Preimplantation development in European mink is continuous, without implantation delay. Table 6 summarises the observations of its progress in the females examined during the 2004 breeding season when 26 embryos flushed from the females on days 2 - 6 of pregnancy and seven embryos flushed from females on days 7 - 11 were not used for embryo transfer, but instead were randomly chosen to investigate preimplantation development. In addition, two trophoblastic vesicles were flushed from one female on day 12 pc (already at implantation) and one of these vesicles was also processed and measured.
Ova or embryos were readily flushed from oviducts and uterine horns in all the pregnant females studied between 2 and 11 dpc, but on the first day pc no embryos/eggs were flushed and on day 12 pc flushing of the uterine horns was usually no longer possible as implantation occured.
No embryos or ova were flushed from the reproductive tract on day 1 pc, although motile spermatozoa were observed in both the oviducts and uterine horns. However, on day 2 pc, live spermatozoa were flushed from the oviducts, but not from the uterine horns. On this day, ovulated eggs and two-cell embryos were observed in the oviductal flushings (Figure 8A, Figure 9A). The absence of ovulated eggs in one animal on day 1 pc and their presence in one animal on day 2 pc suggests that ovulation in this species occurs during the night between the first and the second days pc.
On days 3 and 4 pc, there were no motile spermatozoa in the oviducts and uterine horns. Cleavage-stage embryos, mostly containing eight cells, were flushed from the oviducts on day 3 pc (Figure 8B, Figure 9B). On day 4 pc, embryos of different cleavage stages (up to 16 cells) were flushed from the oviducts (Figure 8C, Figure
9C). On day 5 pc, morula-stage embryos were flushed from the oviducts (Figure 8D), with cell numbers ranging from 19 to 43 (Figure 9D).
Migration of European mink embryos from the oviducts into the uterus occurs on day 6 of their development. Two females were flushed on day 6 pc and, in both cases 50
% of the embryos were still in the oviducts and 50 % had already migrated into the uterine horns (two and two and one and one, respectively). The stage of the embryos on this day was either morula or early blastocyst (Figure 8E), with the number of cells ranging from 60 to 70 (morula) and from 76 to 108 (blastocysts) (Figure 9E).
Table 6. Changes in the reproductive tract of the European mink females during first two
PF, preovulatory follicles; CL, corpora lutea; ov, oviduct; ut, uterus Cleav, cleavage stages; M, morula; EB, early blastocyst; B, blastocyst ExB, expanded blastocyst; EG, early gastrulation; IMP, implantation;
nd – not detected; ns – not studied; np – not possible
* In one donor animal two trophoblastic vesicles were accidentally flushed by extra pressure
Figure 8. Preimplantation embryo development in the European mink from ovulation until implantation. A. Ovulated egg and two-cell embryo, oviduct, 2 dpc. B. Cleavage-stage embryo, oviduct, 3 dpc. C. Cleavage-Cleavage-stage embryo, oviduct, 4 dpc. D. Morula, oviduct, 5 dpc. E. Bottom: morula, oviduct; top: early blastocyst, uterine horn, 6 dpc.
F. Blastocyst, uterine horn, 7 dpc. G. Expanding blastocyst, uterine horn, 8 dpc. H.
Blastocyst with initial stages of endoderm differentiation, uterine horn, 10 dpc. This blastocyst is slightly collapsed with a visible gap between the zona pellucida and trophoblast layers. I. Blastocyst with continuous endoderm differentiation, uterine horn, 11 dpc. (Unstained; light microscopy; scale bar = 100 µm). Modified from III.
Figure 9. Preimplantation embryo development in the European mink from ovulation until implantation. A. Ovulated egg and two-cell embryo, oviducts, 2 dpc. B. Cleavage-stage embryo, oviduct, 3 dpc. C. Cleavage-Cleavage-stage embryo, oviduct, 4 dpc. D. Morula, oviduct, 5 dpc. E. Early blastocyst, uterine horns, 6 dpc. F. Blastocyst, uterine horn, 7 dpc. G. Expanding blastocyst, uterine horn, day 8 dpc. H. Blastocyst with initial stages of endoderm differentiation, uterine horn, 10 dpc. I. Blastocyst with continuous endoderm differentiation, uterine horns, 11 dpc. (DAPI: 4c,6c-diamidino-2-phenylindole staining (2 µg mL–1), fluorescent microscopy. Scale bar = 100 µm). Modified from III.
Blastocysts of different sizes were recovered in the European mink from the uterine horns on day 7 pc (Figure 8F) and onwards. The number of cells was slightly higher than 100 for day-7 blastocysts (Figure 9F). On day 8 pc (Figure 8G) the blastocysts markedly expanded in size, although they still contained ICM without signs of differentiating. The number of cells was approximately 300, sometimes even more (Figure 9G). On day 10 pc some differentiation started and the formation of a distinct layer of cells in the ICM was observed (Figure 8H, Figure 9H), this process being more prominent on day 11 pc (Figure 8I, Figure 9I).
The diameters of cleavage-stage embryos, morulae and early blastocysts did not differ until day 7 pc. On day 8 pc, there was visible about two-fold increase in the size of blastocysts, as until 7 dpc the embryos were about the same size as were ovulated eggs (Figure 10). The size of the embryos continued to progressively increase on days 9 - 10 and particularly on day 11 pc, when the blastocysts had achieved a size of 1.5 mm. From day 12 pc, signs of implantation were evident in pregnant European mink females. However, trophoblastic vesicles (without ICM) were flushed from the uterine horns of one of these (12 dpc) females when the flushing pressure was increased. It was not possible to flush the uterine horns of the other two females, and implantation was confirmed by studying the fixed uterus. For all three day-12 females, the sites of implantation were clearly visible (see section 5.3.3 for more details).
0 1 2 3 4 5 6 7 8 9 10 11
Figure 10. Embryonic growth in the European mink during the preimplantation period.
All flushed embryos were measured and studied on days 2 - 6 pc. One randomly selected embryo from day-7 donor was studied. Three randomly selected blastocysts from three different donor females were studied on day 8 pc. One randomly selected embryo was studied per donor female on each of days 9 - 11. One trophoblastic vesicle which was accidentally flushed from donor female at implantation (12 dpc) was studied. Diameter of embryos at each day of pregnancy is represented as M ± SEM of the sample of embryos as it was on days 2 - 6 and 8 pc. See Table 6 for more details on the numbers of embryos/donor females. Modified from III.
All the European mink blastocysts flushed during the preimplantation period were enveloped by intact embryonic coats. Even one day before implantation, 11 dpc embryos were well inside of the coats and no signs of hatching were observed.
Shrinking of the trophoblastic layer away from the coats, i.e. partial collapse of the blastocyst, was observed in some (although not all) of these blastocysts when they were flushed from uterus (Figure 8H).