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Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos
Journal Pre-proof Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos Xiaolin Li, Liqin Wang, Chen Yin, Jiapeng Lin, Yangsheng Wu, Dayong Chen, Chunjuan Qiu, Bin Jia, Juncheng Huang, XiangJu Jiang, Lan Yang, Li Liu PII:
S0011-2240(19)30612-1
DOI:
https://doi.org/10.1016/j.cryobiol.2020.02.001
Reference:
YCRYO 4182
To appear in:
Cryobiology
Received Date: 29 November 2019 Revised Date:
2 February 2020
Accepted Date: 3 February 2020
Please cite this article as: X. Li, L. Wang, C. Yin, J. Lin, Y. Wu, D. Chen, C. Qiu, B. Jia, J. Huang, X. Jiang, L. Yang, L. Liu, Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos, Cryobiology (2020), doi: https://doi.org/10.1016/j.cryobiol.2020.02.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
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Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of
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vitrified in vitro sheep embryos
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Xiaolin Li1,2#, Liqin Wang2#, Chen Yin1,2#,Jiapeng Lin2, Yangsheng Wu2, Dayong
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Chen3, Chunjuan Qiu3, Bin Jia1*, Juncheng Huang2*, XiangJu Jiang4, Lan Yang1, Li Liu1
6 7 8
1
College of Animal Science and Technology, Shihezi University, Shihezi, 832003, China
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2
Key Laboratory of Genetics Breeding and Reproduction of Grass Feeding Livestock, Ministry of
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Agriculture and Rural affairs, P.R.China, Urumqi. 830000, China
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4 HouBo College of Xinjiang Medical University, Karamay, 834000, China
Inner Mongolia Sino Sheep Technology Co. Ulanchap，011800，China
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14
#
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Abbreviations: ApAFP914, The antifreeze protein from Anatolica polita; FBS, Fetal bovine serum;
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EG, ethylene glycol; DMSO, Dimethyl sulfoxide.
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* Corresponding author.
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E-mail addresses: [email protected] (X. L. Li), [email protected] (L. Q. Wang),
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[email protected] (Y. Chen), [email protected] (J. P. Lin), [email protected] (Y. S. Wu),
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[email protected](D.Y. Chen), [email protected](C. J. Qiu), h_ [email protected] (J. C.
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Huang), [email protected] (B. Jia), [email protected](X. J. Jiang).
These authors contributed equally to the present work
1
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[email protected](L. Yang), [email protected](L. Liu).
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AB STRACT
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Embryo cryopreservation is an important tool to preserve endangered species. As a
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cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914)
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has demonstrated utility. In the present study, the effects of controlled slow freezing and
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vitrification methods on the survival rate of sheep oocytes fertilized in vitro after
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freezing-thawing were compared. Different ApAFP914 concentrations were added to
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the vitrification liquid for exploring the effect of antifreeze protein on the warmed
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embryos. The results showed that the survival and hatching rates of in vitro derived
31
embryos were significantly higher than that of the slow freezing method. Furthermore,
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among the cryopreserved embryos at different developmental stages, the survival and
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hatching rates of the expanded blastocyst were significantly higher than those of the
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blastocysts, early blastocysts and morula. The survival and the hatching rates of the
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fast-growing embryos were both significantly higher than that of the slow-growing
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embryos. Additionally, treatment of ApAFP914 (5-30 µg/mL) did not increase the
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freezing efficiency of the 6-6.5 d embryos. However, addition of 10 µg/mL of
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ApAFP914 significantly increased the hatching rate of slow-growing embryos. In
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conclusion, our study suggests that the vitrification is better than the slow freezing
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method for the conservation of in vitro sheep embryos, and supplementation of
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ApAFP914 (10 µg/mL) significantly increased the hatching rate of slow-growing
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embryos after cryopreservation.
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Keywords: Embryo cryopreservation; Blastocyst; Vitrification; Controlled slow
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freezing; Antifreeze protein
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1. Introduction
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Cryopreservation of embryos is a safe and effective method of protecting the elite
47
germplasm of endangered animals [27]. In the process of embryo freezing, osmotic
48
shock, intracellular ice crystal formation, and cryoprotectant toxicity are the main
49
factors leading to embryonic cell damage [1,9]. During the freezing process,
50
minimizing the embryo damage is the key to improve the efficiency of embryo
51
cryopreservation. To date, there are mainly two cryopreservation methods, including
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conventional method, which consists in slow cooling, and vitrification, which consists
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in rapid cooling. Controlled slow freezing requires the use of a slow freezing apparatus,
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gradually cools the embryos, and produces a stable freezing effect with high viability.
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In contrast, vitrification requires a simple operation, short time, and low cost. There are
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a few reports on the application of these two methods in sheep embryos, especially with
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respect to the freezing time of in vitro embryo cryopreservation, the selection criteria of
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frozen in vitro embryos, and the use of antifreeze proteins. Controlled slow freezing
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and vitrification are commonly used for embryo cryopreservation. Therefore, the
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present study aimed to compare the effects of these two freezing methods on in vitro
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embryos, in order to explore a freezing method suitable for in vitro sheep embryos.
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Antifreeze protein (AFP) is a newly characterized cryoprotectant that exhibits lower
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cytotoxicity than other cryoprotectants [7]. This protein inhibits the formation of ice
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crystals during the freezing process and protects cell membranes from damage [21].
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Antifreeze proteins have been isolated from plants, insects, bacteria, and fungi.
5
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Although these proteins show similar functions, their amino acid sequences, protein
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structures and the mechanisms vary widely among the species. For example, insect
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antifreeze proteins bind to the ice crystal basal plane and avoid the formation of
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hexagonal pyramid ice crystals. Furthermore, the heat lag activity of insect AFP is
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generally 10-100 times than that of fish [14,22]. In general, the amino acids, which
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repeat inside the molecule are arranged spatially to form a β-helical structure. The TXT
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motif structure in the repeat sequence matches the facet and base surface of the ice
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crystal and can adsorb onto the surface of the ice crystal to inhibit further crystal growth
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[12,18,37].
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Anatolica polita is distributed throughout the Gurbantunggut Desert of China, which
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is the second-largest desert in northwestern China. A. polita maintains a low
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supercooling point by increasing the ratio of bound water to free water for balancing the
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ratio of antifreeze protein and glycerol, which increases their cold tolerance [41]. The A.
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polita antifreeze protein (ApAFP914) acts as a cryoprotectant that binds to ice crystals
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for preventing further growth, thereby reducing the physical damage to the cells. The
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inhibition of ice crystal formation helps the cells to maintain the stability of the
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membrane-bound organelles, mount a low-temperature reaction, and regulate the
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expression of their related genes [5,17,23,]. There have been several reports on the
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application of ApAFP914 in the cryoprotection of oocytes, although research on the
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low-temperature tolerance of in vitro derived embryos in the presence of ApAfp914 is
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lacking [30]. Therefore, the main objective of the present study was to compare the
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effects of different concentrations of ApAFP914 on cryopreservation of IVF sheep 6
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embryos.
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2 Materials and methods
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2.1. Preparation of recombinant ApAFP914
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The recombinant plasmid pET28a-ApAFP914 containing A. polita antifreeze protein
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gene, ApAFP914 (GenBank no. GU358704) was transformed into E. coli BL21 (DE3).
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The transformants expressed the fusion protein upon induction with 0.8 mM isopropyl
96
β-d-1-thiogalactopyranoside (IPTG). Analysis by SDS-PAGE indicated that the
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recombinant protein, ApAFP914 was highly expressed in the form of an inclusion body.
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The lysates were sonicated and then centrifuged. The supernatant was then washed with
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2% TritonX-100, 0.2% deoxycholic acid and 2 M urea, dissolved in 8 M urea and 2+
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purified by Ni
-NTA affinity chromatography in a refolding solution (20 mM
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Tris-HCl, 1 mM EDTA, 0.2 mM GSH, and 0.02 mM GSSG; pH 8.0) at 4℃. After the
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renaturation, the suspension was concentrated and purified by gel filtration
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chromatography using Superdex 75. High-purity His-ApAFP914 was obtained, and the
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molecular weight was about 20 kDa. Purified His-ApAFP914 was identified by
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SDS-PAGE and immunoblot analysis. The initial concentration of protein for the
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cryoprotection assay was 0.1 mg/mL.
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2.2. Oocyte collection and in vitro maturation (IVM)
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The ovaries used in this study were collected from ewes at a local abattoir and were
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prepared as described previously [39]. The cumulus-oocyte complexes (COCs) with
112
uniform cytoplasm and intact cumulus cells were selected for IVM according to the
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classification method by Lee et al. (2015) [17]. The oocyte recovery solution, IVM
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composition, and culture conditions were used according to Wang et al, (2012) [39].
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Briefly, After washing with IVM medium (bicarbonate-buffered TCM-199 culture
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medium supplemented with 10% FBS, 5 µg/mL porcine (p) FSH, 5 µg/mL LH, 1
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µg/mL 17b-oestradiol, 0.8 mM sodium pyruvate and 50 µg/ mL gentamicin) for three
118
times, groups of 15-20 COCs were cultured in 100-µL droplets of IVM medium
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overlaid with sterile mineral oil in 35-mm diameter Petri dishes for 24 h in 5% CO2
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with maximum humidity at 38.58°C.
121
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2.3. Mature oocytes in vitro fertilization (IVF)
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After 22-24 h of culture, cumulus cells were denuded by pipetting in H-199
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containing 0.1% (w/v) hyaluronidase (2IU/mL, Sigma-Aldrich, St. Louis, MO, USA)
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and 10% (v/v) FBS for 4–5 min, then washed three times with H-199 containing 10%
127
(v/v) FBS (Sigma-Aldrich, St. Louis, MO, USA). Sperm preparation was performed
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as described previously [4]. A total of 50 μL sperm suspension was added to 450 μ 8
129
L of the medium containing oocytes to make a final concentration of 1×106/ mL
130
spermatozoa. Gametes were co-incubated for 21 h. Sperm were removed by pipetting
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and oocytes were washed with embryo culture medium.
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2.4. In vitro embryo culture
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Presumptive zygotes were washed three times with synthetic oviductal fluid (SOF)
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and then cultured in 50 µL drops of SOF supplemented with 10% FBS for 72 h (10
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zygotes per well) and transferred into SOF supplemented with 10% serum and 1.5 mM
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glucose for 96 h. The embryos were incubated at 38.5℃with 5% CO2, and a humidified
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environment. At 48 h post-fertilization, the cleavage rate was counted, and single-cell
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embryos were discarded. The blastocyst development was counted on days 6-8
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post-fertilization and frozen.
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2.5. Controlled slow freezing and thawing
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The embryos were prepared for freezing by washing 2 times with freezing medium
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containing sucrose (0.4 M) (VIGRO, Canada, Vetoquinol N. A. Inc). Three to five
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embryos were loaded into the middle portion of each straw, and placed in the cooling
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chamber of the programmable freezer (CL8800i; Cryologic, Blackburn, Vic., Australia) 9
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for 10 min. Embryos were cooled from room temperature (RT) to 0℃ at a rate of 2℃
150
/min and then cooled from 0℃ to -7℃ at a rate of 2℃/min, held at -7℃ for 10 min
151
and then subjected to manual seeding. After seeding, straws were kept for 5 min at -7℃.
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Embryos were then cooled at a rate of 1℃/min to -30℃, held at -30℃ for 10 min,
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cooled at a rate of 0.5℃/min to -35℃maintained at -35℃ for 5 min and then immersed
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in liquid nitrogen.
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2.6. Thawing after slow freezing
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For warming, the straws were removed from the liquid nitrogen exposed to RT for 10
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s, and immersed in a 35℃ water bath for 10 s. The contents of the straws were poured
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into a 35-mm dish containing T20 and 0.5 M sucrose solution and then transferred into
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0.25 M sucrose. Subsequently, blastocysts were transferred to medium containing 0.15
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M sucrose for 5 min and finally transferred to T20 without sucrose for 10 min.
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Blastocysts with an intact zona pellucida that regained shape and had an uniform
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membrane after warming were considered normal and were transferred into in vitro
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culture (IVC) medium (SOF with 10% serum and 1.5 mM glucose). Hatching was
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assessed after 24 h culture in the IVC medium.
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2.7. Vitrification and warming
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The vitrification media used were shown in Table 1. A 3-step vitrification process was used (Table 2).
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The embryos were then thawed using a 2-step process. Thawing media used were as
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follows: T1: 0.5M sucrose in HM; T2: 0.25 M sucrose in HM. The whole process was
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conducted on a 38.6℃ heating table. The thawing media T1, T2 and HM were preheated
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in advance. A vitrified embryo was placed into a culture dish containing T1 and was
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gently agitated to facilitate rapid thawing. The embryos were incubated in T1 for 5 min,
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T2 for 5 min, and then washed with HM for 3 times, followed by 3 washes in balanced
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IVC solution (above processes were completed on a 38.6℃ heating table). The
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cultures were maintained for 48 h in the IVC droplets, in a humidified, 38.6℃, and 5%
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CO2 incubator.
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2.8. Experimental design
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Blastocysts were distributed into five experimental groups, with at least three
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replicates in each group. The experimental design was shown in Table 3. In
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experiments 4 and 5, a group of blastocysts was maintained for 8 days after IVC as a
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control to determine blastocyst and hatching rates.
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2.9. Statistical analysis 11
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Statistical analysis was done using Statistical Package for the Social Sciences (SPSS)
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ver. 19.0, and the results were expressed as mean ± standard deviation (SD). For data
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passing the homogeneity test of variance, an independent sample t-test or one-way
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analysis of variance (ANOVA) was used. A non-parametric Krukal-Wallish test was
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used when the variance homogeneity test was not satisfied. Differences were
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statistically significant at P < 0.05.
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3. Results
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3.1. Effects of freezing methods on the freezing efficiency of in vitro fertilized sheep
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embryos
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The expanded blastocysts of 6-6.5 d postfertilization were selected and
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cryopreserved by either vitrification or slow freezing method. The survival and
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post-development after thawing are presented in Table 4. The survival rate of the
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blastocysts after vitrification was 97.17%, which was significantly higher than that of
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the slow freezing method (72.47%; P<0.01). The hatching rate after 24 h of thawing
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was also significant (58.74%; P<0.01) compared to the slow freezing method
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(35.03%). 12
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3.2. Effect of in vitro embryo development on freezing efficiency
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Embryos at different developmental stages, such as blastocyst or expanded
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blastocyst stages, were frozen by vitrification. The data shown in Table 5 revealed that
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from morula, early blastocysts, blastocysts and expanded blastocysts, the survival and
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hatching rates of the frozen embryos gradually increased. The hatching rate of the
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expanded embryos was significantly higher than that of the other stages (P<0.01). The
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survival rate of the expanded embryos (96.57%) was significantly higher than that of
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the blastocyst 80.86% (P<0.05) and was also significantly higher than that of the
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morula and early blastocysts (P<0.01).
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3.3. Effect of in vitro embryo development speed on freezing effect
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The expanded embryos of 6-6.5 d fertilized eggs were classified as the
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fast-developing group. Those reaching the expanded embryo stage at 7-8 d after
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fertilization were classified as the slow-developing group. The two sets of expanded
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embryos were frozen by vitrification. The data are presented in Table 6. The survival
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rate of the embryos in the fast-developing group was significantly higher than that of
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the slow-developing group (P<0.05). The incubation rate after continued culture was 13
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59.37%, which was significantly higher than 25.28% of the slow-developing group
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(P<0.01).
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3.4. Effects of ApAFP914 on development of sheep embryos
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Expanded embryos of the fast-developing group were selected for the experiment.
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Final concentrations of 5-30 µg/mL of ApAFP914 were added to the vitrification
236
solutions (VS1 and VS2). The untreated group was used as control. The results showed
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that ApAFP914 had no effect on the freezing of the embryos in the fast-developing
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group after thawing (Table 7). However, thawing of the slow-developing embryos
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treated with 10 µg/mL of ApAFP914 significantly increased the hatching rate
240
compared to the control or other treatment groups (P<0.05). However, there was no
241
effect on the survival rate (Table 8).
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4. Discussion 14
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4.1. Vitrification and slow freezing of sheep embryos
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Conventional or slow freezing method is used to preserve the embryos of livestock to
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enhance production [35]. However, compared to the slow freezing method, vitrification
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is cheaper, simpler, faster, and most commonly used method of embryo
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cryopreservation [11,32].
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Rall and Fahy, (1985) reported the application of the cryopreservation method in
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embryo cryopreservation of rats and cattle [26,33]. Recently, the use of vitrified frozen
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sheep embryo technology has also been increased. Bettencourt et al. (2009) compared
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controlled slow freezing, conventional vitrification, and open pulled straw (OPS)
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vitrification methods for the preservation of Portuguese merino sheep embryos and
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observed no difference in embryo survival and pregnancy rates by either vitrification or
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slow freezing method [3]. In contrast, the survival rate of vitrified embryos (97.17%)
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was significantly higher than that of slow freezing (72.47%). Similarly, Balaban et al.
263
(2008) examined the effect of vitrification on human embryos [2]. Bhat et al. (2015)
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analyzed the effect of vitrification media in combination with a slow freezing protocol
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on sheep embryos [4]. The highest recovery and hatching rates of frozen OPS were
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92.2% and 65.8%, respectively in vitrification method, while the hatching rate was
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30% in the slow freezing method, which are similar to our results. This may be related
268
to the poor quality of in vitro embryos and sensitivity to freezing time. The slow 15
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freezing method requires approximately 2 hours of freezing time, and a large number of
270
ice crystals are easily formed during the freezing process, which results in damage to
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the internal structure of the embryo during the cooling and re-warming processes. On
272
the other hand, in the vitrification method, a few ice crystals formed, resulting in
273
increased embryo recovery rates. Therefore, the application of the vitrification method
274
can preserve in vitro sheep embryos to a greater extent and increase the success rate of
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frozen-thawed embryo transfer.
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4.2. Cryopreservation of in vitro fertilized embryos at different developmental stages
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The cold tolerance of an embryo depends on the species, cryopreservation method,
280
and the developmental stage of the embryo [20,28]. In livestock production, early
281
embryos and recipient cytoplasm exchange nutrients, and rejection rates are typically
282
lower when more developed embryos are used. As a result, fresh embryos are often
283
used for direct transplantation.
284
Currently, there are a few reports on the cryopreservation of embryos in vivo.
285
Westhusin et al. (1991) found that bovine nuclear transfer embryos were very sensitive
286
to low temperatures. The embryos used for freezing were typically in the morula,
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blastocyst, or expanded blastocyst stages. The embryos at different developmental
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stages had different appearances, cell numbers, and tolerance to freezing. In livestock
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production, in vivo fertilized embryos at the compacted morula and early blastocyst 16
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stages are generally used for cryopreservation. In the present study, the efficacy of the
291
vitrification method was analyzed using in vitro produced sheep embryos at different
292
developmental stages. The survival and hatching rates of morula or cleavage (16-32
293
cells) were significantly lower than those of early blastocysts, blastocysts and dilated
294
embryos (P<0.01). As the embryo developed in the developmental stages, the viability
295
after freezing highly improved. These data indicate that the expanded blastocyst is the
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most suitable stage for the vitrification of in vitro fertilized embryos. Previous studies
297
reported that embryos that develop to the blastocyst stage were resistant to freezing,
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and it was most convenient to expand blastocysts on day 7 [34,42]. Freezing of late
299
embryos highly improved the survival and hatching rates relative to early embryos. The
300
possible reason for this is that late and dilated blastocysts have more cells than morula
301
and blastocysts, which result in small inner cell mass fraction. Compared with small
302
cells, large cells are not easily dehydrated.
303
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4.3. Freezing effect on embryos
305
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Morphology is only one of the important criteria used for judging the quality of an
307
embryo. As the embryo develops, quality cannot be judged by the shape.
308
Developmental speed is one of the key factors in assessing embryo quality.
309
Mínguezalarcón et al. (2015) [24] reported that embryos exhibiting slow cleavage and
310
development rates were more likely to have abnormal chromosomes (chromosome 17
311
aneuploidy, chimerism, polyploid, etc.) than normal embryos. Embryos of reduced
312
quality often result in the stagnation of development, loss of implantation potential, and
313
high abortion rates after attachment. Fenwick et al. (2002) [8] observed that the embryo
314
blastocyst formation rate of the first cleavage on the first day after in vitro fertilization
315
of human embryos was significantly higher than that of non-early cleaved embryos. It
316
was reported that the ability of early cleaved embryos to develop into blastocysts after
317
fertilization was significantly higher than that of late cleavage embryos [35]. The
318
fertilization rate after embryo transfer within 24 h cleavage was significantly higher
319
than that of embryos with 27 h and 27-48 h cleavage (unpublished data). According to
320
Ruiz et al. (2011), blastomeres were divided into 2 groups at 3 d post IVF. Embryos
321
were classified as rapidly developing embryos if they had at least 6 cells [31]. Those
322
with less than 6 cells were considered to be slow-developing embryos. Pregnancy rates
323
when fast-developing embryos implanted were significantly higher than that of
324
embryos with less than 6 cells. It was reported that the embryogenic potential during the
325
2-cell phase was significantly lower than that of 4-cell embryos [45], which is
326
consistent with our results. In the present study, embryos at 6-6.5 d post IVF were
327
fast-developing. Embryos that developed to 7-8 d after fertilization were recorded as
328
slow-developing. Also, the frozen survival rate and late developmental ability of the
329
embryos with fast-development were significantly higher than those of the
330
slow-developing group.
331
18
332
4.4. Effect of adding ApAFP914 to embryo vitrification media
333
334
Previous studies have shown that the high sensitivity of embryonic cell membranes
335
and organelles to hypothermia is a major factor in decreasing embryo viability [19]. In
336
vitro fertilized embryos are sensitive to cryopreservation. The addition of
337
cryoprotectants can effectively improve the efficiency of in vitro derived embryos. Bhat
338
et al. (2015) reported that the re-expansion and hatching rates of embryos were higher
339
than those frozen in media supplemented with 33% ethylene glycol (EG) or 33%
340
dimethyl sulfoxide (DMSO) during the vitrification process [4]. The addition of
341
caffeine during in vitro maturation did not have any significant effect on the quality and
342
developmental capacity of embryos after vitrification [25]. The use of cryoprotective
343
agents is a crucial aspect of all cryopreservation protocols from slow freezing to
344
vitrification. AFPs can be suitable cryoprotectants to protect cells from injuries [29].
345
Antifreeze protein is highly present in Arctic fish, as it can reduce the freezing point
346
of the body fluids, change the ice crystal formation, and inhibit recrystallization to
347
protect the fish from freezing [43]. In this study, AFP had an effect on cryopreservation
348
of animal embryos. In mammalian embryos, AFP supplementation can improve cell
349
mass, embryonic development, oocyte survival and embryo cleavage rates in mice [16].
350
However, Lagneaux at al. (1997) reported that the addition of AFP to horse embryos at
351
4°C for cryopreservation had no difference between AFP and mixed glycerol
352
supplemented medium [15]. 19
353
The number of regularly spaced TXT motifs produced by A. polita can directly affect
354
the hysteresis activity of antifreeze proteins, which is positively correlated with the
355
degree of regularity of the TXT motif. To the best of our knowledge, this study for the
356
first time used ApAFP914 to preserve in vitro derived sheep embryos. The results
357
showed that the addition of 10 ug/mL ApAFP914 to the freezing media significantly
358
increased the hatching rate of slow-growing embryos after freezing. This may be
359
because ApAFP914 acts as a cryoprotectant that binds to ice crystals, which prevents
360
further formation of the crystals during intracellular freezing, and reduces the physical
361
damage to cellular membranes [6,10]. ApAFP914 directly interacts with the membrane
362
structure of the cell, which allows the oocyte to adapt to the low temperature by
363
maintaining the stability of the cell membrane structure. Also, AFP functions by
364
regulating the cellular expression of cryo-response genes. In this study, the in vitro
365
fertilized embryo freezing efficiency did not increase significantly with the increase of
366
ApAFP914 (30 µg/mL), although there was a slight tendency towards improved
367
responses.
368
369
5. Conclusions
370
371
The effect of the freezing process on cell morphology and metabolism still limits the
372
successful rate of cryopreservation, although there is an acceptable survival and
373
hatching rates of sheep embryos. The conventional methods of cryopreservation do not 20
374
protect the cells effectively and damage cells. Vitrification of the in vitro fertilized
375
embryos can protect the embryos, and increase the success rate of frozen-thawed
376
embryo transfer. Also, the embryonic developmental stage has a significant effect on
377
the fertility rate. The survival and hatching rates of sheep following the expansion of
378
blastocysts are significantly higher than the morula or cleavage (16-32 cells), early
379
blastocysts, and blastocysts. Therefore, expanded blastocysts appear to be the most
380
suitable for embryo vitrification. Developmental speed is one of the key factors in the
381
assessment of embryo quality. The expanded blastocysts at 6-6.5 d postfertilization
382
appeared to be the best time for cryopreservation. As a cryoprotectant, ApAFP914 can
383
increase the hatching rate, indicating that AFP can be used for sheep embryo
384
cryopreservation. However, high concentrations of AFP tend to reduce developmental
385
efficiency.
386
387
Funding sources
388
389
This work was supported by the National Natural Science Foundation of China
390
(Project number: 31660659 and 31860646) and Major science and technology projects
391
of Inner Mongolia autonomous region.
392
393
Acknowledgements 21
394
395
The authors wish to thank M. J. Liu for reviewing this English version of the
396
manuscript. Thanks to Dr X. F. Mao from Xinjiang University for providing
397
antifreeze protein.
398 399 400
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401
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transfer, Hum. Reprod. 13(1998):178-181
26
Table 1 Solution preparation of vitrification method for sheep embryo Media name
Media composition
HM VS1 VS2
TCM199 + 10% FBS 80% HM + 10% EG + 10% DMSO 50% HM + 20% EG + 20% DMSO + 10% 0.5 M sucrose
Table 2 The procedure of cryopreservation of embryo by vitrification NO
Detailed steps
1
Embryos were washed and exposed with HM for 10 min at room temperature Transfer 5-8 embryos to VS 1 and leave for 3 mins Transfer embryo from VS 1 to VS 2 then leave for 20 second Take embryos with 5uL volume of VS 2 then drop it onto LN Collected and placed in a cryotube for further storage after the frozen embryos sank to the bottom of the LN.
2 3 4 5
Table 3 Experimental design Experiment
No. embryos
1
288
2
334
3
218
4
670
5
318
Detailed treatment
Total 165 blastocysts were cryopreserved using controlled slow freezing and 123 blastocysts using the vitrification method Total 116 embryos in expanded period, 88 embryos in blastocyst, 86 embryos in early blastocyst and 44 embryos in morula or cleavage (16-32 cells) phase were cryopreserved Total 141 embryos with 6-6.5 days to develop blastocysts were cryopreserved and 77 embryos with 7-8 days to develop blastocysts were cryopreserved Total 68, 239, 103, 134 rapid development in vitro embryos were cryopreserved with different concentration of AFP914 (5, 10, 15, 30 µg/mL) Total 64, 72, 59, 63 slow development in vitro embryos were cryopreserved with different concentration of AFP914 (5, 10, 15, 30 µg/mL)
Table 4 Effects of freezing methods on freezing efficiency of sheep embryos in vitro. Freezing method
No. thawed embryos
No. surviving embryos
No. Hatched embryos
Survival （%）
rate
Vitrification Slow freezing
123 165
119 117
70 60
97.17±1.65A 72.47±3.24B
Hatching （%）
rate
58.74±5.70A 35.03±4.55B
Note: Different capital letters in the same column, the difference is extremely significant (p <0.01).
Survival rate = number of embryos recovered in the blastocyst cavity / number of thawed embryos * 100%; hatching rate = number of hatching embryos / number of thawed embryos * 100%. The same below.
Table 5 Effect of sheep in vitro embryo development on freezing efficiency (repeated 10 times) Embryonic developme nt Expanded embryo Blastocyst Early blastocyst Morula or cleavage (16-32 cells).
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate（%） Hatching rate（%）
116
112
63
96.57±2.14Aa
57.94±4.77A
88 86
74 61
19 10
80.86±5.66ABb 67.40±7.37Bc
20.32±5.74B 12.51±3.07B
44
6
0
12.96±6.68C
0B
Note: Different lowercase letters in the same column, the difference is significant (p <0.05), the same below.
Table 6 Effect of in vitro embryo development speed on freezing effect (repeated 4 times) Time of development to expansion blastocyst 6-6.5d 7-8d
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate （%）
Hatching （%）
rate
141 77
137 75
83 21
97.52±1.46a 89.39±3.00b
59.37±5.24A 25.28±4.63B
Table 7 Effect of ApAFP914 on the rapid development of in vitro embryos in sheep Concentratio No.thawed n（ µg/ mL） embryos
No. surviving embryos
No. hatching embryos
Survival rate （%）
Hatching rate （%）
Control 5 10 15 30
122 64 227 97 125
72 41 138 53 74
95.14±2.55 96.23±2.46 95.09±1.74 94.12±1.87 93.50±2.13
56.74±5.51 66.16±6.13 62.92±5.79 55.56±3.93 51.86±5.81
126 68 239 103 134
Table 8 Effect of ApAFP914 concentration on slower in vitro embryo freezing efficiency in sheep (repeated 3 times) Concentration （µg/ mL）
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate（%）
Hatching （%）
Control 5 10 15 30
60 64 72 59 63
48 51 60 48 54
11 12 24 5 5
77.37±3.32 78.66±1.92 84.95±5.55 82.43±2.12 85.62±2.89
22.95±5.95a 23.22±0.81a 35.63±7.59b 9.11±3.23a 7.95±1.62a
rate
Highlights Vitrification is superior to the slow freezing method for in vitro sheep embryos. The hatching and survival rate of the expanded embryos cryopreservation was optimal. ApAFP914 significantly increased the hatching rate of slow-growing embryos.
Declaration of interest All authors declared that they do not have any potential conflict of interest.
S0011-2240(19)30612-1
DOI:
https://doi.org/10.1016/j.cryobiol.2020.02.001
Reference:
YCRYO 4182
To appear in:
Cryobiology
Received Date: 29 November 2019 Revised Date:
2 February 2020
Accepted Date: 3 February 2020
Please cite this article as: X. Li, L. Wang, C. Yin, J. Lin, Y. Wu, D. Chen, C. Qiu, B. Jia, J. Huang, X. Jiang, L. Yang, L. Liu, Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos, Cryobiology (2020), doi: https://doi.org/10.1016/j.cryobiol.2020.02.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
1
Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of
2
vitrified in vitro sheep embryos
3 4
Xiaolin Li1,2#, Liqin Wang2#, Chen Yin1,2#,Jiapeng Lin2, Yangsheng Wu2, Dayong
5
Chen3, Chunjuan Qiu3, Bin Jia1*, Juncheng Huang2*, XiangJu Jiang4, Lan Yang1, Li Liu1
6 7 8
1
College of Animal Science and Technology, Shihezi University, Shihezi, 832003, China
9
2
Key Laboratory of Genetics Breeding and Reproduction of Grass Feeding Livestock, Ministry of
10
Agriculture and Rural affairs, P.R.China, Urumqi. 830000, China
11
3
12
4 HouBo College of Xinjiang Medical University, Karamay, 834000, China
Inner Mongolia Sino Sheep Technology Co. Ulanchap，011800，China
13
14
#
15
Abbreviations: ApAFP914, The antifreeze protein from Anatolica polita; FBS, Fetal bovine serum;
16
EG, ethylene glycol; DMSO, Dimethyl sulfoxide.
17
* Corresponding author.
18
E-mail addresses: [email protected] (X. L. Li), [email protected] (L. Q. Wang),
19
[email protected] (Y. Chen), [email protected] (J. P. Lin), [email protected] (Y. S. Wu),
20
[email protected](D.Y. Chen), [email protected](C. J. Qiu), h_ [email protected] (J. C.
21
Huang), [email protected] (B. Jia), [email protected](X. J. Jiang).
These authors contributed equally to the present work
1
22
[email protected](L. Yang), [email protected](L. Liu).
2
23
AB STRACT
24
Embryo cryopreservation is an important tool to preserve endangered species. As a
25
cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914)
26
has demonstrated utility. In the present study, the effects of controlled slow freezing and
27
vitrification methods on the survival rate of sheep oocytes fertilized in vitro after
28
freezing-thawing were compared. Different ApAFP914 concentrations were added to
29
the vitrification liquid for exploring the effect of antifreeze protein on the warmed
30
embryos. The results showed that the survival and hatching rates of in vitro derived
31
embryos were significantly higher than that of the slow freezing method. Furthermore,
32
among the cryopreserved embryos at different developmental stages, the survival and
33
hatching rates of the expanded blastocyst were significantly higher than those of the
34
blastocysts, early blastocysts and morula. The survival and the hatching rates of the
35
fast-growing embryos were both significantly higher than that of the slow-growing
36
embryos. Additionally, treatment of ApAFP914 (5-30 µg/mL) did not increase the
37
freezing efficiency of the 6-6.5 d embryos. However, addition of 10 µg/mL of
38
ApAFP914 significantly increased the hatching rate of slow-growing embryos. In
39
conclusion, our study suggests that the vitrification is better than the slow freezing
40
method for the conservation of in vitro sheep embryos, and supplementation of
41
ApAFP914 (10 µg/mL) significantly increased the hatching rate of slow-growing
42
embryos after cryopreservation.
43
Keywords: Embryo cryopreservation; Blastocyst; Vitrification; Controlled slow
3
44
freezing; Antifreeze protein
4
45
1. Introduction
46
Cryopreservation of embryos is a safe and effective method of protecting the elite
47
germplasm of endangered animals [27]. In the process of embryo freezing, osmotic
48
shock, intracellular ice crystal formation, and cryoprotectant toxicity are the main
49
factors leading to embryonic cell damage [1,9]. During the freezing process,
50
minimizing the embryo damage is the key to improve the efficiency of embryo
51
cryopreservation. To date, there are mainly two cryopreservation methods, including
52
conventional method, which consists in slow cooling, and vitrification, which consists
53
in rapid cooling. Controlled slow freezing requires the use of a slow freezing apparatus,
54
gradually cools the embryos, and produces a stable freezing effect with high viability.
55
In contrast, vitrification requires a simple operation, short time, and low cost. There are
56
a few reports on the application of these two methods in sheep embryos, especially with
57
respect to the freezing time of in vitro embryo cryopreservation, the selection criteria of
58
frozen in vitro embryos, and the use of antifreeze proteins. Controlled slow freezing
59
and vitrification are commonly used for embryo cryopreservation. Therefore, the
60
present study aimed to compare the effects of these two freezing methods on in vitro
61
embryos, in order to explore a freezing method suitable for in vitro sheep embryos.
62
Antifreeze protein (AFP) is a newly characterized cryoprotectant that exhibits lower
63
cytotoxicity than other cryoprotectants [7]. This protein inhibits the formation of ice
64
crystals during the freezing process and protects cell membranes from damage [21].
65
Antifreeze proteins have been isolated from plants, insects, bacteria, and fungi.
5
66
Although these proteins show similar functions, their amino acid sequences, protein
67
structures and the mechanisms vary widely among the species. For example, insect
68
antifreeze proteins bind to the ice crystal basal plane and avoid the formation of
69
hexagonal pyramid ice crystals. Furthermore, the heat lag activity of insect AFP is
70
generally 10-100 times than that of fish [14,22]. In general, the amino acids, which
71
repeat inside the molecule are arranged spatially to form a β-helical structure. The TXT
72
motif structure in the repeat sequence matches the facet and base surface of the ice
73
crystal and can adsorb onto the surface of the ice crystal to inhibit further crystal growth
74
[12,18,37].
75
Anatolica polita is distributed throughout the Gurbantunggut Desert of China, which
76
is the second-largest desert in northwestern China. A. polita maintains a low
77
supercooling point by increasing the ratio of bound water to free water for balancing the
78
ratio of antifreeze protein and glycerol, which increases their cold tolerance [41]. The A.
79
polita antifreeze protein (ApAFP914) acts as a cryoprotectant that binds to ice crystals
80
for preventing further growth, thereby reducing the physical damage to the cells. The
81
inhibition of ice crystal formation helps the cells to maintain the stability of the
82
membrane-bound organelles, mount a low-temperature reaction, and regulate the
83
expression of their related genes [5,17,23,]. There have been several reports on the
84
application of ApAFP914 in the cryoprotection of oocytes, although research on the
85
low-temperature tolerance of in vitro derived embryos in the presence of ApAfp914 is
86
lacking [30]. Therefore, the main objective of the present study was to compare the
87
effects of different concentrations of ApAFP914 on cryopreservation of IVF sheep 6
88
embryos.
89
2 Materials and methods
90
91
2.1. Preparation of recombinant ApAFP914
92
93
The recombinant plasmid pET28a-ApAFP914 containing A. polita antifreeze protein
94
gene, ApAFP914 (GenBank no. GU358704) was transformed into E. coli BL21 (DE3).
95
The transformants expressed the fusion protein upon induction with 0.8 mM isopropyl
96
β-d-1-thiogalactopyranoside (IPTG). Analysis by SDS-PAGE indicated that the
97
recombinant protein, ApAFP914 was highly expressed in the form of an inclusion body.
98
The lysates were sonicated and then centrifuged. The supernatant was then washed with
99
2% TritonX-100, 0.2% deoxycholic acid and 2 M urea, dissolved in 8 M urea and 2+
100
purified by Ni
-NTA affinity chromatography in a refolding solution (20 mM
101
Tris-HCl, 1 mM EDTA, 0.2 mM GSH, and 0.02 mM GSSG; pH 8.0) at 4℃. After the
102
renaturation, the suspension was concentrated and purified by gel filtration
103
chromatography using Superdex 75. High-purity His-ApAFP914 was obtained, and the
104
molecular weight was about 20 kDa. Purified His-ApAFP914 was identified by
105
SDS-PAGE and immunoblot analysis. The initial concentration of protein for the
106
cryoprotection assay was 0.1 mg/mL.
107
7
108
2.2. Oocyte collection and in vitro maturation (IVM)
109
110
The ovaries used in this study were collected from ewes at a local abattoir and were
111
prepared as described previously [39]. The cumulus-oocyte complexes (COCs) with
112
uniform cytoplasm and intact cumulus cells were selected for IVM according to the
113
classification method by Lee et al. (2015) [17]. The oocyte recovery solution, IVM
114
composition, and culture conditions were used according to Wang et al, (2012) [39].
115
Briefly, After washing with IVM medium (bicarbonate-buffered TCM-199 culture
116
medium supplemented with 10% FBS, 5 µg/mL porcine (p) FSH, 5 µg/mL LH, 1
117
µg/mL 17b-oestradiol, 0.8 mM sodium pyruvate and 50 µg/ mL gentamicin) for three
118
times, groups of 15-20 COCs were cultured in 100-µL droplets of IVM medium
119
overlaid with sterile mineral oil in 35-mm diameter Petri dishes for 24 h in 5% CO2
120
with maximum humidity at 38.58°C.
121
122
2.3. Mature oocytes in vitro fertilization (IVF)
123
124
After 22-24 h of culture, cumulus cells were denuded by pipetting in H-199
125
containing 0.1% (w/v) hyaluronidase (2IU/mL, Sigma-Aldrich, St. Louis, MO, USA)
126
and 10% (v/v) FBS for 4–5 min, then washed three times with H-199 containing 10%
127
(v/v) FBS (Sigma-Aldrich, St. Louis, MO, USA). Sperm preparation was performed
128
as described previously [4]. A total of 50 μL sperm suspension was added to 450 μ 8
129
L of the medium containing oocytes to make a final concentration of 1×106/ mL
130
spermatozoa. Gametes were co-incubated for 21 h. Sperm were removed by pipetting
131
and oocytes were washed with embryo culture medium.
132
133
2.4. In vitro embryo culture
134
135
Presumptive zygotes were washed three times with synthetic oviductal fluid (SOF)
136
and then cultured in 50 µL drops of SOF supplemented with 10% FBS for 72 h (10
137
zygotes per well) and transferred into SOF supplemented with 10% serum and 1.5 mM
138
glucose for 96 h. The embryos were incubated at 38.5℃with 5% CO2, and a humidified
139
environment. At 48 h post-fertilization, the cleavage rate was counted, and single-cell
140
embryos were discarded. The blastocyst development was counted on days 6-8
141
post-fertilization and frozen.
142
143
2.5. Controlled slow freezing and thawing
144
145
The embryos were prepared for freezing by washing 2 times with freezing medium
146
containing sucrose (0.4 M) (VIGRO, Canada, Vetoquinol N. A. Inc). Three to five
147
embryos were loaded into the middle portion of each straw, and placed in the cooling
148
chamber of the programmable freezer (CL8800i; Cryologic, Blackburn, Vic., Australia) 9
149
for 10 min. Embryos were cooled from room temperature (RT) to 0℃ at a rate of 2℃
150
/min and then cooled from 0℃ to -7℃ at a rate of 2℃/min, held at -7℃ for 10 min
151
and then subjected to manual seeding. After seeding, straws were kept for 5 min at -7℃.
152
Embryos were then cooled at a rate of 1℃/min to -30℃, held at -30℃ for 10 min,
153
cooled at a rate of 0.5℃/min to -35℃maintained at -35℃ for 5 min and then immersed
154
in liquid nitrogen.
155
156
2.6. Thawing after slow freezing
157
For warming, the straws were removed from the liquid nitrogen exposed to RT for 10
158
s, and immersed in a 35℃ water bath for 10 s. The contents of the straws were poured
159
into a 35-mm dish containing T20 and 0.5 M sucrose solution and then transferred into
160
0.25 M sucrose. Subsequently, blastocysts were transferred to medium containing 0.15
161
M sucrose for 5 min and finally transferred to T20 without sucrose for 10 min.
162
Blastocysts with an intact zona pellucida that regained shape and had an uniform
163
membrane after warming were considered normal and were transferred into in vitro
164
culture (IVC) medium (SOF with 10% serum and 1.5 mM glucose). Hatching was
165
assessed after 24 h culture in the IVC medium.
166
167
2.7. Vitrification and warming
168
10
169 170
The vitrification media used were shown in Table 1. A 3-step vitrification process was used (Table 2).
171
The embryos were then thawed using a 2-step process. Thawing media used were as
172
follows: T1: 0.5M sucrose in HM; T2: 0.25 M sucrose in HM. The whole process was
173
conducted on a 38.6℃ heating table. The thawing media T1, T2 and HM were preheated
174
in advance. A vitrified embryo was placed into a culture dish containing T1 and was
175
gently agitated to facilitate rapid thawing. The embryos were incubated in T1 for 5 min,
176
T2 for 5 min, and then washed with HM for 3 times, followed by 3 washes in balanced
177
IVC solution (above processes were completed on a 38.6℃ heating table). The
178
cultures were maintained for 48 h in the IVC droplets, in a humidified, 38.6℃, and 5%
179
CO2 incubator.
180
181
2.8. Experimental design
182
183
Blastocysts were distributed into five experimental groups, with at least three
184
replicates in each group. The experimental design was shown in Table 3. In
185
experiments 4 and 5, a group of blastocysts was maintained for 8 days after IVC as a
186
control to determine blastocyst and hatching rates.
187
188
2.9. Statistical analysis 11
189
190
Statistical analysis was done using Statistical Package for the Social Sciences (SPSS)
191
ver. 19.0, and the results were expressed as mean ± standard deviation (SD). For data
192
passing the homogeneity test of variance, an independent sample t-test or one-way
193
analysis of variance (ANOVA) was used. A non-parametric Krukal-Wallish test was
194
used when the variance homogeneity test was not satisfied. Differences were
195
statistically significant at P < 0.05.
196
197
3. Results
198
199
3.1. Effects of freezing methods on the freezing efficiency of in vitro fertilized sheep
200
embryos
201
202
The expanded blastocysts of 6-6.5 d postfertilization were selected and
203
cryopreserved by either vitrification or slow freezing method. The survival and
204
post-development after thawing are presented in Table 4. The survival rate of the
205
blastocysts after vitrification was 97.17%, which was significantly higher than that of
206
the slow freezing method (72.47%; P<0.01). The hatching rate after 24 h of thawing
207
was also significant (58.74%; P<0.01) compared to the slow freezing method
208
(35.03%). 12
209
210
3.2. Effect of in vitro embryo development on freezing efficiency
211
212
Embryos at different developmental stages, such as blastocyst or expanded
213
blastocyst stages, were frozen by vitrification. The data shown in Table 5 revealed that
214
from morula, early blastocysts, blastocysts and expanded blastocysts, the survival and
215
hatching rates of the frozen embryos gradually increased. The hatching rate of the
216
expanded embryos was significantly higher than that of the other stages (P<0.01). The
217
survival rate of the expanded embryos (96.57%) was significantly higher than that of
218
the blastocyst 80.86% (P<0.05) and was also significantly higher than that of the
219
morula and early blastocysts (P<0.01).
220
221
3.3. Effect of in vitro embryo development speed on freezing effect
222
223
The expanded embryos of 6-6.5 d fertilized eggs were classified as the
224
fast-developing group. Those reaching the expanded embryo stage at 7-8 d after
225
fertilization were classified as the slow-developing group. The two sets of expanded
226
embryos were frozen by vitrification. The data are presented in Table 6. The survival
227
rate of the embryos in the fast-developing group was significantly higher than that of
228
the slow-developing group (P<0.05). The incubation rate after continued culture was 13
229
59.37%, which was significantly higher than 25.28% of the slow-developing group
230
(P<0.01).
231
232
3.4. Effects of ApAFP914 on development of sheep embryos
233
234
Expanded embryos of the fast-developing group were selected for the experiment.
235
Final concentrations of 5-30 µg/mL of ApAFP914 were added to the vitrification
236
solutions (VS1 and VS2). The untreated group was used as control. The results showed
237
that ApAFP914 had no effect on the freezing of the embryos in the fast-developing
238
group after thawing (Table 7). However, thawing of the slow-developing embryos
239
treated with 10 µg/mL of ApAFP914 significantly increased the hatching rate
240
compared to the control or other treatment groups (P<0.05). However, there was no
241
effect on the survival rate (Table 8).
242
243
244
245
246
247
4. Discussion 14
248
249
4.1. Vitrification and slow freezing of sheep embryos
250
251
Conventional or slow freezing method is used to preserve the embryos of livestock to
252
enhance production [35]. However, compared to the slow freezing method, vitrification
253
is cheaper, simpler, faster, and most commonly used method of embryo
254
cryopreservation [11,32].
255
Rall and Fahy, (1985) reported the application of the cryopreservation method in
256
embryo cryopreservation of rats and cattle [26,33]. Recently, the use of vitrified frozen
257
sheep embryo technology has also been increased. Bettencourt et al. (2009) compared
258
controlled slow freezing, conventional vitrification, and open pulled straw (OPS)
259
vitrification methods for the preservation of Portuguese merino sheep embryos and
260
observed no difference in embryo survival and pregnancy rates by either vitrification or
261
slow freezing method [3]. In contrast, the survival rate of vitrified embryos (97.17%)
262
was significantly higher than that of slow freezing (72.47%). Similarly, Balaban et al.
263
(2008) examined the effect of vitrification on human embryos [2]. Bhat et al. (2015)
264
analyzed the effect of vitrification media in combination with a slow freezing protocol
265
on sheep embryos [4]. The highest recovery and hatching rates of frozen OPS were
266
92.2% and 65.8%, respectively in vitrification method, while the hatching rate was
267
30% in the slow freezing method, which are similar to our results. This may be related
268
to the poor quality of in vitro embryos and sensitivity to freezing time. The slow 15
269
freezing method requires approximately 2 hours of freezing time, and a large number of
270
ice crystals are easily formed during the freezing process, which results in damage to
271
the internal structure of the embryo during the cooling and re-warming processes. On
272
the other hand, in the vitrification method, a few ice crystals formed, resulting in
273
increased embryo recovery rates. Therefore, the application of the vitrification method
274
can preserve in vitro sheep embryos to a greater extent and increase the success rate of
275
frozen-thawed embryo transfer.
276
277
4.2. Cryopreservation of in vitro fertilized embryos at different developmental stages
278
279
The cold tolerance of an embryo depends on the species, cryopreservation method,
280
and the developmental stage of the embryo [20,28]. In livestock production, early
281
embryos and recipient cytoplasm exchange nutrients, and rejection rates are typically
282
lower when more developed embryos are used. As a result, fresh embryos are often
283
used for direct transplantation.
284
Currently, there are a few reports on the cryopreservation of embryos in vivo.
285
Westhusin et al. (1991) found that bovine nuclear transfer embryos were very sensitive
286
to low temperatures. The embryos used for freezing were typically in the morula,
287
blastocyst, or expanded blastocyst stages. The embryos at different developmental
288
stages had different appearances, cell numbers, and tolerance to freezing. In livestock
289
production, in vivo fertilized embryos at the compacted morula and early blastocyst 16
290
stages are generally used for cryopreservation. In the present study, the efficacy of the
291
vitrification method was analyzed using in vitro produced sheep embryos at different
292
developmental stages. The survival and hatching rates of morula or cleavage (16-32
293
cells) were significantly lower than those of early blastocysts, blastocysts and dilated
294
embryos (P<0.01). As the embryo developed in the developmental stages, the viability
295
after freezing highly improved. These data indicate that the expanded blastocyst is the
296
most suitable stage for the vitrification of in vitro fertilized embryos. Previous studies
297
reported that embryos that develop to the blastocyst stage were resistant to freezing,
298
and it was most convenient to expand blastocysts on day 7 [34,42]. Freezing of late
299
embryos highly improved the survival and hatching rates relative to early embryos. The
300
possible reason for this is that late and dilated blastocysts have more cells than morula
301
and blastocysts, which result in small inner cell mass fraction. Compared with small
302
cells, large cells are not easily dehydrated.
303
304
4.3. Freezing effect on embryos
305
306
Morphology is only one of the important criteria used for judging the quality of an
307
embryo. As the embryo develops, quality cannot be judged by the shape.
308
Developmental speed is one of the key factors in assessing embryo quality.
309
Mínguezalarcón et al. (2015) [24] reported that embryos exhibiting slow cleavage and
310
development rates were more likely to have abnormal chromosomes (chromosome 17
311
aneuploidy, chimerism, polyploid, etc.) than normal embryos. Embryos of reduced
312
quality often result in the stagnation of development, loss of implantation potential, and
313
high abortion rates after attachment. Fenwick et al. (2002) [8] observed that the embryo
314
blastocyst formation rate of the first cleavage on the first day after in vitro fertilization
315
of human embryos was significantly higher than that of non-early cleaved embryos. It
316
was reported that the ability of early cleaved embryos to develop into blastocysts after
317
fertilization was significantly higher than that of late cleavage embryos [35]. The
318
fertilization rate after embryo transfer within 24 h cleavage was significantly higher
319
than that of embryos with 27 h and 27-48 h cleavage (unpublished data). According to
320
Ruiz et al. (2011), blastomeres were divided into 2 groups at 3 d post IVF. Embryos
321
were classified as rapidly developing embryos if they had at least 6 cells [31]. Those
322
with less than 6 cells were considered to be slow-developing embryos. Pregnancy rates
323
when fast-developing embryos implanted were significantly higher than that of
324
embryos with less than 6 cells. It was reported that the embryogenic potential during the
325
2-cell phase was significantly lower than that of 4-cell embryos [45], which is
326
consistent with our results. In the present study, embryos at 6-6.5 d post IVF were
327
fast-developing. Embryos that developed to 7-8 d after fertilization were recorded as
328
slow-developing. Also, the frozen survival rate and late developmental ability of the
329
embryos with fast-development were significantly higher than those of the
330
slow-developing group.
331
18
332
4.4. Effect of adding ApAFP914 to embryo vitrification media
333
334
Previous studies have shown that the high sensitivity of embryonic cell membranes
335
and organelles to hypothermia is a major factor in decreasing embryo viability [19]. In
336
vitro fertilized embryos are sensitive to cryopreservation. The addition of
337
cryoprotectants can effectively improve the efficiency of in vitro derived embryos. Bhat
338
et al. (2015) reported that the re-expansion and hatching rates of embryos were higher
339
than those frozen in media supplemented with 33% ethylene glycol (EG) or 33%
340
dimethyl sulfoxide (DMSO) during the vitrification process [4]. The addition of
341
caffeine during in vitro maturation did not have any significant effect on the quality and
342
developmental capacity of embryos after vitrification [25]. The use of cryoprotective
343
agents is a crucial aspect of all cryopreservation protocols from slow freezing to
344
vitrification. AFPs can be suitable cryoprotectants to protect cells from injuries [29].
345
Antifreeze protein is highly present in Arctic fish, as it can reduce the freezing point
346
of the body fluids, change the ice crystal formation, and inhibit recrystallization to
347
protect the fish from freezing [43]. In this study, AFP had an effect on cryopreservation
348
of animal embryos. In mammalian embryos, AFP supplementation can improve cell
349
mass, embryonic development, oocyte survival and embryo cleavage rates in mice [16].
350
However, Lagneaux at al. (1997) reported that the addition of AFP to horse embryos at
351
4°C for cryopreservation had no difference between AFP and mixed glycerol
352
supplemented medium [15]. 19
353
The number of regularly spaced TXT motifs produced by A. polita can directly affect
354
the hysteresis activity of antifreeze proteins, which is positively correlated with the
355
degree of regularity of the TXT motif. To the best of our knowledge, this study for the
356
first time used ApAFP914 to preserve in vitro derived sheep embryos. The results
357
showed that the addition of 10 ug/mL ApAFP914 to the freezing media significantly
358
increased the hatching rate of slow-growing embryos after freezing. This may be
359
because ApAFP914 acts as a cryoprotectant that binds to ice crystals, which prevents
360
further formation of the crystals during intracellular freezing, and reduces the physical
361
damage to cellular membranes [6,10]. ApAFP914 directly interacts with the membrane
362
structure of the cell, which allows the oocyte to adapt to the low temperature by
363
maintaining the stability of the cell membrane structure. Also, AFP functions by
364
regulating the cellular expression of cryo-response genes. In this study, the in vitro
365
fertilized embryo freezing efficiency did not increase significantly with the increase of
366
ApAFP914 (30 µg/mL), although there was a slight tendency towards improved
367
responses.
368
369
5. Conclusions
370
371
The effect of the freezing process on cell morphology and metabolism still limits the
372
successful rate of cryopreservation, although there is an acceptable survival and
373
hatching rates of sheep embryos. The conventional methods of cryopreservation do not 20
374
protect the cells effectively and damage cells. Vitrification of the in vitro fertilized
375
embryos can protect the embryos, and increase the success rate of frozen-thawed
376
embryo transfer. Also, the embryonic developmental stage has a significant effect on
377
the fertility rate. The survival and hatching rates of sheep following the expansion of
378
blastocysts are significantly higher than the morula or cleavage (16-32 cells), early
379
blastocysts, and blastocysts. Therefore, expanded blastocysts appear to be the most
380
suitable for embryo vitrification. Developmental speed is one of the key factors in the
381
assessment of embryo quality. The expanded blastocysts at 6-6.5 d postfertilization
382
appeared to be the best time for cryopreservation. As a cryoprotectant, ApAFP914 can
383
increase the hatching rate, indicating that AFP can be used for sheep embryo
384
cryopreservation. However, high concentrations of AFP tend to reduce developmental
385
efficiency.
386
387
Funding sources
388
389
This work was supported by the National Natural Science Foundation of China
390
(Project number: 31660659 and 31860646) and Major science and technology projects
391
of Inner Mongolia autonomous region.
392
393
Acknowledgements 21
394
395
The authors wish to thank M. J. Liu for reviewing this English version of the
396
manuscript. Thanks to Dr X. F. Mao from Xinjiang University for providing
397
antifreeze protein.
398 399 400
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Table 1 Solution preparation of vitrification method for sheep embryo Media name
Media composition
HM VS1 VS2
TCM199 + 10% FBS 80% HM + 10% EG + 10% DMSO 50% HM + 20% EG + 20% DMSO + 10% 0.5 M sucrose
Table 2 The procedure of cryopreservation of embryo by vitrification NO
Detailed steps
1
Embryos were washed and exposed with HM for 10 min at room temperature Transfer 5-8 embryos to VS 1 and leave for 3 mins Transfer embryo from VS 1 to VS 2 then leave for 20 second Take embryos with 5uL volume of VS 2 then drop it onto LN Collected and placed in a cryotube for further storage after the frozen embryos sank to the bottom of the LN.
2 3 4 5
Table 3 Experimental design Experiment
No. embryos
1
288
2
334
3
218
4
670
5
318
Detailed treatment
Total 165 blastocysts were cryopreserved using controlled slow freezing and 123 blastocysts using the vitrification method Total 116 embryos in expanded period, 88 embryos in blastocyst, 86 embryos in early blastocyst and 44 embryos in morula or cleavage (16-32 cells) phase were cryopreserved Total 141 embryos with 6-6.5 days to develop blastocysts were cryopreserved and 77 embryos with 7-8 days to develop blastocysts were cryopreserved Total 68, 239, 103, 134 rapid development in vitro embryos were cryopreserved with different concentration of AFP914 (5, 10, 15, 30 µg/mL) Total 64, 72, 59, 63 slow development in vitro embryos were cryopreserved with different concentration of AFP914 (5, 10, 15, 30 µg/mL)
Table 4 Effects of freezing methods on freezing efficiency of sheep embryos in vitro. Freezing method
No. thawed embryos
No. surviving embryos
No. Hatched embryos
Survival （%）
rate
Vitrification Slow freezing
123 165
119 117
70 60
97.17±1.65A 72.47±3.24B
Hatching （%）
rate
58.74±5.70A 35.03±4.55B
Note: Different capital letters in the same column, the difference is extremely significant (p <0.01).
Survival rate = number of embryos recovered in the blastocyst cavity / number of thawed embryos * 100%; hatching rate = number of hatching embryos / number of thawed embryos * 100%. The same below.
Table 5 Effect of sheep in vitro embryo development on freezing efficiency (repeated 10 times) Embryonic developme nt Expanded embryo Blastocyst Early blastocyst Morula or cleavage (16-32 cells).
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate（%） Hatching rate（%）
116
112
63
96.57±2.14Aa
57.94±4.77A
88 86
74 61
19 10
80.86±5.66ABb 67.40±7.37Bc
20.32±5.74B 12.51±3.07B
44
6
0
12.96±6.68C
0B
Note: Different lowercase letters in the same column, the difference is significant (p <0.05), the same below.
Table 6 Effect of in vitro embryo development speed on freezing effect (repeated 4 times) Time of development to expansion blastocyst 6-6.5d 7-8d
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate （%）
Hatching （%）
rate
141 77
137 75
83 21
97.52±1.46a 89.39±3.00b
59.37±5.24A 25.28±4.63B
Table 7 Effect of ApAFP914 on the rapid development of in vitro embryos in sheep Concentratio No.thawed n（ µg/ mL） embryos
No. surviving embryos
No. hatching embryos
Survival rate （%）
Hatching rate （%）
Control 5 10 15 30
122 64 227 97 125
72 41 138 53 74
95.14±2.55 96.23±2.46 95.09±1.74 94.12±1.87 93.50±2.13
56.74±5.51 66.16±6.13 62.92±5.79 55.56±3.93 51.86±5.81
126 68 239 103 134
Table 8 Effect of ApAFP914 concentration on slower in vitro embryo freezing efficiency in sheep (repeated 3 times) Concentration （µg/ mL）
No. thawed embryos
No. surviving embryos
No. hatching embryos
Survival rate（%）
Hatching （%）
Control 5 10 15 30
60 64 72 59 63
48 51 60 48 54
11 12 24 5 5
77.37±3.32 78.66±1.92 84.95±5.55 82.43±2.12 85.62±2.89
22.95±5.95a 23.22±0.81a 35.63±7.59b 9.11±3.23a 7.95±1.62a
rate
Highlights Vitrification is superior to the slow freezing method for in vitro sheep embryos. The hatching and survival rate of the expanded embryos cryopreservation was optimal. ApAFP914 significantly increased the hatching rate of slow-growing embryos.
Declaration of interest All authors declared that they do not have any potential conflict of interest.