In vitro development and post-thaw survival

of blastocysts derived from delipidated

zygotes from domestic cats

Ni Wayan Kurniani Karja a, Takeshige Otoi a,*,Pimprapar Wongsrikeao a, Masako Murakami a,Budiyanto Agung a, Mokhamad Fahrudin a,

Takashi Nagai b

aLaboratory of Animal Reproduction, Department of Veterinary Sciences,

Yamaguchi University, Yamaguchi 753-8515, JapanbDepartment of Research Planning and Coordination, National Institute of

Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan

Received 6 March 2005; accepted 10 April 2005

Abstract

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the

survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline

zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of

cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated

zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of

delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or

control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing

and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were

not significantly different between the both groups. In conclusion, in vitro-produced feline blas-

tocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not

impair their in vitro developmental competence (up to the blastocyst stage), and reduction of

www.journals.elsevierhealth.com/periodicals/the

Theriogenology 65 (2006) 415–423

* Corresponding author. Tel. +81 83 933 5904; fax: +81 83 933 5904.

E-mail address: otoi@yamaguchi-u.ac.jp (T. Otoi).

0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2005.04.029

cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts

after cryopreservation.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Cryopreservation; Intracellular lipids; Delipidated embryo; Blastocyst; Cat

1. Introduction

Cryopreservation of mammalian embryos has important implications for the long-term

storage of embryos, propagation and transport of valuable genotypes of agricultural and

zoological interest, and also for in vitro fertilization (IVF) programs. The ability to

cryopreserve embryos of domestic cats produced in vitro represents a potential method of

retaining valuable genetic material of endangered felids [1]. The domestic cat may be a

useful model for developing assisted reproduction techniques for application to threatened

and endangered species of non-domestic cats [2].

In vitro-fertilized embryos from domestic cats have been successfully produced using

either in vitro- [3,4] or in vivo-matured oocytes [2,5]. The increasing availability of

embryos from domestic cats offers new opportunities for cryopreservation. However, in

vitro-produced (IVP) bovine embryos were considered to be much more sensitive to

freezing than their in vivo-produced counterparts [6,7]. This high sensitivity of in vitro-

derived embryos to cryopreservation may be related to morphological, cellular, metabolic,

and physiological differences between the two categories of embryos [7–9]. In vitro-

produced embryos had a darker cytoplasm and an increased lipid content [7,10], well

known to result in lower cryotolerance [6–8]. Recently, several studies demonstrated that

removal of intracellular lipids increased the tolerance to cryopreservation of in vitro-

derived porcine [11,12] and bovine embryos [13,14]. Lipid vesicles within the cytoplasm

have a close spatial arrangement with the smooth endoplasmic reticulum within the

embryo [15]; they help provide nutrition to the cell, as well as modifying the physical

properties and functions of the cellular plasma membranes [16]. Therefore, removal of the

cytoplasmic lipids may eliminate potential cytoplasmic elements and disrupt subcellular

localization of organelles [11], such as mitochondria.

In earlier studies of feline embryo cryopreservation, the first birth of live kittens after

embryo transfer of cryopreserved embryos was reported with in vivo-derived embryos at

the late morula or blastocyst stages [17]. Subsequently, cryopreservation of feline embryos

produced by IVF of in vivo-matured oocytes at the 2- to 4-cell stage was demonstrated by

Pope et al. [18]. Furthermore, feline embryos produced by IVF of in vitro-matured oocytes

have been successfully frozen at the 8- to 16-cell stage (Days 2–3) and at the morula stage

(Days 4–5) of IVC [1,19]. Furthermore, kittens have been produced after transfer of both

morula- and blastocyst-stages embryos previously frozen at premorula or morula stages

[1,20]. To date, however, there have been no reports concerning the cryopreservation of

IVP-derived feline blastocysts after removal of intracellular lipids. It has been reported that

one distinct feature of feline oocytes is a uniformly dark appearance of the oocytes and

embryos, indicating a high concentration of intracellular lipids [21]. Therefore, it was

hypothesized that removal of intracellular lipids from feline embryos should promote post-

N.W.K. Karja et al. / Theriogenology 65 (2006) 415–423416

thaw survival. The objectives of the present study were to examine the developmental

ability of delipidated feline zygotes produced in vitro, and to investigate the in vitro

survival of IVP blastocysts obtained after freezing and thawing.

2. Materials and methods

2.1. Recovery and culture of oocytes

Feline oocytes were matured and fertilized according to procedures previously

described by Karja et al. [22]. In brief, ovaries were obtained from local veterinary clinics

following routine ovariohysterectomy. Ovaries were kept in physiological saline at 35 8Cbefore oocyte recovery. Each ovary was sliced repeatedly with a scalpel blade to release

cumulus-oocyte complexes (COCs) in a 90-mm culture dish containing modified-PBS

(mPBS; Embryotech, Nihon Zenyaku kogyo, Japan). Only COCs exhibiting uniform,

darkly-pigmented ooplasm and an intact cumulus cell investment were used for further

culture. The COCs were cultured for 24 h in a 100-mL drop of maturation medium,

consisting of tissue culture medium (TCM) 199 with Earle’s salts (Gibco, Grand Island,

NY, USA), supplemented with 0.4% bovine serum albumin (BSA; Sigma, St. Louis, MO,

USA), 0.1 IU/mL human menopausal gonadotropin (Teikokuzoki, Tokyo, Japan), 10 IU/

mL human chorionic gonadotropin (Teikokuzoki), 1 mg/mL 17b-estradiol (Sigma), and

50 mg/mL gentamicin (Sigma). All cultures were performed at 38 8C in a humidified

incubator containing 5% CO2 in air.

2.2. Sperm collection and cryopreservation for IVF

Testes were collected from adult male cats following castration at local veterinary

clinics. They were kept in physiological saline and maintained at room temperature before

sperm collection. The epididymis was removed from the testes and sliced repeatedly with a

scalpel blade to release spermatozoa in a 90-mm culture dish containing m-PBS at 37 8C.The released spermatozoa were washed in m-PBS by centrifugation at 500 � g for 5 min.

After removal of the supernatant, the suspension (about 500 mL) of spermatozoa was

cryopreserved according to the method described by Pursel and Johnson [23], with minor

modifications. Before cryopreservation, a small amount of sperm suspension was subjected

to analysis of motility immediately after centrifugation. Only semen samples with >50%

progressively motile spermatozoa were used for cryopreservation. Then, the suspension of

spermatozoa was diluted at room temperature with 450 mL of the first extender, that

consisted of 8.8% (w/v) lactose (Wako Pure Chemical, Osaka, Japan), 200 mg/mL

ampicillin (Mitaka, Tokyo, Japan) and 20% (v:v) egg yolk in distilled water. The diluted

spermatozoa were equilibrated in a water bath at 4 8C for 2 h. After equilibration, 250 mL

of the second extender, i.e. the first extender supplemented with 6% (v:v) glycerol (Wako)

and 1.48% (v:v) orvus ES paste (Miyazaki Kagaku, Tokyo, Japan), which had been cooled

to 4 8C, was added. The spermatozoawere then equilibrated at 4 8C for an additional 5 min.

At the end of the equilibration period, the same volume (250 mL) of the second extender

was added at 4 8C. The spermatozoa were immediately loaded into a 0.25 mL French straw

N.W.K. Karja et al. / Theriogenology 65 (2006) 415–423 417

(I.V.M., France) and frozen by placing the straw on a styrofoam plate in liquid nitrogen

vapor for 20 min (4 cm above the surface of liquid nitrogen), and subsequently stored in

liquid nitrogen. On the day of insemination, the straw was placed in air for 5 s, and then

submerged into a 30 8Cwater bath for 30 s. After thawing, only semen samples with>50%

progressivelymotile spermatozoawere used for IVF. The frozen-thawed spermatozoa from

the same donors were used for IVF in this experiment, to exclude effects of a donor

difference.

2.3. In vitro fertilization

Frozen-thawed spermatozoa were washed twice in Brackett-Oliphant medium (BO

medium) [24] supplemented with 137 mg/mL sodium pyruvate and 50 mg/mL gentamicin

by centrifugation at 500 � g for 5 min. The supernatant was removed and the sperm pellet

was diluted in 500 mL of the BO medium. The sperm concentration was adjusted to

4 � 106 spermatozoa/mL in the BO medium, and further diluted with additional BO

medium supplemented with 0.6% BSA and 20 mg/mL heparin (Novo Industry A/S, Osaka,

Japan) to a final concentration of 2 � 106 spermatozoa/mL. After 24 h of in vitro culture,

oocytes were transferred separately into 100 mL of the sperm microdrops (each with 3–5

COCs) for fertilization and co-incubated for 12 h. After the co-incubation with

spermatozoa, cumulus cells surrounding putative zygotes were removed mechanically

with a small-bore pipette. Denuded zygotes were equally allocated into three groups, and

then they were cultured inMK-1 medium [25] supplemented with 0.4% BSA (BSA-MK-1).

2.4. Removal of cytoplasmic lipid droplets and embryo culture

We applied previously described methods used for porcine embryos to polarize

intracellular lipids by centrifugation [11,12,26]. Briefly, putative zygotes cultured for 20 h

after insemination were centrifuged at 15,000 rpm for 15 min in Dulbecco’s phosphate-

buffered saline (PBS; Gibco) supplemented by 0.3% BSA and 5 mg/mL cytochalasin B

(Sigma) using a high-speed, refrigerated centrifuge (MR-150: Tomy Seiko, Tokyo, Japan).

The intracellular lipid layer was then removed by micromanipulation using a bevelled

suction pipette (30 mm in diameter). Sham-operated putative embryos were centrifuged,

and then the zona pellucida of the zygote was penetrated by the aspiration pipette, but not

delipidated. During lipid removal and sham-operated treatments, the embryos were held in

the same medium used for centrifugation. After lipid removal or sham operation, the

putative embryos were continuously cultured in BSA-MK-1. As the control group, intact

putative zygotes were directly cultured in BSA-MK-1 after insemination. At 72 h of

culture, all cleaved embryos were transferred to fresh MK-1 medium supplemented with

5% FBS (FBS-MK-1) for an additional 3 days to evaluate their ability to develop to

blastocysts.

2.5. Freezing and thawing of embryos

Briefly, blastocysts obtained at Day 6 of culture were washed three times in PBS

supplemented with 0.3% BSA (BSA-PBS) and then suspended in the cryopreservation

N.W.K. Karja et al. / Theriogenology 65 (2006) 415–423418

medium at room temperature. The cryopreservation medium consisted of 10% (v:v)

ethylene glycol (Wako), 5% (v:v) polyvinylpyrrolidone (PVP) (Denka, Tokyo, Japan) and

0.05 M trehalose (Wako) in BSA-PBS. Embryos were placed in the cryopreservation

medium and loaded immediately into 0.25 mL straws (I.V.M.), with a maximum of six

embryos/straw. The straws were then placed horizontally into the cooling chamber of an

alcohol-freezer (ET-1, Fujihira Industry Co., Tokyo, Japan) and cooled from 0 to�7 8C at a

rate of 1 8C/min. Straws were seeded at�7 8C, held at that temperature for 15 min, cooled

to�30 8C at a rate of 0.3 8C/min, and finally plunged into liquid nitrogen. After storage in

liquid nitrogen for 3–4 weeks, thawing of frozen embryos was done by holding the straws

in air for 5 s and then in a 30 8C water bath until the ice in the straws disappeared. The

content of each straw was expelled into a 35-mm culture dish. The embryos were washed

twice in BSA-PBS and incubated for 5 min. The embryos were then washed twice and

cultured in FBS-MK-1. The morphology of embryos was evaluated microscopically at 24-

h intervals up to 72 h. Survival rates were assessed by re-expansion of the blastocoelic

cavity after 24 h of post-thaw culture.

2.6. Statistical analysis

The percentages of embryo cleaved and developed to the morula or blastocyst stages

were subjected to arc sine transformation before analysis, and then were tested by Scheffe’s

F-test. The survival rates of blastocysts after thawing was analyzed by Chi Square test.

Differences at a probability P � 0.05 were considered significant.

3. Results

Intracellular lipids of 150 putative zygotes were removed by micromanipulation for

assessment of the developmental competence of feline zygotes after delipidation (Table 1).

More than 67% of the delipidated zygotes cleaved and 36% of them developed to the

morula stage. Microscopic pictures clearly showed the difference in cytoplasmic darkness

at morula stage between control, sham-operated, and delipidated embryos (Fig. 1). The rate

of the delipidated zygotes that developed to the blastocyst stage (26%) was similar to that

of sham-operated (30.5%) or control embryos (31.3%). The development to the hatching

N.W.K. Karja et al. / Theriogenology 65 (2006) 415–423 419

Table 1

Developmental competence of delipidated IVP cat embryosa

Group No. putative

zygotes examined

No. (%) of

cleaved embryos

No. (%)b (%)c of embryos that developed to

Morula Blastocyst Hatching

blastocysts

Delipidated 150 101 (67.3) 54 (36.0) (53.5) 39 (26.0) (38.6) 23 (15.3) (22.8)

Sham 131 95 (72.5) 57 (43.5) (60.0) 40 (30.5) (42.1) 11 (8.4) (11.6)

Control 131 90 (68.7) 54 (41.2) (60.0) 41 (31.3) (45.6) 12 (9.2) (13.3)a Eight or nine replicate trials were conducted.b Percentage of developed embryos as a proportion of putative zygotes examined.c Percentage of developed embryos as a proportion of cleaved embryos.

blastocyst stage tended to increase in the delipidated group compared with sham-operated

and control groups. However, the rates of development to the blastocyst and hatching

blastocyst stages of the delipidated zygotes did not differ among groups (P > 0.05).

The in vitro survival rates of blastocysts derived from delipidated zygotes after freezing

and thawing were investigated (Table 2). Although the in vitro survival rate of delipidated

blastocysts (81.8%) after freezing and thawingwas slightly higher than that of sham-operated

(74.2%) or control embryos (60.6%), rates did not differ among the groups (P > 0.05).

4. Discussion

This study is the first to report that feline IVP blastocysts can be successfully preserved

and that delipidated zygotes can develop into the blastocyst stage with a similar

development rate as control or sham-operated embryos. Therefore, removal of the

cytoplasmic lipid content after centrifugation in feline zygotes did not impair in vitro

developmental competence (to the blastocyst stage). Similar studies in other species

demonstrated that porcine [11] and bovine embryos [27] retained their potential for

development in vitro into the blastocyst stage after lipid removal by centrifugation and

extraction. Although it is known that cytoplasmic lipid droplets in embryos are a source of

energy and contain metabolic products and structural elements [13], it appears that the

cytoplasmic lipid droplets are not essential for the development of embryos at early

cleavage stages [13] or that the embryos are able to synthesize additional lipids [28].

Removal of intracellular lipids from embryos prior to cryopreservation increased the

tolerance of embryos to low temperatures in pigs [11] and cattle [13,29]. However, in the

present study, the survival rate of blastocysts derived from delipidated zygotes following

N.W.K. Karja et al. / Theriogenology 65 (2006) 415–423420

Fig. 1. In vitro fertilized feline embryos at morula stage: intact control embryos (a), sham operated embryos (b)

and delipidated embryos (c) (magnification 100�).

Table 2

Survival rates of IVP cat blastocysts after freezing and thawing

Group No. embryos

examined

No. (%) of

embryos survived

No. (%) frozen-thawed blastocysts that

developed to hatching blastocysts

Delipidated 33 27 (81.8) 4 (12.1)

Sham 31 23 (74.2) 7 (22.6)

Control 33 20 (60.6) 2 (6.1)

freezing and thawing was slightly but not significantly higher than that of sham-operated or

control embryos. Our results indicated that the presence of the intracellular lipid in feline

embryos may have no direct effect on embryo survival during cryopreservation. Moreover,

previous studies in domestic cats demonstrated that 90% of IVF-derived feline embryos

cryopreseved at early cleavage stages resumed development in vitro and the frequency of

blastocyst development was not different from that of non-frozen controls [1,2]. Therefore,

the role of intracellular lipids on the freezing tolerance in embryos from domestic cats may

be different from that of other two species.

The choice of cryoprotective medium used to suspend the embryosmay have affected the

in vitro survival of embryos after freezing and thawing. The present study demonstrated that

feline blastocysts could be successfully frozen using ethylene glycol. In a previous study,

Swanson et al. [30] demonstrated that cryopreservation of early cleavage stages (2- to 8-cell)

IVP-derived feline embryos with ethylene glycol resulted in superior post-thaw embryo

survival, compared to other cryoprotectants (e.g. propylene glycol and glycerol). Although

we did not compare ethylene glycol with other cryoprotectants in the present study, 60–80%

ofblastocysts survived after freezing in ethyleneglycol. In the present study, a combination of

ethylene glycol, PVP, and trehalose appeared to have beneficial effects on the invitro survival

of feline blastocysts after freezing and thawing. Ethylene glycol can readily diffuse out of

embryonic cells without causing gross cellular damage [31]. When ethylene glycol entered

the cells, trehalose concurrently helped to dehydrate the embryonic cell, a process that is very

important for successful vitrification [32]. Trehalose also facilitated the removal of

intracellular cryoprotectives during dilution and may have reduced toxicity by causing

embryos to shrink rapidly [33] and maintaining a high osmotic pressure in the extracellular

medium. On the other hand, PVP, a polymeric solute, prevented the seeding of supercooled

water inside the cells or coated sensitive membranes to prevent denaturation by a strong salt

solution [34]. All of those protective activities might have contributed to achievement of a

high rate of cryosurvival of the frozen-thawed IVF feline blastocysts in this study.

We expected that the rate of cryo-induced injuries would be higher in the delipidated

embryos, since a small incision was made in the zona pellucida of each embryo during lipid

extraction. However, survival rate of frozen-thawed of blastocysts was not detrimentally

affected by either an incision in the zona pellucida (sham-operated) or removal of

intracellular lipids (delipidated). Therefore, our results indicated that removal of

cytoplasmic lipids had neither a negative nor a positive effect on the post-thaw in vitro

viability of feline blastocysts.

In conclusion: (1) IVP feline blastocysts can be cryopreserved; (2) the removal of the

cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental

competence up to the blastocyst stage; and (3) the reduction of cytoplasmic lipids by

aspiration had no apparent effects on the survival of in vitro-derived feline blastocysts after

cryopreservation.

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