Animal Reproduction Science xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Successful pregnancies from vitrified embryos in the dromedary
camel: Avoidance of a possible toxic effect of sucrose on embryos
⁎
M. Herrid , M. Billah, J.A. Skidmore
Camel Reproduction Centre, PO Box 79914, Dubai, United Arab Emirates
AR TI CLE I NF O
AB S T R A CT
Keywords:
Camel
Embryo
Vitrification
Sucrose
Successful embryo cryopreservation facilitates the wider application of assisted reproduction
technologies and also provides a useful method for gene banking of valuable genetics.
Unfortunately attempts to establish an effective cryopreservation protocol for camelid embryos
have been unsuccessful. In the current study, a modified vitrification protocol with three steps
was investigated, whereby embryos were exposed to solutions containing increasing amounts of
glycerol and ethylene glycol for fixed time periods. Embryos were then loaded into an Open Pull
Straw (OPS) and plunged directly into liquid nitrogen for storage. Three experiments were designed to investigate the effect of 1) artificial shrinkage (AS) of embryos, 2) the addition of
sucrose to the vitrification solutions, and 3) the replacement of sucrose by galactose in the
warming solution, on the outcome of vitrification. The results showed that neither AS of hatched
embryos prior to vitrification, nor the addition of sucrose into vitrification solutions improves the
outcome of vitrification, while replacement of sucrose with galactose in warming solution increases the survival and developmental rates of vitrified embryos in culture. Transfer of vitrified
embryos that were warmed in galactose resulted in a pregnancy rate of 42.8% per embryo or
46.1% per recipient. Collectively, these results suggest a possible species-specific toxic effect of
sucrose on camel embryos, and that avoiding its use either in vitrification or warming solution is
critical for establishing an effective protocol. This study may also be applicable to the vitrification of embryos of other camelid species including alpaca and llamas.
1. Introduction
Assisted reproduction technologies (ART), such as fresh embryo transfer (ET), have been widely practiced in dromedary camel
breeding programs to quickly disseminate valuable genetics, or overcome the problems associated with low reproductive efficiency.
The inability to successfully cryopreserve hatched blastocysts has, however, hampered the wider applications of ART in this species
(Anouassi and Tibary, 2012; Herrid et al., 2016, 2017).
Vitrification has been considered as an effective approach to cryopreserving embryos because of its simplicity of use and efficacy
(Edgar and Gook, 2012). Two types of cryoprotectants are commonly used to displace intracellular water of embryos or oocytes and
thereby reduce the formation of ice crystals during cryopreservation. These are referred to as permeating cryoprotectants (glycerol
(GLY), propandiol (PROH), dimethylsulfoxide (DMSO) and ethylene glycol (EG)), or non-permeating cryoprotectants (sucrose, trehalose and galactose; Best, 2015). Although all cryoprotectants can facilitate removal of intracellular water from the cells via osmotic
gradients, the major difference between the two types is that permeating cryoprotectants are capable of permeating into the cell
⁎
Corresponding author.
E-mail address: mherrid@gmail.com (M. Herrid).
http://dx.doi.org/10.1016/j.anireprosci.2017.10.015
Received 10 August 2017; Received in revised form 15 October 2017; Accepted 25 October 2017
0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Herrid, M., Animal Reproduction Science (2017), http://dx.doi.org/10.1016/j.anireprosci.2017.10.015
Animal Reproduction Science xxx (xxxx) xxx–xxx
M. Herrid et al.
membrane, while non-permeating cryoprotectants cannot (Best, 2015). Toxicities of permeating cryoprotectants have previously
been recognized in different type of cells and tissues from a variety of animal species (Best, 2015) and it is evident that there are
differences between species in the way oocytes and embryos respond to these chemicals (Skidmore et al., 2004). For example, DMSO
is widely used for human, cattle and mouse embryo cryopreservation, but it appears to have a relatively greater toxicity to camelid
embryos (Skidmore et al., 2004). Replacing DMSO with EG in protocols commonly used for other species has been a focus of freezing
camelid embryos (Skidmore et al., 2004; Herrid et al., 2016, 2017). There have been fewer studies on the toxicity of non-permeating
cryoprotectants as these are generally used only to control the osmolarity of the vitrification or warming solutions (Arav et al., 1993;
Somfai et al., 2015). An example of the different protective effects of sugars on vitrification of immature cattle oocytes is where
trehalose was, however, shown to be less harmful than sucrose (Arav et al., 1993).
The embryo size also influences the outcome of cryopreservation, there being a lesser survival rate for large embryos (Best, 2015;
Herrid et al., 2017; Sanchez et al., 2017). The primary reason for this is the formation of ice crystals within embryonic cells, which is
associated with a slower penetration of cryoprotectants and subsequent incomplete dehydration of larger embryos (Edgar and Gook,
2012). One way to overcome this problem and to improve viability of human, pig and horse embryos after vitrification has been to
adopt a process of artificial shrinkage (AS), which can be achieved by either the physical removal of the blastocoel fluid via needle
puncture (Mukaida et al., 2006; Choi et al., 2011; Diaz et al., 2016) or by incubating embryos in high osmotic solutions (Barfield
et al., 2009). For example, Diaz et al. reported the use of AS and the removal of 95% to 99% of blastocoel fluid to achieve successful
vitrification of expanded blastocysts of horses with a mean diameter of 821 μm, resulting in a pregnancy rate of 83% (5/6; Diaz et al.,
2016).
Camel embryos at Day 7 or 8 of development are of a similar size to embryos of horses (150–1150 μm in diameter) collected the
same time after ovulation (Barfield et al., 2009; Herrid et al., 2016). Thus, the object of the current study was to assess the impact of
1) AS of hatched blastocysts, 2) the addition of sucrose into vitrification solutions and 3) warming embryos in galactose solution on
the survival rate and developmental potential of vitrified embryos.
2. Material and methods
2.1. Experimental animals
Mature females (n = 72) aged 5–14 years, and three mature male dromedary camels were used. The males were maintained in
pens, each of an area of approximately 20 square meters, while the females were housed in much larger pens in groups of 20–30. All
animals had access to water ad libitum, and were fed a diet of mixed concentrates and lucerne hay once a day.
2.2. Superovulation and embryo production
Ovaries and uteri of donor camels were examined by trans-rectal ultrasonography on alternate days during the winter breeding
season in Dubai, United Arab Emirates using an Aloka Model 500 real-time scanner with a 5-MHz linear array transducer (Al Carmal,
Dubai, UAE), as previously described (Skidmore et al., 2004). Each donor (female) camel was injected with 20 μg of the GnRH
analogue, buserelin, when a mature follicle between 1.3 and 1.7 cm in diameter was detected in the ovaries. On day 5 after the
injection of GnRH, each donor was further treated over a 4-day period to induce superovulation. A dose of 2000 IU equine chorionic
gonadotrophin (eCG; Folligon; Intervet Laboratories, Cambridge, UK) was administered as a single intramuscular injection on day 1
of the treatment period, and porcine follicle-stimulating hormone (pFSH; Folltropin; Vetrepharm Canada Inc., Belleville, ON, Canada)
was administered twice daily by intramuscular injections over 4 days with decreasing doses each day (i.e., 2 × 80; 2 × 60; 2 × 40;
2 × 20 mg).
All treated camels responded with development of at least four ovarian follicles being detected. When the majority of these
follicles had reached a mature size (1.3–1.8 cm diameter), the donors were mated twice, 24 h apart, by one of three available mature
males. Camels are an induced ovulating species and ovulation is known to occur between 24 and 48 h after mating. Accordingly, the
day of ovulation (Day 0) was defined as the day after the first mating.
On either 7, 8 or 8.5 days after first mating, the uteri of the donor camels were flushed non-surgically by trans-cervical uterine
lavage using 20-Fr catheters (Benkat Instruments, Hertford, Hertfordshire, UK) and embryo flushing medium (IMV Technologies,
L’Aigle, France), as described previously (Skidmore et al., 2004). Each embryo was identified, morphologically assessed, and graded
based on a criterion established previously (Skidmore et al., 2004). Only grade A (excellent, perfectly spherical with a smooth
surface) and B (good/fair, spherical with some irregularities in contour) embryos were used in the following cryopreservation experiments and randomly assigned to the various groups. There was no difference in the developmental rate in culture between fresh
Grade A and B embryos in a preliminary experiment.
2.3. Preparation of holding, vitrification, warming and culture solutions
Holding solution (HS): Dulbeccos’s phosphate buffered saline without calcium chloride and magnesium chloride (D-PBS; SigmaAldrich, St. Louis, MO) was used as the base medium for all vitrification solutions. The D-PBS was supplemented with 0.3 mM sodium
pyruvate (Sigma-Aldrich, St. Louis, MO), 3.3 mM glucose (Sigma-Aldrich, St. Louis, MO) and 20% fetal bovine serum (v/v) (HyClone
Inc., Logan, UT).
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2.3.1. Vitrification solution (VS)
Three vitrification solutions each containing differing concentrations of glycerol and ethylene glycol (Sigma-Aldrich) were used.
The VS1 was composed of 1.4 M glycerol, VS2: 1.4 M glycerol + 3.6 M ethylene glycol and VS3: 3.4 M glycerol + 4.6 M ethylene
glycol.
2.3.2. Warming solution (WS)
Two warming solutions each containing differing concentrations of sucrose (Sigma-Aldrich) were used. These were WS1: HS
containing 0.5 M sucrose, and WS2: HS containing 0.25 M sucrose).
2.3.3. Culture solution
The culture solution contained TCM 199 + 5% FCS + MEM Non-Essential Amino Acid Solution (100×; Sigma-Aldrich) + MEM
Amino Acids (50x; Sigma-Aldrich).
2.4. Process of vitrification, warming and culture
Vitrification was performed essentially according to a protocol designed for day 8 embryos of horses (Diaz et al., 2016) with an
Open Pulled Straw (OPS) modification (Vajta et al., 1998). Embryos from each donor were divided into two groups of equal numbers.
One group was used as treatment group and subjected to one of the procedures described in the experimental design. Another group
was used as fresh control for in vitro culture. On occasion, embryos collected from two donors on the same day were pooled before
dividing them into the two groups for the procedures.
For equilibration, embryos were washed briefly in one drop (50 μl) of HS, then treated in sequence with 500–700 μl of VS1 and
VS2 for 5 min each. The embryos were then exposed to two drops of VS3 for 20–30 s each at room temperature before being loaded
into the OPS straw and plunged into liquid nitrogen. Based on the number of embryos collected each day, either one or two embryos
were cryopreserved in a single straw.
For warming, the straw was held in air for 3 s and then directly submerged into 1 ml WS1 in a 4-well culture dish at 37 °C, and the
warmed blastocysts were then incubated for 1 min. Subsequently, the blastocysts were transferred into 1 ml of WS2 at room temperature (∼24 °C) in a second 4-well dish. After 5 min, the embryos were transferred to 1 ml HS for an additional 5 min.
This vitrification and warming protocol was termed as the “Original Protocol”. The vitrified embryos were stored in liquid
nitrogen between 1 and 30 days. After the warming process, each embryo was re-graded, and then cultured in CS in a CO2 incubator
for 5 days in each experiment. Re-expansion of embryos was morphologically assessed after 2, 24, 48, 72 and 96 h in culture for in
vitro survival and developmental potential, respectively. Embryos with normal and spherical shape, without lysis, and not shrunken,
swollen, or with blackened cytoplasm were regarded as surviving.
2.5. Experimental design
2.5.1. Experiment 1: comparison of physical AS using different sized needles
The current study was designed to compare methods of AS and to assess relative benefits to the survival and development of
vitrified camel hatched blastocysts. The approach was to simplify and reduce the financial expense of the procedure by assessing the
efficacy of acupuncture needle puncturing as compared with micromanipulator needle puncturing.
For this technique an 8 μm micro-needle puncture was used that has been described previously (Diaz et al., 2016). Briefly, the
expanded blastocysts were washed three times in HS and then placed in a 50 μl drop of VS1. The blastocyst was held with a holding
pipette connected to the micromanipulator, and the inner cell mass (ICM) was placed at the “6 or 12 o’clock” position. A glass microneedle was pushed through the trophectoderm into the blastocoel cavity. After removing the micro-needle, the contraction of the
blastocoele was observed for several minutes. For AS using a 50 μm acupuncture needle, embryos were placed in 100 μl HS in a
60 mm culture dish and manually punctured with observation with a stereomicroscope. During the process, the ICMs were avoided to
prevent damage to the embryo. After 5 min incubation in VS1, including the puncturing procedure, the embryo was transferred to
VS2 and the remainder of the original protocol was followed. For the control group, embryos were treated in HS for 5 min and then
vitrified using the original protocol.
2.5.2. Experiment 2: effect of reducing permeating cryoprotectant concentrations, or of adding sucrose in the final vitrification solution on
vitrification outcome
The concentrations of permeating cryoprotectants in VS2 and VS3 of the Original Protocol were reduced to (1.4 M glycerol
+ 1.6 M ethylene glycol) and (2.2 M glycerol + 2.8 M ethylene glycol), respectively, while VS1 remained as described – this is
referred to as “Modified Protocol 1”. In a second treatment group, sucrose was added to a final concentration of 0.5 M in VS3, while
VS1 and VS2 were retained as in the Modified Protocol 1–this is referred to as “Modified Protocol 2”.
The vitrification and warming procedures, together with the viability assessments in those two methods were otherwise the same
as for the Original Protocol.
2.5.3. Experiment 3: effect on the survival and developmental rate using different sugars in the warming solution
Embryos were vitrified using the Original Protocol, and then either warmed with the original warming protocol, or by using a
modified warming solution in which sucrose was replaced by the same concentration of galactose. This modified warming protocol
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Table 1
Summary of experimental design.
Experiment No
Experimental groups
Vitrification
Warming
1
Original Protocol
VS1 (1.4 M GLY) X 5 min;
VS2 (1.4 M GLY + 3.6 EG) X 5 min;
VS3 (3.4 M GLY + 4.6 M EG) X 45 s
Same as above
Same as above
N/A
WS1 (0.5 M Suc) X 1 min, 37C°; WS2 (0.25 M Suc) X 5 min, RT;
HM X 5 min, RT
AS with 8 μm
AS with 50 μm
Fresh Control
2
Original Protocol
Modified Protocol 1
Modified Protocol 2
3
Same as above
Same as above
N/A
Same as in Experiment 1
VS1 (1.4 M GLY) X 5 min;
VS2 (1.4 M GLY + 1.6 EG) X 5 min;
VS3 (2.2 M GLY + 2.5 M EG) X 45 s
VS1 (1.4 M GLY) X 5 min;
VS2 (1.4 M GLY + 1.6 EG) X 5 min;
VS3 (2.2 M GLY + 2.5 M EG + 0.5 M Suc) X 45 s
Original Protocol
Galactose Warming
Same as in Experiment 1
Same as in Experiment 1
Fresh Control
N/A
Same as in Experiment 1
Same as in Experiment 1
Same as in Experiment 1
Same as in Experiment 1
WS1 (0.5 M Gal) X 1 min, 37C°; WS2 (0.25 M Gal) X 5 min, RT;
HM X 5 min, RT
N/A
VS: vitrification solution; WS: warming solution; HM: holding medium; GLY: Glycerol; EG: Ethylene glycol; Suc: Sucrose; Gal: Galactose.
was defined as “Galactose Warming”.
As well as the standard viability assessments in this experiment, the physical size of each embryo was also measured at the start of
the procedure to compare survivability with size.
2.5.4. Experiment 4: pregnancy rate with embryo transfer
On the day of embryo transfer, vitrified embryos were warmed according to the various protocols in Experiments 1–3. All
embryos were transferred non- surgically in HS within 1 h after warming, by passing the embryo transfer gun through the cervix into
the uterus of the recipient camel on Day 5 or 6 after ovulation as previously described (Skidmore et al., 2004). Pregnancy was first
diagnosed by ultrasonic assessment of the uterus18 to 20 days after ovulation, and subsequently confirmed by detecting a fetus with a
heart-beat between days 27 and 32 of gestation.
Details of each procedure, including reagent concentrations, the timing of each step and temperature, are summarized in Table 1.
2.6. Statistical analysis
Using Statistical Analysis System (SAS) software (SAS Institute Inc, Cary, NC, USA), embryo re-expansion rate at 2, 48, 96 h and
pregnancy rate were analyzed using a chi-square test for independence. Level of significance was set P < 0.05.
3. Results
Superovulation procedures were used to treat 72 donor camels, and 347 Grade A and B embryos were selected and assigned to
different experimental treatments.
3.1. Effect of different physical AS methods on the survival and developmental rate of vitrified embryos
The embryos punctured either by micromanipulator (n = 39) or acupuncture needle (n = 28) started the process of shrinkage in
VS1, while the timing of shrinkage was delayed in non-punctured embryos (n = 45) with the initiation of shrinkage occurring in VS2.
There was, however, no difference in the amount of final shrinkage of embryos between the three groups in VS3 (Fig. 1).
The AS resulting from membrane puncturing resulted in a decrease in the survival and developmental potential of embryos
(micromanipulator; 51.2%) or (acupuncture needle; 46.4%) following vitrification, while the re-expansion rates of vitrified control
embryos was greater using the Original Protocol (80.0%) and was also greater for fresh embryos (82.6%). The embryo size appeared
to have an impact on the outcome of vitrification in the treatment groups, with larger embryos (300–850 μm; 64.0%) having a greater
developmental rate than that of smaller embryos (< 300 μm; 28.5%). This effect was, however, not observed in the original group,
where the development rate of smaller embryos (< 300 μm; 73.3%) was not different from the larger embryos (300–850 μm; 80.0%).
In addition, no difference was observed in the survival rate and developmental rate between the original groups (80.0%) and fresh
control (82.6%) during the first 48 h culture.
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Fig. 1. Image of fresh embryos in holding solution (a) or the shrinkage of non-punctured embryos when three vitrification solutions were used with the Original
Protocol (b).
3.2. Effect of reduction of permeating cryoprotectants concentrations or addition of sucrose in VS3 on the survival and developmental rate of
vitrified embryos
The survival and developmental rates of the vitrified embryos in the three groups after 2 h of culture (Table 3) were not significantly different from each other. The reduction of cryoprotectants concentration (Modified Protocol 1; n = 35; 51.4%) or the
addition of sucrose in VS3 (Modified Protocol 2; n = 25; 12.0%), however. reduced the survival and developmental rates at 48 h
culture when compared with the Original Protocol (n = 60; 80.0%; P < 0.05). Furthermore, addition of sucrose in VS3 with use of
the Modified Protocol 1 (12.0%) resulted in a lesser survival rate compared to the use of the Original (80.0%) and Modified 1 (51.4%)
Protocols at 48 h culture (P < 0.05; Table 3).
3.3. Effect of warming solution of different sugars on the survival and developmental rate of vitrified embryos
At 2 h of culture after warming, the survival and developmental rates of the vitrified embryos did not differ between the three
groups, either warmed with the sucrose (n = 56) or galactose (n = 35), or the fresh embryo group (n = 83; Table 4). At 48 h culture,
a lesser number of embryos survived in the groups treated with sucrose for warming (78.3%; P < 0.05) compared with the fresh
embryo control (87.5%), but there was no difference between control and galactose groups (85.7%). At 96 h culture, this trend of
differences between these two groups was even greater and the developmental rate of embryos in the group where there was warming
with sucrose (24.0%) was less than that of the group where there was warming with the galactose solution (71.4%; P < 0.05) and
fresh control group (85.7%; P < 0.05). The developmental rate of galactose-warmed embryos (71.4%) was less than those of the
fresh control (85.7%; P < 0.05).
3.4. Pregnancy rates after vitrified compared with fresh embryo transfer
Data for pregnancy rates after embryo transfers of vitrified and freshly hatched blastocysts are shown in Table 5. The pregnancy
rate with fresh embryos (65.0%) was the greatest among all groups, while the pregnancy rate of embryos vitrified with the Original
Protocol and warmed in galactose was the greatest of the treatment groups. Of the 21 embryos warmed in galactose solutions, five
embryos were transferred into five recipients and two of these were confirmed pregnant (2/5; 40.0%), while 16 embryos were
transferred in pairs into eight recipients and one single embryo pregnancy and three twin embryos pregnancies were diagnosed
(42.8% per embryo and 46.1% per recipient). After AS was performed with micromanipulators, one pregnancy was detected at day
Table 2
Effect of artificial shrinkage on the viability and in vitro development of vitrified embryos after 48 h in culture.
Method
Original Protocol
AS with micromanipulator (8 μm)
AS with acupuncture needle (50 μm)
Fresh Control
Developmental rate (%)
< 300 μm
300–850 μm
11/15 (73.3%)a
4/14 (28.5%)b
2/9 (22.2%)b
12/16 (75.0%)a
25/30
16/25
11/19
26/30
Values with different superscripts in the same column are different (P < 0.05).
5
(83.3%)a
(64.0%)b
(57.8%)b
(86.7%)a
Total
36/45
20/39
13/28
38/46
(80.0%)a
(51.2%)b
(46.4%)b
(82.6%)a
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Table 3
Effect of addition of sucrose to the vitrification solution on the viability and in vitro development of vitrified camel
embryos after 2 and 48 h of culture.
Method
Developmental rate (%)
2h
A
48 h
a
48/60 (80.0%)a
18/35 (51.4%)b
3/25 (12.0%)c
55/60 (91.6%)
30/35 (85.7%)a
24/25 (96.0%)a
Original Protocol
Modified Protocol 1
Modified Protocol 2
Values with different superscripts in the same column are different (P < 0.05).
A
Embryos (n = 45) were frozen in Experiment 1 (see Table 2 the Original Protocol) and 15 embryos were frozen
in Experiment 2 (n = 45 + 15 = 60).
Table 4
Effect of different sugars added to the warming solution on the viability and in vitro development of vitrified camel embryos after 2, 48 and 96 h in culture.
Method
Developmental rate (%)
2h
A
48 h
a
72/83 (86.7%)
31/35 (88.5%)a
51/56 (91.1%)a
Original Protocol
Galactose Warming
Fresh ControlB
96 h
a
65/83 (78.3%)
30/35 (85.7%)ab
49/56 (87.5%)b
20/83 (24.0%)a
25/35 (71.4%)b
48/56 (85.7%)c
Values with different superscripts in the same column are different (P < 0.05).
A
Embryos (n = 45) were frozen in Experiment 1 (see Table 2 the Original Protocol) and 15 were frozen Experiment 2 (see Table 3 the Original Protocol) and 23
embryos were frozen in Experiment 3.
B
Embryos (n = 46) were frozen in Experiment (see Table 2 the Fresh Control) 1 and 10 embryos were frozen in Experiment 3.
Table 5
Pregnancy rates after transfer of fresh and vitrified embryos.
Method
AS with micromanipulator
AS with acupuncture needle
Original Protocol
GalactoseA
Fresh Embryo (control)
No. of recipients
4
4
6
13
80
No. of embryos
8
8
12
21
80
Pregnancy rate (%)
Per embryo
Per recipient
1/8 (12.5%)a
0/8 (0)a
0/12 (0)a
9/21 (42.8%)b
52/80 (65.0%)c
1/4 (25%)a
0/4 (0)a
0/6 (0)a
6/13 (46.1%)b
52/80 (65.0%)c
Values with different superscripts in the same column are different (P < 0.05).
A
Grade A embryos (n = 21) were selected and transferred single into five and pairs into eight recipients. All other groups, embryos were transferred in pairs.
20, but it was confirmed as a trophoblast vesicle without a fetus at examination on day 27. This vesicle was detected for the next 5
weeks with twice weekly ultrasonic assessments, however, was not detected at day 70.
4. Discussion
The current research has demonstrated an effective vitrification protocol (using OPS) for hatched camel blastocysts, with a 46.1%
pregnancy rate. The results also confirmed that artificial shrinkage of blastocoels using either a mico-needle (8 μm) or an acupuncture
needle (50 μm) prior to vitrification is not beneficial to the survival rate or pregnancy outcome. Most importantly, the results of the
current study indicate that is a possible toxic effect of sucrose on camel embryos, which is consistent with finding in the previous
research where there were failed attempts to cryopreserve camelid embryos when sucrose was a component of either in the vitrification or warming solution (Herrid et al., 2016).
One of common strategies to reduce toxicity of permeating cryoprotectants to embryos/oocytes is to reduce the cryoprotectant
concentrations (Rail, 1987). Camel embryos were found to withstand much greater concentrations of permeating cryoprotectants
than many other species and this is similar to what has been previously observed for horse embryos which are also resistant to
damage with cryoprotectant treatments (Diaz et al., 2016). Camel emrbyos are, however, more resistant to cryoprotectant damage
than those of mice (Rail, 1987), pigs (Cuello et al., 2016) and humans (Fasano et al., 2014). With the protocol used in the present
study, the final concentration of GLY (3.4 M, ∼25%) and EG (4.6 M, ∼25%) is 8.0 M (∼50%). Except for horse embryos, however,
the final combined concentration of permeating cryoprotectants such as EG (16%) and DMSO (16%) in the vitrification solutions for
most mammals is generally less than 35% (Rail, 1987; Fasano et al., 2014; Cuello et al., 2016). The demonstration of an inverse
relationship between cryoprotectant permeability and embryo capsule thickness helps to explain the use of greater concentrations of
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cryoprotectants for horse embryo vitrification (Diaz et al., 2016) as compared with that used in most other species. Furthermore,
because hatched blastocysts of camels lack a zonae pellucidae, one would expect that these embryos would be much more sensitive to
osmolarity changes or toxicity of cryoprotectants compared with non-hatched embryos in other species (Rail, 1987; Fasano et al.,
2014; Cuello et al., 2016) The capacity of hatched embryos of camels to withstand greater concentrations of cryoprotectants may
indicate that the dehydration of embryos by changing osmolarity is less in camelid species at similar osmolarities than that for most
other species. Camel erythrocytes contain the greatest osmotically unresponsive (bound) water fraction (58%) compared with swine
(33%), cattle (32%), and human (26%) erythrocytes (Bogner et al., 1998). Similarly, camel embryos may contain a greater percentage of osmotically inactive cellular water, which would then require large osmolarity variations created by a greater concentration of cryoprotectants to replace the water. The replacement of intracellular water with permeating cryoprotectants is the only
way to prevent ice crystal formation during vitrification (Fasano et al., 2014; Herrid et al., 2016). Greater concentrations of cryoprotectants is, therefore, critical for successful vitrification of hatched blastocysts of camels. Indeed, the survival and developmental
rate decreased from 80.1% to 51.4% when the final concentrations of GLY and EG was reduced from 8.0 M to 4.7 M in the VS3 in
Experiment 2.
Another common strategy to reduce toxicity of permeating cryoprotectants is to add non-permeating cryoprotectants in the
vitrification solutions (Legrand et al., 2002). Considering the increased osmolarity of the vitrification solution by non-permeating
cryoprotectants, the final concentrations of GLY and EG in VS3 were reduced from 8.0 to 4.7 M whilst 0.5 M sucrose was added to the
final vitrification solution. The results of the present study indicate that the addition of sucrose decreased embryo survival and
developmental rates, indicating that there is a toxicity of sucrose to camel embryos. Because sucrose is widely used in human (Edgar
and Gook, 2012; Fasano et al., 2014), cattle (Cuello et al., 2016) and mice (Rail, 1987) embryo vitrification, it is proposed that there
is a species-specific toxicity of sucrose to camel embryos. The viability of two-cell rabbit embryos treated with trehalose was greater
than for those with sucrose treatments, but the same effect was not observed with one-cell stage embryos (Smorag et al., 1990). In
addition, the positive effect of trehalose on the freezing capacity of one-cell embryos of cattle was not demonstrated as there was no
difference in viability between embryos treated with trehalose or sucrose (Smorag et al., 1990). As a result, a species and cell-stage
dependent beneficial effect of trehalose on embryo cryopreservation was suggested. Similarly, no difference was found between
trehalose and sucrose in facilitating vitrification of pig oocytes (Aller et al., 2002). Taken together, these results confirm an existence
of species-specific toxicity of sucrose to camel embryos. This might have been a second major problem in establishing an effective
cryopreservation protocol for camelid species because the majority of the previous studies utilized sucrose in vitrification solutions
(Matsuo et al., 2013; Herrid et al., 2016).
To further investigate the hypothesis in the present study of a possible toxic effect of sucrose for camel embryos, while trying to
optimize the protocol, vitrified embryos were warmed in two different solutions, 0.5 M sucrose or 0.5 M galactose. While no differences were observed at 48 h, the survival and developmental rate of vitrified embryos warmed in galactose was greater than that of
the sucrose group at 96 h of culture. This indicated a slow and delayed toxic effect of sucrose. It has been reported that there is no
difference in the osmotic response of human embryos to sucrose and trehalose, but trehalose is more effective in stabilizing cell
membranes during dehydration compared with sucrose (Vanderzwalmen et al., 2002). Similarly, galactose might provide greater cell
membrane protection to embryos than sucrose. Most importantly, the detrimental effect of sucrose on embryo development becomes
more evident after long term culture (96 h). This result supports observations in previous studies of the outcome of embryo transfers
using embryos that were vitrified and warmed in sucrose solutions. Although these embryos were morphologically intact at warming
and re-expanded in culture for a short period of time, none of these embryos when transferred to recipients resulted in pregnancies
after embryo transfer (Herrid et al., 2016, 2017). Likewise, no pregnancies were obtained from embryos vitrified or warmed in
sucrose solutions in the current study, lending further evidence to a possible toxic effect of sucrose on camel embryos.
In contrast to previous reports that AS of blastocysts prior to vitrification improves survival rate and pregnancy outcome in
different species (Arav et al., 1993; Bollen et al., 2002; Hinrichs and Choi, 2016), the current study provided evidence that AS
treatment actually compromises the survival of vitrified embryos. There is a relationship with the size of embryos, where larger
embryos (300–850 μm) survived the treatment to a greater extent compared with the smaller embryos (< 300 μm). This may be due
to the difficulty of precisely controlling the needle to avoid damage to the inner cell mass during the procedure. After transfer, the
trophoblast development of a vitrified embryo (300–850 μm) from the AS by micromanipulator group may indicate that this type of
mechanical injury could happen to larger embryos as well. It is difficult to identify the inner cell mass in hatched camel blastocysts.
Amongst the different methods of AS, aspiration of blastocoel fluid appears to produce more desirable results compared with other
methods, such as micro-pipetting and puncture. In the present study, however, camel embryos were always fully collapsed when
punctured and then were placed in vitrification solution without any aspiration. Furthermore, at the majority of human IVF clinics,
there are desirable pregnancy rates when vitrified embryos are used without AS (Vanderzwalmen et al., 2002; Edgar and Gook,
2012).
5. Conclusions
Using an OPS vitrification protocol, the results of the current study demonstrated that 1) hatched camel blastocysts can withstand
a greater concentration (8.0 M, ∼50%) of cryoprotectants and can be successfully cryopreserved; 2) artificial shrinkage of hatched
embryos prior to vitrification does not improve the outcome of vitrification; and 3) the inclusion of sucrose in vitrification or warming
solution may have a toxic effect on camel embryos cryopreservation and subsequent development.
7
Animal Reproduction Science xxx (xxxx) xxx–xxx
M. Herrid et al.
Competing interests
None of the authors of this article have any conflict of interest.
Acknowledgments
Authors acknowledge the technical assistances of A Rehman. A Ahmed, and A. Billah in superovulation and embryo flushing, and
critical reading of the manuscript by Professor G Vajta and Dr. EG Crichton.
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