light to early cleavage stages.

Keywords: light; room temperature; preimplantation embryos; cell proliferation; rabbit

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B. Fischer and B. D. Bavister

Oxygen tension was measured using flexible polarographic microelectrodes within the oviductal and uterine lumen in rhesus monkeys (n = 9), golden hamsters (n = 21) and rabbits (n = 6), during the reproductive cycle (monkey), during oestrus and pseudopregnancy (hamsters, rabbits) and during pregnancy (hamsters). In general, oxygen tensions in each species were much less than half of atmospheric O2, ranging from high values of about 60 mm Hg (8.7% O2) in the rabbit oviduct, rabbit and hamster uterus, to as low as 11 mm Hg (1.5% O2) in the monkey uterus. Oxygen tensions did not vary significantly between left and right sides of the reproductive tracts (all species), nor between pregnant and pseudopregnant states nor between oviduct and uterus (hamsters). Differences owing to reproductive stage were found in the monkey oviduct, hamster oviduct and uterus, and rabbit uterus. Oxygen tensions were consistently very low (11–14 mm Hg) in the monkey uterus throughout the menstrual cycle. In hamsters and rabbits, intrauterine O2 decreased significantly at about the normal time of blastocyst formation and implantation, to 37 mm Hg (5.3% O2) and 24 mm Hg (3.5% O2), respectively. This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period. The data have implications for investigations of embyro metabolism and for improving embryo development in vitro.

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B. Fischer and C. E. Adams

Summary. The possible selection of spermatozoa for fertilization by the female genital tract was investigated using genetically homogeneous, numerically adjusted 'cervix-selected' and 'unselected' rabbit spermatozoa. Samples of spermatozoa were marked by 15 min exposure to 1·3 mg TEPA/ml and then washed for 15 min. TEPA-treated and control samples were inseminated alternately into the vagina (= 'cervix-selected' or uterine horns (= 'unselected') of prospective donors. After 6 h spermatozoa were recovered from the uterine horns of the donors. Equal numbers of 'selected' and 'unselected' spermatozoa were inseminated either into the uterine horns (24 does) or oviducts (25 does) of recipients. The fertilization rates were 48 and 72% respectively. Significantly more eggs were fertilized by untreated than by TEPA-treated spermatozoa. Almost identical fertilization rates, however, were observed between 'cervix-selected' and 'unselected' spermatozoa. It is concluded, therefore, that in the rabbit no selection of (preincubated) spermatozoa for fertilization takes place at the cervical level.

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B. Gosch, T. Bartolomaeus, and K. Fischer

Summary. No basic differences in size (mean ± s.d. for at least 300 spermatozoa), shape and ultrastructure of the spermatozoa of fallow deer were detected (1) in comparison to other artiodactyls, (2) between different fallow bucks, and (3) between different months of the fertile season.

The total length of the normal spermatozoon was 67·2 ± 1·2 μm. The flat, paddle-shaped head was 8·2 ± 0·3 μm long, 4·4 ± 0·2 μm for the greatest width, 1·9 ± 0·2 μm for basal width and, ∼0·7 μm in thickness. The tail measurements were 13·7 ± 0·3 μm for the midpiece, 0·5 ± 0·1 μm for the diameter of the midpiece, 42·6 ± 0·9 μm for the principal piece, and 2·7 ± 0·6 μm for the endpiece. Spermatozoa with abnormalities such as cytoplasmic remnants and droplets, bent and coiled tails, as well as microcephalic forms were observed.

Keywords: fallow deer; scanning electron microscopy; light microscopy; spermatozoa; morphology

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B. Fischer, E. Winterhager, and L. C. Busch

Summary. Ovulation was induced in rabbits between Days 14 and 18 of pseudopregnancy by an intravenous injection of hCG. Induction of ovulation from Day 16 onwards led to normal progestational endometrial transformation. In rabbits injected on Day 14 or 15, a normal preimplantation endometrial morphology developed, but not earlier than 7 days after hCG (Day 14/15 +7). Uteroglobin secretion was advanced during the second pseudopregnancy. After mating or artificial insemination, fertility was greatest on Day 18 of pseudopregnancy. Conception failed on Day 14 and embryo transfers were unsuccessful on Day 14 +1. Transfers performed on Day 14 +3, however, led to implantation and offspring, even though endometrial morphology did not correspond to the normal Day 3 preimplantational morphology at the time of transfer.

We conclude that (1) endometrial transformation typical of normal pseudopregnancy can be induced by ovulation during the regeneration phase of pseudopregnancy from Day 16 onwards; (2) fertilization and implantation can be achieved as early as Day 15 of pseudopregnancy; (3) an oestrous period with high mating activity and fertility occurs about 3 days later; and (4) Day 14 after hCG represents a limited time of functional change from pseudopregnancy to a fertile uterine cycle in the rabbit.

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R Augustin, P Pocar, C Wrenzycki, H Niemann, and B Fischer

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.