1. Effect of vitamin K and co­enzyme Q on cleavage rates and developmental
potential of mice embryos in an in vitro fertilization setting
Kezia Emeny­Smith and Taylor Reynolds
California Polytechnic State University,​ ​San Luis Obispo, California
March 10, 2015
______________________________________________________________________________
Abstract
During preimplantation embryonic development, the mitochondrion undergoes
significant structural and functional changes [1]. Mitochondria play important roles within the
body, including ATP generation and many other cellular processes; therefore, mitochondrial
function contributes to embryo developmental confidence [1]. When used to supplement
standard ​in vitro ​culture media, vitamin K and co­enzyme Q have been shown to repair
mitochondrial damage, thus improving embryo development potential [2,3]. The present study
compares cleavage rates and developmental potential of mice embryos with or without the
addition of vitamin K and co­enzyme Q to the culture media. The results show no significant
difference in developmental potential when vitamin K and co­enzyme Q are added to the
fertilization media.

2.
Introduction
In vitro​ fertilization (IVF) is an assisted reproductive technology in which fertilization is
performed outside of the body, typically in a dish or tube, and requires sufficient oocyte
maturation, sperm separation, and sperm capacitation. In animals, IVF is mainly used for
research purposes but can also be used in cases of abnormalities, semen sexing, to address
limited sperm supply, to fertilize multiple eggs at once, and to overcome poor sperm motility.
The present study was conducted to determine if the addition of vitamin K and co­enzyme Q to
in vitro ​culture media improves cleavage rates and developmental potential of mice embryos.
Mitochondria are organelles that play a role in normal cell function, such as apoptosis
regulation, lipid metabolism, steroid synthesis, calcium homeostasis, and energy production [1].
Currently, their role in ATP production is the most investigated topic; however, it has become
apparent that mitochondria play a significant role in the developmental potential of oocytes and
preimplantation stage embryos [4]. Creating a culture media that replicates the ​in vivo
environment has been attempted but ​in vitro​ conditions disrupt adequate mitochondrial function,
causing embryos produced ​in vitro ​to be of lower quality [1,4]. Previous studies using bovine
embryos have shown that the manipulation of oocyte conditions within culture media improves
blastocyst formation rates and expression of genes related to mitochondrial function [5].
Mitochondria are maternally inherited and create a balance between ATP production by
the electron transport chain and oxidative stress. Preimplantation embryos rely on maternally
inherited mitochondria for energy to develop [6]. ​In vitro ​conditions often result in compromised
mitochondrial function in embryos; however, the presence of co­enzyme Q and vitamin K in
culture media has been shown to rescue mitochondrial function. Vitamin K acts as a
membrane­bound mitochondrial electron carrier during ATP production by the electron transport
chain, and co­enzyme Q is an electron carrier crucial for electron transfer within the
mitochondrial membrane and plays a role in protecting biological membranes from oxidative
damage [3]. Vitamin K and co­enzyme Q are electron carriers that aid in the movement of
protons throughout the electrochemical gradient of the electron transport chain. When
supplemented with both of these electron carriers, the electron transport chain ultimately
produces more ATP, thus increasing available energy for embryos to utilize in their
preimplantation stage development [3]. Previous studies using bovine models show that addition
of vitamin K to culture media is most effective when done 72 hours after fertilization, resulting
in embryos of higher morphological quality, and significantly increasing the percentages of
blastocysts and expanded blastocysts [2]. Additionally, vitamin K supplementation significantly
improved mitochondrial activity and had a notable influence on embryonic gene expression [2].
Based on the previous study performed by Baldoceda, et al. 2014 [2], and the similar
function of coenzyme Q to vitamin K, we hypothesized that the addition of vitamin K and
co­enzyme Q to culture media at 72 hours post­fertilization would increase cleavage rates and
development of mice embryos to the blastocyst stage. In the present study, we intended to

3. investigate if the addition of vitamin K and coenzyme Q at 72 hours after the ​in vitro​ fertilization
of mouse embryos would improve embryonic mitochondrial function and result in more
developmentally competent embryos when compared to embryos cultured in non­supplemented
media.
2) Material and Methods:
2.1. ​Superovulation and Animal Sacrificing
Mature female mice (3 per trial, 4 trials total) were each injected with 0.1ml pregnant
mare’s serum gonadotropin (PMSG) 67.5 hours pre­fertilization (Table 3) to induce follicular
growth, mimicking the effect of the body’s natural hormone, FSH. Exactly 47 hours after
receiving PMSG (Table 3), female mice received 0.1ml human chorionic gonadotropin (hCG) to
induce ovulation, mimicking the effect of the body’s natural hormone, LH. All injections were
administered intraperitoneally using 25 gauge needles and 1 ml syringes. 20.5 hours post­hCG
injection, the 3 female mice and 1 male mouse were humanely euthanized via carbon dioxide
administration in an induction box, followed by cervical dislocation. Animal handling and
euthanasia were performed in accordance with California Polytechnic State University
Institutional Animal Care and Use Committee guidelines.
2.2. ​Collection and Preparation of Gametes
A. Female
The ovaries and the oviduct of each female mouse were excised and placed in a 60mm
dish containing G­MOPS™ Plus, and the oocytes were collected from the oviduct using a 25
gauge needle. The oocytes were washed and sorted in six 30 ​µ​l drops of pre­warmed G­MOPS™
Plus holding media under mineral oil. A total of four trials were performed.
B. Male
The testis and a portion of the vas deferens were excised and placed in a 60mm dish
containing G­MOPS™ Plus, where the epididymis was removed. Spermatozoa from the caudal
epididymis were placed in a sterile tube of 500 ​µ​l pre­warmed G­IVF™ Plus media. The sperm
were incubated and underwent a swim­up for a minimum of thirty minutes. Once complete, a
400 ​µ​l supernatant was removed and placed into a new, sterile tube for fertilization. A total of
four trials were performed. Due to consistently low sperm concentrations, hemocytometer
procedures were excluded from the all four trials.
2.3. ​In Vitro Fertilization
Following washing of collected oocytes in G­MOPS™ Plus, oocytes were placed in a 30
µ​l drop of pre­incubated G­IVF™ Plus media under mineral oil. 10 ​µ​l of spermatozoa were then
added to the G­IVF™ Plus drop for fertilization. Oocytes and sperm were co­incubated at 37.5
degrees Celsius with 5% CO2. A total of four trials were performed.

4. 2.4. ​Embryo Culture
One day post­​in vitro​ fertilization, presumptive zygotes were transferred to G­1™ Plus
media and incubated at 37.5​°​ Celsius under 5% CO2. After 2 days of culture, embryos were
sorted into a control and treatment group and evaluated for cleavage. Half of the embryos were
placed in a new culture dish containing a 30​µ​l drop of G­2™ Plus media and half of the embryos
were placed in a new culture dish containing a 30​µ​l drop of G­2™ Plus media with the addition
of Vitamin K and Co­enzyme Q. On day 4 post­fertilization, embryos were evaluated for stage of
development using the International Embryo Transfer Society guidelines.
2.5​ Assessment of Fertilization and Quality of Embryos
Embryos were assessed for fertilization and normality one day post­in vitro fertilization.
Normal fertilization was defined as the presence of 2 pronuclei in the ooplasm. Any embryos that
deviated from these requirements (abnormal or unfertilized) were discarded from the trial and
excluded from further analysis. Stage­development and quality grading of embryo was done
using the International Embryo Transfer Society (IETS) guidelines (Table 1 and Table 2).
Table 1. IETS Guidelines for Assessing Embryo Stage Development
Stage Stage of Development
1 Unfertilized
2 2­12 cells
3 Early morula
4 Morula
5 Early blastocyst
6 Blastocyst (blastocoel capsule formed, ICM formed)
7 Expanded blastocyst (ZP thins, ICM, and embryo is symmetrical)
8 Hatched blastocyst
9 Expanded blastocyst
Table 2. IETS Guidelines for Grading Embryo Quality
Grade Embryo Quality
1 Excellent/Good (<10% not good)
2 Fair (70% ok, 30% not good)

5. 3 Poor (>30% not good)
4 Dead/Degenerating
2.6. ​Experimental Design
The objective of the current experiment was to determine the effect of vitamin K and
co­enzyme Q on cleavage rate and developmental potential of mice oocytes. Drops containing
day 3 embryos were incubated with or without vitamin K and co­enzyme Q added to the
fertilization media, G­2™ Plus. The experiment was replicated a total of four times.
Table 3. Experiment Procedural Timeline
Sunday
9:30PM
Tuesday
8:30PM
Wednesday
5­7PM
Thursday
11AM­1P
M
Saturday
10­11AM
Sunday
11AM
PMSG 3​♀ hCG​ 3♀
∙ Sacrifice
3​♀ &​ 1​♂
∙ IVF
Check for
pronuclei
∙ Check 2­cell
cleavage
∙ Add CoQ/Vit. K
to trial group
∙ 8­cell
cleavage
∙ Assess
3) Results
Oocytes (n=155) were retrieved from twelve superovulated female mice throughout four
trials. During the four trials, a total of sixty­five oocytes (41.9%) were fertilized one day after the
in vitro ​fertilization procedure (Table 4). This corresponds to approximately 13 oocytes retrieved
per female mouse. Fertilization rates were calculated by dividing the number of fertilized
oocytes day 1 post ​in vitro ​fertilization by the total amount of collected oocytes for each trial.
Embryos were assessed 24 hours, 72 hours, and 96 hours post­​in vitro ​fertilization for
development and cleavage rate. At 96 hours post­​in vitro ​fertilization, embryos were analyzed
and assigned a stage and grade for each treatment group (Figure 3 and 4). In trial 1, 4 embryos
were assigned to the treatment group and 1/ 4(25%) remained unfertilized, while 3/ 4 (75%) were
stage 2, grade 4 embryos. In trial 1, 3 embryos were assigned to the control group and 1/ 3
embryos progressed to the blastocyst stage, while 2/3 (66.7%) were stage 2, grade 4 embryos. In
trial 2, 14 embryos were assigned to the treatment group and 14/14 (100%) were stage 2, grade 4
embryos. In trial 2, 13 embryos were assigned to the control group and 13/13 (100%) embryos
were stage 2, grade 4 embryos. In trial 3, 6 embryos were assigned to the treatment group and
6/6 (100%) were stage 2, grade 4 embryos. In trial 3, 7 embryos were assigned to the control
group and 7/7 (100%) embryos were stage 2, grade 4 embryos. In trial 4, 9 embryos were
assigned to the treatment group and 9 / 9 (100%) were stage 2, grade 4 embryos. In trial 4, 9

6. embryos were assigned to the control group and 1/ 9 (11%) remained unfertilized, while 8/ 9
(89%) were stage 2, grade 4 embryos.
Figure 1. Stages of mouse preimplantation development (Saiz et al., 2012) [7]
Table 4. Fertilization Rates for Trial 1 ­ Trial 4
Trial # Presumptive Zygotes from Collected
Oocytes, Day 1 Post­Fertilization
Fertilized (%)
1 7/16 43.8%
2 27/62 43.5%
3 13/26 50.0%
4 18/51 35.3%
Average​= 43.2%

7.
Figure 3. Stage and grade of embryos 96h post­fertilization (control).
Figure 4. Stage and grade of embryos 96h post­fertilization (treatment).

8.
A. Stage 2, Grade 4 embryo 5 days post­​in vitro ​fertilization
B. Stage 1 embryo 5 days post­​in vitro ​fertilization
4) Discussion
Vitamin K and co­enzyme Q are often added to culture medium to improve embryo
developmental competence [2,3]. The objective of this study was to investigate if the addition of
vitamin K and coenzyme Q at 72 hours post­​in vitro ​fertilization of mouse embryos would
improve embryonic mitochondrial function and result in more developmentally competent
embryos when compared to embryos cultured in non­supplemented media. It was hypothesized
that embryos cultured with vitamin K and co­enzyme Q may have increased mitochondrial
function leading to increased cleavage rate and overall higher developmental potential [1­4].
However, in the present study, the addition of vitamin K and co­enzyme Q did not improve
cleavage rates and embryo development. Reasons for the discrepancies between this study and
previous successful studies are difficult to resolutely pinpoint. However, there are a wide array of
potential causes for the present study’s deviation from the expected outcome.
Throughout the experimental set­up, there were several factors that could have
contributed to the unexpected outcome of the study’s results. The fertilization media used
throughout this study was the G­series from Vitrolife. When revising the guidelines provided by
the manufacturer of the media, we found that it was suggested to pre­incubate media between 6
to 18 hours prior to use [8]. Instead, this study only incubated media for approximately 30­45
minutes before a transfer; however, during trial 1 the G­2 Plus plates were prepared and
incubated 12 hours prior to transfer. This trial led to the only blastocyst formation throughout all
four trials. Although this is not a largely significant result, it offers some evidence that
pre­incubating media for an extended period of time can improve developmental potential of ​in
vitro ​produced mice embryos. Although a direct correlation cannot be made based upon one
incidence alone, it seems plausible that with longer equilibration times, media more closely
mimics the uterine environment in terms of pH and temperature, leading to further embryo
developmental progression. A previous study that was successful in obtaining a significant
amount of blastocysts using vitamin K supplemented culture media, explained that oocytes were

9. fertilized with semen after 15­18 hours of incubation at 38.5​°​ C containing 5% CO2 [2]. This
evidence implies that equilibrating oocytes in culture media for an extended period of time
enhances blastocyst rates. In addition, Vitrolife also recommends washing the embryos through a
plate of G­MOPS™ Plus before transferring between medias [8]. Due to economic restraints this
step was omitted from the present study; however, given the resources we believe this could
enhance the outcome in future trials.
One factor of experimental set­up that we were extremely meticulous about was
maintaining a warm environment for gametes while manipulating and transferring ​in vitro.
Throughout all trials, mature oocytes were used for fertilization, meaning that the meiotic spindle
was formed. Meiotic spindles are formed from microtubules and are extremely sensitive to
temperature fluctuations [5]. If temperatures dropped below or went above the desirable 38.5​°​C,
it is likely that the spindle could have disassembled, thereby negatively affecting fertilization
rates and embryo developmental potential. In efforts to minimize this potential hazard, a heated
glass microscope stage was utilized and a heating plate at 38.5​°​C was kept close to the
workspace at all times in efforts to maintain the temperature of an ​in vivo ​environment and
stabilize the meiotic spindle formation.
According to the Jackson Laboratory, when fresh mouse sperm is used for ​in vitro
fertilization, the average fertilization rate is 65% [9]. Throughout all four trials, fertilization rates
were much lower than this average, 43.8%, 43.5%, 50.0%, and 35.3%, respectively (table 4).
Many factors could have contributed to the consistently low fertilization rates present throughout
the present study. As mentioned previously, if the temperatures were not ideal throughout oocyte
handling, the meiotic spindle could have been disassembled [5]. Throughout all four trials the
cd1 strain of mice were used. It is reasonable to hypothesize that this strain could be low
performing mice in terms of reproductive potential. All females ovulated mature oocytes during
each trial, while sperm concentrations were consistently low. Given the resources, investigating
this strain would be something to further investigate for future trials, especially male fertility
potential. In total, 155 oocytes were collected from 12 mature, female mice averaging about 13
collected oocytes per mouse. In addition to low fertilization rates, there was a consistent issue
with unnaturally low epididymal sperm counts from the male mice used in the trials. The mice
used across the four trials were smaller than average, leading us to speculate that the male mice
potentially had lower than normal testosterone levels, which could have had a detrimental effect
on sperm production. In addition, when sperm were viewed under a high magnification scope,
many abnormalities were present. In particular, many cytoplasmic droplets were observed,
indicating immature spermatozoa [10]. Due to the consistently low sperm counts, our
experiment was confounded on two fronts. In the first two trials, 1ml of G­IVF ™ Plus was used
for the sperm swim­up and resulted in very low sperm counts when semen was analyzed using
the hemocytometer. In an effort to increase sperm concentration, the following two trials utilized
.5mL G­IVF ™ Plus for the swim­up. Although the reduction of the G­IVF ™ Plus volume
hypothetically should have had a favorable effect on sperm concentrations, the reduction of

10. volume made it more difficult to remove the desirable supernatant produced by the swim­up. The
hurdle posed by having to remove only the desired 400​µ​l of supernatant from a 500​µ​l total
volume subsequently increased the difficulty in achieving the appropriate concentration of sperm
to use for ​in vitro​ fertilization. Theoretically, the swim­up would have led to capacitated, viable
sperm remaining at the top portion of the supernatant; however, the difficulty of removing the
desired 400 ul of supernatant could have resulted in non capacitated sperm being utilized for
fertilization, furthering less than ideal fertilization rates (table 4). Due to the excessively low
sperm counts, we were unable to use the hemacytometer effectively to adjust sperm counts to the
desired concentrations. We ultimately decided to pipette 400​µ​l of supernatant into a new tube,
and from this new tube fertilized each oocyte­containing drop with 10​µ​l of non­concentration
adjusted motile sperm. This unexpected hindrance to our experimental design must be
considered as a possible influence on the less than ideal results of the present study. For future
trials, it could be advisable to sacrifice an additional male for each trial in order to maximize the
amount of sperm available to use for the fertilization of oocytes.
Throughout all four trials, embryo development did not conform to the desired outcome.
Besides the one blastocyst formation during trial 1, all fertilized oocytes did not progress past the
2­16 cell stage (stage 2) when analyzed post­​in vitro ​fertilization. Figure 1 illustrates the stages
of mouse preimplantation development [5]. It was expected that on day 1 post­​in vitro
fertilization, embryos would be at a two cell stage, developing into blastocysts by day 4 post­​in
vitro ​fertilization [5]. However, the majority of embryos were stage 2, grade 4 embryos when
assessed day 4 post­​in vitro ​fertilization as indicated by figure 5A. Reasons for the discrepancies
have been previously discussed.
In conclusion, the present study found that when vitamin K and co­enzyme Q are added
to ​in vitro ​fertilization media, cleavage rates and embryo developmental potential are not
improved.

11.
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