Fertilization, embryo development and pregnancy|

Abstract. With the aim of recovering oocytes and two cell embryos from oviducts, 40 Mus musculus
and 10 Mastomys coucha were injected with PMSG and hCG for inducing superovulation. Results were
satisfactory for Mus musculus superovulation, while for Mastomys coucha more advances need to be done.
Regarding mating for in vivo fertilization in Mus musculus and obtaining of two-cell embryos, results
were not as good as for oocytes obtaining. Handling of junior embryologist should be consider when
looking at data variability between individuals.

Key Words. Superovulation, Mus musculus, Mastomys coucha, PMSG, hCG, oocytes, two cell embryos

Material and Methods. Ovulation was induced in 40 Mus musculus and 10 Mastomys coucha females.
For that, Mus musculus were injected with 5 IU of PMSG and Mastomys coucha with 25 IU of PMSG.
Forty-eight hours later, both mice were injected with a hCG (Ovitrelle©) concentration of 5 UI
for Mus musculus and 50 IU for Mastomys coucha. Once here, mice were separated in two groups; one
group of 20 Mus musculus and 10 Mastomys coucha was killed after twenty-four hours for recovering
oviduct oocytes. The other group, of 20 Mus musculus, was mated with males (1 female:2males) for
the obtaining of two-cell embryos forty-eight hours later. For both groups, female mice were
slaughtered with CO2 (see figure 1).

For recovering the oocytes from oviduct, 17 Mus musculus and 10 Mastomys coucha were dissected
and both oviducts extracted using surgical scissors and bistoury. Once placed in a plate, were
washed and maintained wet during all the procedure with PBS. Under the stereromicroscope,
ampulla (Figure 2) was located and separated from the rest
of the oviduct. Ampulla was broken carefully with bistoury
for recovering oocyte-cumulus complexes (Figure 3) with
glass Pasteur pipettes. Once recovered, cumulus cells were
placed in fresh PBS and hyaluronidase was added to
removing cumulus cells. For obtaining the embryos
resulting from the presumptive mating between Mus
musculus, the 20 mice left were dissected and the same
procedure than in the other was applied: prior to ovary,
oviduct and uterus extraction, mice were killed. Under the
stereomicroscope, the ampulla-istmus region was localized
in the oviduct and dissected. Then, this tissue was gently
cut with bistoury for allowing the exit of 2-cell embryos
located there. Figure 1. Machine exchanging O2 for C02
progressively.

Figure 2. Where to found oocytes in the ampula Figure 3. Oocytes from the ampula
(Source: Maillo-Sevilla, 2016)
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Results and Discussion. The results of superovulation induction in Mus musculus and Mastomys
coucha were evaluated by means of the quantity and the quality of oocytes and embryos obtained.
Results are shown in Table 1 and 2 and grouped for each mouse. The first aspect to analyse the
wide variety between quantity of oocytes obtained in each mouse. As each mouse was assigned
to one student, results are also grouped by each student handling. That fact explains the
differences between quantity of oocytes obtained, due to the heterogeneous ability within
operators. Besides that, general results about quantity of oocytes obtained is good and it can be
affirmed superovulation protocol has functioned. Although statistical treatment of data should
be need, it can be affirmed there are differences between Mus musculus and Mastomys coucha oocyte
quantity. All Mus musculus ovulated, while only three Mastomys coucha did it. The reason could rely
on the high fat content of Mastomys coucha, which could complicate hormones diffusion along
tissues for reaching the ovary. Moving to embryo quantity, interpreting of results is slightly
different, due to more factors are needed for succeeding. As it is shown in table 2, not in all Mus
musculus embryos were found but instead, in some cases oocytes were observed. The cause could
be multiple: some females were not mated, or mated but not fertilized. In the second case, the
failure of fertilization may be cause by non-synchronised ovulation. This suggestion is supported
by immature aspect of oocytes obtained this day, with cumulus cells tightly compacted around
the oocyte.
Finally, and in line with quality; in both oocytes and embryos obtained, the presence of
fragmentation is noticed. Fragmentation is associated with poor quality of cells and low yield in
further steps, as could be embryo development or implantation. It is also normally associated to
superovulation treatments, which force the ovaries, put ahead quality to quantity and recruit
follicles that otherwise would not have been ovulated.
Table 1. Quantity of oocytes collected on Thursday 03/11/2016, where Nº is referred to each mouse and
each student handling.
Mus musculus Mastomys coucha
Nº oocytes Nº oocytes
Nº Weight Left ovary Right ovary Nº Weight Left Right
(gr) (gr) ovary ovary
1 27 13 1 56,794 - -
2 - 20 2 69,25 - -
3 4 19 3 68,26 11 7
4 7 5 4 78,79 - -
5 - 11 5 80,07 11 16
6 8 6 6 81,41 - -
7 10 (8 fragm.) 9 (4 fragm.) 7 60,31 1 3
8 16 fragm. 20 (4 fragm.) 8 70,87 - -
9 8 14 9 75,55 - -
10 25 (23 35 (34 10 76,7 12 18
fragm.) fragm.)
11 5 14
12 13 7
13 6 13
14 11 -
15 25 -
16 0 5
17 11 not dissected

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Fertilization, embryo development and pregnancy|

Table 2. Number of of 2-cell embryos and oocytes obtained on Friday 04/11/2016, where Nº is referred to
each mouse and each operator
Mus musculus
Nº 2-cell embryos Nº unfertilized
Nº Weight (gr) Left ovary Right ovary Left ovary Right ovary
1 11 14 2 2
2 11 24 3 1
3 0 0 10 7
4 4 4 (1 with 3-cell) Several fragmented
5 0 0 3 fragm. -
6 24 22 - -
7 - - 10 -
8 3 5 1 fragm. -
9 1 - - -
10 21 10 2 3 (1 fragm.)
11 7 5 11 fragm. 4 (1 fragm.)
12 - - - -
13 4 17 - 5 (2 fragm.)
14 4 18 - 7 (4 fragm.)
15 - - - -
16 - - 5 (2 fragm.) 1 fragm.
17 - 8 9 fragm. 3

Conclusions. In base to these results, it can be concluded that PMSG and hCG (Ovitrelle©) at
doses at which they were administered, are suitable for oocyte superovulation in Mus musculus.
However, results for Mastomys coucha superovulation were under what it was expected. More
work need to be done in this last species to develop better treatment, varying hormone principles
or doses.
References.

Malillo-Sevilla, V. (2016). Embryo culture: generalities and problems derived from in vitro culture. INIA.

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