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Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts
Cryobiology xxx (2015) xxx–xxx
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Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts q Alper Kocyigit a,⇑, Mesut Cevik b a b
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Cumhuriyet University, Sivas, Turkey Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey
a r t i c l e
i n f o
Article history: Received 14 April 2015 Revised 25 May 2015 Accepted 26 May 2015 Available online xxxx Keywords: Bovine blastocyst Growth factor Cytokine Vitrification Cryotolerance
a b s t r a c t The present study examined the developmental capacity and cryotolerance of cultured bovine embryos in defined media (synthetic oviduct fluid, SOF) supplemented with insulin-like growth factor I (IGF-I) and leukemia inhibitor factor (LIF). The objectives of the present study were: (1) to examine the effects IGF-I and LIF on bovine embryo development potential and (2) to investigate the cryotolerance and survivability of vitrified blastocysts obtained from embryos cultured in a defined media. We studied the development of bovine embryos produced in vitro and cultured (in four different treatments) until Day 7 after fertilization. In Experiment 1, zygotes were cultured to the blastocyst stage and differentially stained for determine the count of cells. In Experiment 2, zygotes were vitrified before staining. LIF alone or combined with IGF-I was significantly effective on in vitro bovine embryo development especially ratio to reach blastocyst. The cells for both ICM and TE decreased by the effect of freezing in all treatment groups in the Experiment 2 compared with Experiment 1. Interestingly, the LIF treatment showed fewest variations. In addition to this, for average number of ICM and TE cells, LIF treatment showed fewest variation compared with other treatments (ICM: 23.5 vs 19.5, TE: 53.6 vs 51). These results are the first to demonstrate that the addition of IGF-I along with LIF to the culture medium was found to be beneficial for bovine embryonic development based on cellular cryotolerance after vitrification. Ó 2015 Elsevier Inc. All rights reserved.
Introduction The improvement of in vitro culture systems are important for production of embryos with high developmental competence that are used in embryo transfer programs and biomedical researches [17]. Although with the advancements of culture procedures, in vitro produced (IVP) embryos it usually shows low quality and viability than developed in vivo counterparts. Besides, IVP embryos are more sensitive to cryoinjury than in vivo-derived embryos, such as shown by lower cell numbers, less compaction and a lower number of tight-junctions compared to embryos in vivo [11,37,38,42]. Tight-junction formation is a prerequisite for blastocoel formation, expansion of the blastocyst and final differentiation of two distinct cell populations: the trophectoderm (TE) and the inner cell mass (ICM). It is well known that the ratio and allocation of these cells is considered to be a potential indicator on embryo
q Statement of funding: This work was funded by the Research Foundation of Ondokuz Mayis University, Samsun/TURKEY. This article was produced by the same name PhD thesis study. Project ID: PYO.VET.1904.11.011. ⇑ Corresponding author. E-mail address: [email protected] (A. Kocyigit).
quality and crucial for implantation [40,44]. Therefore, developing an optimal culture system is essential to improve quality of embryos produced in vitro. There are several ways to improve blastocysts quality and cryotolerance during in vitro culture. One way to improve blastocysts quality is supplementation of culture media by growth factors and cytokines. Insulin-like growth factor I (IGF-I) have been reported to be mitogens inducing a positive effect on development of embryos [41]. Moreover, growth factors are involved in the compaction and formation of the blastocyst, activation of transport systems responsible for the uptake of glucose, inhibition of apoptosis, enhancement of endocytosis, probably protein turnover [14,27]. One of the cytokines is leukemia inhibitory factor (LIF), a pleiotropic member of the interleukin-6 (IL-6) family. Leukemia inhibitory factor receptors (LIFR) have been detected in embryos of different species that is considered essential for blastocyst development such as hatching rate, cell count and cryotolerance [2,4,16,26,45]. LIF was originally implicated in regulating the early development of the embryo and promoted the development of bovine embryos and increased the viability and cryotolerance of bovine blastocysts. Cryotolerance may be a useful indicator of blastocyst quality [31,36]. As a functional criterion for the evaluation of blastocysts
http://dx.doi.org/10.1016/j.cryobiol.2015.05.068 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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cryotolerance after vitrification, a post-thaw re-expansion capacity and blastocysts cell allocation have been used [24]. Numerous studies, with contrary views, reported that IGF-I combination with cytokines and added to SOF medium has improved development and cryotolerance of bovine blastocysts [16,32,39]. The objectives of the present study were: (1) to examine the effects IGF-I and LIF on bovine embryo development potential and (2) to investigate the cryotolerance and survivability of vitrified blastocysts obtained from embryos cultured in a defined media.
(10 ll/ml), penicillin (100 U/ml) and streptomycin (100 lg/ml) on the day of use a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 °C. In the current study, LIF was added to the culture medium at Day 4 post insemination (p.i.), while IGF-1 was supplemented in the culture medium at Day 0 p.i (fertilization day). A concentration of 100 ng/ml was unprompted selected for addition to culture media following our study of previous reports.
Materials and methods
In Experiment 1, effect of IGF-I and LIF on the bovine embryo development competence rate studied. Presumptive zygotes were cultured in four different treatments (six replicates). Growth factor and cytokine added to the culture medium were as follows; treatment I: SOF alone, treatment II: SOF plus 100 ng/ml LIF, treatment III: SOF plus 100 ng/ml IGF-I, treatment IV: SOF plus 100 ng/ml IGF-I and 100 ng/ml LIF. The growth factor and cytokine used in our study were human recombinants. Zygotes were cultured to the blastocyst stage and assessed for morphology approximately 168 h (day 7) post insemination, immediately prior to differential staining.
All chemicals used in this study were purchased from Sigma– Aldrich (Istanbul, TURKEY), except where otherwise indicated. Collection and in vitro maturation of oocytes Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory at approximately 34 ± 2.0 °C in physiological saline solution supplemented with gentamycin sulfate (0.1 ll/ml). Cumulus oocyte complexes (COCs) were recovered from follicles 2–8 mm in diameter by aspiration. The COCs were collected in 3–4 ml Hepes-buffered Medium-199 containing Earle’s salts and supplemented with 1% v/v antibiotic–antimycotic solution. Before in vitro maturation, the COCs were assessed morphologically and only oocytes with compact, non-atretic cumulus investment and evenly granulated cytoplasm were selected for maturation. Maturation medium was sodium bicarbonate-buffered Medium-199 with sodium pyruvate (5.5 lg/ml), antibiotic–antimycotic solution (1% v/v), heat-inactivated fetal calf serum (FCS, 10% v/v). The COCs were matured for 22 h in a humidified atmosphere of 5% CO2 in air at 38.5 °C. Spermatozoa preparation and in vitro fertilization After a 22 h maturation period, oocytes were transferred into 44 ll fertilization drops. The fertilization medium was glucose-free modified Tyrode’s albumin lactate pyruvate (TALP) supplemented with bicarbonate (25 mM), Na-lactate (22 mM), Na-pyruvate (1 mM), fatty acid-free bovine serum albumin (BSA) (6 mg/ml), and heparin-sodium salt (184 units/mg heparin, 10 mg/ml) and antibiotic–antimycotic solution (0.5 ll/ml) (pH 7.4 and 280–300 mOsm/kg). Frozen–thawed semen was used for the fertilization of oocytes. Percoll density gradient system was used for the separation of the motile fraction of the frozen–thawed semen [34]. Sperm were then diluted to 50 106 spermatozoon/ml in TL-HEPES, including the 2 106 spermatozoa/ml as final concentration. The fertilization procedure was completed by adding 2 ll of diluted sperm, 2 ll heparin (5 lg/ml) and 2 ll of PHE solution (20 lM penicillamine, 10 lM hypotaurine, and 1 lM epinephrine in final concentration) into the fertilization drops containing oocytes. The oocytes were fertilized with 2 ll diluted semen per fertilization drops for 22 h in a humidified atmosphere of 5% CO2 in air at 38.5 °C. In vitro culture Cumulus cells surrounding the oocytes were removed from presumptive zygotes at approximately 22 h post-insemination by vortexing 3 min. The zygotes were transferred in groups of 20–30 for the culture droplets. Embryos were cultured in the synthetic oviduct fluid (SOF) medium supplemented with pyruvate (0.4 mM), fatty acid free (BSA-FAF) (8 mg/ml), 100 minimum essential medium (MEM) (20 ll/ml), 50 basal medium eagle (BME)
Experiment 1
Experiment 2 This part of the study, the effect of IGF-I and LIF on the subsequent development of bovine blastocysts after freezing and thawing was examined. Presumptive zygotes maturated and fertilized using standard procedures were cultured in four treatments as in Experiment 1 (four replicates). Early blastocysts were harvested on day 6 or day 7 of in vitro culture. After two initial washes in SOF, blastocysts were placed in a 10% glycerol solution for 1 min. Embryos then transferred to 20% ethylene glycol (EG) + 10% glycerol solution for 1 min. Finally embryos then transferred to %25 EG + %25 glycerol for 25 s. The blastocysts were immediately drawn by capillary action into sterile straws, along with a maximum of 1–2 ml medium. Blastocysts were vitrified using the simple method of directly submerging embryos into liquid nitrogen after brief exposure to a cryoprotectant solution. In the thawing procedure (one day after cryopreservation), straws were thawed in a water bath at 35 °C for 15 s, and their contents were released into 0.5 M sucrose solution. Blastocysts were recovered and equilibrated in respectively 0.5 M and 0.25 M sucrose for 5 min, then washed once in SOF medium containing BSA-FAF (8 mg/ml) and cultured in SOF drops at 24 h. The blastocysts were considered viable if they re-expanded or hatched from the zona pellucidae. Determination of the numbers of ICM and TE cells Finally expanded blastocysts from the non-vitrified in Experiment 1 and vitrified in Experiment 2 to determine the differential count of cells in the ICM and TE. Expanding blastocysts characterized by zona pellucidae showing signs of thinning and with slightly increased embryo diameters by day 7 or 8 was stained as described by Van Soom et al. [40]. Briefly, zona intact blastocysts were incubated with 10 mM picrylsulphonic acid (Sigma, in cold Ca2+-free PBS) for 5 min in the refrigerator (4 °C). Then they were washed and incubated for 30 min at 39 °C in anti-dinitrophenyl antibody (Sigma) diluted to 30% (v/v) with Ca+2 free PBS. Embryos were then transferred into guinea pig complement (55852, ICN biochemicals, Irvine, CA, USA) diluted to 20% (v/v) in Ca2+-free PBS containing 50 lg/ml propidium iodide, 12.5 lg/ml bisbenzimide, and 50 lg/ml RNAse a for 30 min at 39 °C. Finally, embryos were fixed in 2% paraformaldehyde in PBS for 1–2 min at room temperature. Before mounting on slides with a 10 ll drop of 0.2 M 1,4 diazabicyclo-octane in 50% glycerol (v/v) in Ca2+-free
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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PBS as an anti-fading solution. Embryos were examined in whole mount under a fluorescence microscope (Nikon Invert Microscope Eclipse Ti-FL, 340–380 nm excitations and 430 nm suppression). Thus, ICM nuclei labeled with bisbenzimide appeared blue and TE nuclei labeled with both bisbenzimide and propidium iodide appeared pink to red. Numbers of ICM and TE nuclei were counted directly under the fluorescence microscope.
and embryos consisted average of 104.9 cells in total, with 31 cells in the ICM and 73.8 cells in the TE. In treatment III, 53 embryos counted and embryos consisted average of 77 cells in total with 23.5 cells in the ICM and 53.6 cells in the TE. In treatment IV, 45 embryos counted and embryos consisted of an average of 99.8 cells in total with 29.2 cells in the ICM and 70.6 cells in the TE. For ICM/total cell ratio yield, cell ratio increased in favor of the ICM in treatment IGF-LIF, whereas decreased in the other treatments (0.37–0.32, 0.29–0.26, 0.30–0.26 and 0.29–0.31, respectively). As shown in Table 2, their allocation to TE and ICM cell numbers and ICM/Total cell ratio in stained blastocysts. For total cell number yield, treatment BSA showed the highest variation (96.4 vs 72.2). The LIF alone treatment showed fewest variation (77.03 vs 70.56) (Fig. 1). In Experiment 2, 40 blastocysts counted in treatment I and blastocysts consisted average of 72.2 cells in total, with 23.35 cells in the ICM and 48.92 cells in the TE. In treatment II, 39 blastocysts counted and blastocysts consisted average of 88.68 cells in total, with 24 cells in the ICM and 64.68 cells in the TE. In treatment III, 46 blastocysts counted and blastocysts consisted average of 70.56 cells in total with 19.5 cells in the ICM and 51.06 cells in the TE. In treatment IV, 38 blastocysts counted and blastocysts consisted of an average of 81.46 cells in total with 22.53 cells in the ICM and 63.14 cells in the TE. In the Experiment 2, both ICM and TE cell number decreased for all treatment groups compared with Experiment 1. Interestingly, the LIF treatment showed fewest variations for all cell types. In addition to this, for average number of ICM and TE cell, LIF treatment showed fewest variation compared with other treatments (ICM: 23.5 vs 19.5, TE: 53.6 vs 51) (Fig. 2).
Statistical analysis Blastocysts were randomly allocated to each treatment group and all experiments were replicated at least four times. Statistical data analysis was performed by using the general linear models (GLM) procedure of statistical analysis system (SAS) [21]. One-way Analysis of Variance (ANOVA) followed by Duncan’s Multiple Comparison Test was used for statistical comparison of the groups. All values reported as least-squares means ± SEM, and statistical differences were considered as significant when P values were less than 0.05. Results Blastocyst development BSA group had significantly lower percentage of morula rate compared to the other groups. For blastocysts rate yield, that of LIF alone (24.1%, n = 295) or combined with IGF-I (24.0%, n = 249) resulted in higher than BSA alone treatment (18.7%, n = 434). Additionally LIF alone or supplemented with IGF-I yielded similar results in terms of development to the blastocyst stage. For the blastocysts/cleavage rate yield, LIF resulted in highest rate in all groups. IGF-I alone or supplemented with LIF yielded similar results (34.6%, n = 335 and 29.6%, respectively) in terms of development to the blastocysts/cleavage rate. Additionally BSA (26.3%) had the significantly lower than that for all other treatments (P < 0.001). Data from Experiment 1 are shown in Table 1.
Discussion The present study examined the developmental capacity and cryotolerance of cultured bovine embryos in defined media (SOF) supplemented with IGF-I and LIF. The results suggested that cleavage, morula and blastocyst yields were significantly higher in embryos cultured in media containing IGF-I and LIF than in media containing alone BSA. Moreover, the addition of growth factor and cytokines to the culture medium was found to be beneficial for re-expansion rates and cellular cryotolerance of bovine blastocysts after vitrification. In recent years, numerous studies have been performed to improve in vitro culture of embryos by supplementing culture medium with components that may be advantageous for embryo development. These include growth factors, energy substrates, amino acids and albumin [5,18,19,39]. It is known that, embryotrophic factors that have been shown to be released by the co-cultured cells, such as growth factors and cytokines are still absent in these new generation defined culture media compositions [3,7,16,39]. To improve bovine blastocyst development, several growth factors and cytokines have been added to embryo culture media as they are known to be involved in preimplantation developments of mammalian embryos.
Blastocyst viability and cell allocation following vitrification In Experiment 2, no differences were observed between the treatment I and III regarding the blastocyst re-expansion rate after 24 h thawing (38.69%, n = 40 and 37.12%, n = 46). Furthermore no differences were observed between the treatment II and IV (45.76%, n = 39 and 46.11%, n = 38). On the contrary, the blastocyst re-expansion rate of the treatment II and IV was higher than that of the treatment both I and III (P < 0.001). A total of 380 expanded blastocysts were allocated to the same groups of treatments than in the previous experiments; 217 out of them were fixed as fresh for differential staining (Experiment 1), while the remainder 163 ones were vitrified/warmed for differential staining (Experiment 2). In Experiment 1, 54 embryos counted in treatment BSA and embryos consisted average of 154 cells in total, with 48.3 cells in the ICM and 60 cells in the TE. In treatment II, 65 embryos counted
Table 1 Effects of IGF-I and LIF on in vitro developmental competence of bovine embryos. Groups BSA IGF-I LIF IGF-I + LIF D DD
Zygotes (n) 434 335 295 249
Cleavage (% ± SEM)D 71.04 ± 1.3 65.61 ± 2.0 66.76 ± 3.9 73.39 ± 1.7
Morula (% ± SEM)DD b
26.90 ± 3.4 45.80 ± 3.5a 38.62 ± 4.8a 47.90 ± 2.1a
Blastocyst (% ± SEM)DD b
18.72 ± 1.2 22.60 ± 0.9ab 24.17 ± 2.1a 24.03 ± 2.0a
Blastocyst/cleavage (% ± SEM)DD 26.38 ± 1.6b 34.65 ± 1.7ab 37.22 ± 3.5a 29.60 ± 3.1ab
There were no significant differences among any groups (P > 0.01). Values within a column with no common superscripts are significantly different (P < 0.001)
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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Table 2 Effect of LIF and IGF-I on the blastocysts cell allocation following vitrification. Groups
Blastocyst cell number (n ± SEM)D Total cell
BSA IGF-I LIF IGF-I + LIF D
TE
ICM
Non-vitrified
Vitrified
Non-vitrified
Vitrified
Non-vitrified
Vitrified
96.44 ± 2.3b 104.93 ± 1.9a 77.03 ± 1.3c 99.82 ± 2.3ab
72.28 ± 2.1c 88.68 ± 3.2a 70.56 ± 0.7c 81.46 ± 4.4b
60.07 ± 1.6b 73.87 ± 1.3a 53.64 ± 1.1c 70.64 ± 1.0a
48.92 ± 1.5b 64.68 ± 2.3a 51.06 ± 0.8b 63.14 ± 1.3a
36.37 ± 1.3a 31.06 ± 0.8b 23.54 ± 0.7c 29.20 ± 0.6b
23.35 ± 1.3a 24.00 ± 1.3a 19.50 ± 0.6b 22.53 ± 0.5a
Values within a column with no common superscripts are significantly different (P < 0.05).
Fig. 1. Average number of total cells in blastocysts treated with Experiment 1 (non-vitrification) and Experiment 2 (vitrification) at after 24 h warming.
Fig. 2. Average number of TE and ICM cell in blastocysts treated with Experiment 1 (non-vitrification) and Experiment 2 (vitrification) at after 24 h warming.
One of these factors, leukemia inhibitory factor (LIF) plays an important role in early embryonic development and thought to be essential for the maintenance of totipotency of embryonic stem cells and LIF increases the proliferation rate of murine primordial germ cells in vitro [39]. It is known that LIF supplementation of in vitro embryo culture medium enhances embryo development in human [43], murine [29], ovine [35] and bovine [16] models. Furthermore, mLIF or hLIF is able to stimulate implantation in mice [10,29] and to enhance embryo development in vitro in sheep embryos [11] and in frozen/thawed [16], parthenogenetic and in vivo- and in vitro-produced bovine embryos [12]. The role of LIF in bovine embryo culture has been controversial in previous studies [13,26,39] in which the effects of different supplements, such as growth factors, synthetic molecules and BSA were examined. IGF-I is one of the most important members of the growth factor family. Some reports indicate that IGF-I can increase the number of total cells and ICM and decrease the percentage of blastomeres that were apoptotic [19,39,41], especially apoptosis tending to occur predominantly in the ICM [1]. It can be speculated that IGF-I might increase ICM cell numbers by reducing the prevalence of apoptosis within the ICM. Present study results showed that LIF alone or combined with IGF-I was significantly effective on in vitro bovine embryo development especially ratio to reach blastocyst. Our results are in
agreement with the findings of Sirisathien et al. [39] but in contrast to those of Vejlsted et al. [44] reported that the addition of 1000 U/ml LIF to the culture medium was found to be beneficial for bovine in vitro embryonic development. In assessing the viability of the embryos, morphological observations are most widely used as a gross indicator, but blastocyst cell number and the ICM/total cells ratio are valid indicators for the viability of preimplantation embryos, while morphological criteria alone is a poor indicator. Proliferation and differentiation of somatic cells are regulated by autocrine and paracrine secretion of mitogenic growth factors by maternal and embryonic tissues at specific stages of development as evidenced by measurements of total cell numbers and ICM:TE ratios [40,44]. Diaz-Cueto and Gerton [6] reported that IGF-I receptors are only localized in the ICM. Stimulation of cell proliferation is specific to the ICM and the TE is not affected. In consistent with our results, Makarevich and Markkula [24], indicated that the addition of IGF-I to the culture medium increased the total cell number of blastocysts and lowered the apoptotic index by decreasing the number of apoptotic cells per embryo. The ICM/total cell ratio of the bovine blastocysts obtained in the present study were similar to previous studies findings of Choi et al. [5] and Orsi and Leese [33], but lower than studies of Duque et al. [9]. Differences between the present study and others may be related to differences in culture composition such as macromolecule sources, LIF
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
A. Kocyigit, M. Cevik / Cryobiology xxx (2015) xxx–xxx
concentration and laboratory conditions. In agreement with previous studies [23,32,39], our results demonstrated that, IGF-I treated expanded blastocysts were found to have higher total cell number and ICM nuclei than treatment LIF and BSA blastocysts. During the last decade, new strategies to produce embryos with improved ability to resist stressing sources (such as vitrification) have been proposed. The quality of the IVP bovine blastocyst has been classically associated with their sensitivity to cryopreservation [22,31,37] and to the amount and proper allocation of the embryonic cells [40]. Blastocyst cell numbers are usually reduced in IVP embryos as compared to in vivo-derived embryos [20,28]. The viability of animal embryos after cryopreservation can affect efficiency of embryo transfer and birth rate. Therefore, many efforts have been undertaken for several decades to improve post-thaw quality and viability of mammalian embryos. One of approaches is to add various substances with growth-stimulating and/or membrane-stabilizing effects either before freezing or post-thawing or during both periods. Culturing bovine IVP embryos in the presence of varying concentrations of LIF has brought unclear findings with regard to embryo developmental kinetics, cryotolerance and cell count [2,12,13,16,26,39]. Generally, increased blastocyst yield may not be correlated with higher developmental potential. Therefore, other criteria for the evaluation of embryo viability after cryopreservation are required. In our study we used invasive parameters of embryo quality, such as blastocyst cell number, ICM and TE cell allocations. As is known, the ICM/total cell ratio is crucial to the developmental competence of the embryo, as it is assumed that a certain minimal number of ICM cells are required to obtain a pregnancy. It is unknown whether specific traits of ICM and TE could lead to differences in survival to vitrification, although a detrimental effect (such as apoptosis) of vitrification on the numbers of cells on the ICM has been observed [15,30]. The probable reason for reduced embryo viability following freezing and thawing is the disruption of the cytoskeleton, as a result of intracellular ice formation [8,25]. In our study results showed that the re-expansion rate of blastocysts were similar for both types of embryos on treatments BSA and LIF, whereas significantly lower than treatments IGF-I and IGF-I plus LIF. This may be due to the average numbers of cells, because total cell nuclei and ICM nuclei were lower in LIF treated blastocysts compared with other treatments. These results are similar to that reported by Vejlsted et al. [44] but in contrast to that Sirisathien et al. [39] and Neira et al. [32]. Furthermore, Han et al. [16] reported that addition of LIF before freezing improved the cryotolerance of IVF-derived bovine early blastocysts after freezing and thawing. In light of our findings, the data observed suggest that when culture media supplemented with IGF-I and LIF before vitrification, cultures system produces more blastocyst and embryos produced had a higher rate of re-expansion after vitrification. In addition to this, for average cell number yield, LIF treatment showed fewest variations compared with other treatments. In conclusion, the addition of IGF-I along with LIF to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts yields, re-expansion rate and cellular cryotolerance after vitrification. The positive effect of LIF on the cellular cryotolerance of bovine blastocysts after vitrification, as reported here, was not observed in earlier work. Moreover, those measures alone are not enough criterions to evaluate blastocysts cryotolerance, so these results should be supported by the findings of pregnancy.
Conflict of interest None.
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Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts q Alper Kocyigit a,⇑, Mesut Cevik b a b
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Cumhuriyet University, Sivas, Turkey Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey
a r t i c l e
i n f o
Article history: Received 14 April 2015 Revised 25 May 2015 Accepted 26 May 2015 Available online xxxx Keywords: Bovine blastocyst Growth factor Cytokine Vitrification Cryotolerance
a b s t r a c t The present study examined the developmental capacity and cryotolerance of cultured bovine embryos in defined media (synthetic oviduct fluid, SOF) supplemented with insulin-like growth factor I (IGF-I) and leukemia inhibitor factor (LIF). The objectives of the present study were: (1) to examine the effects IGF-I and LIF on bovine embryo development potential and (2) to investigate the cryotolerance and survivability of vitrified blastocysts obtained from embryos cultured in a defined media. We studied the development of bovine embryos produced in vitro and cultured (in four different treatments) until Day 7 after fertilization. In Experiment 1, zygotes were cultured to the blastocyst stage and differentially stained for determine the count of cells. In Experiment 2, zygotes were vitrified before staining. LIF alone or combined with IGF-I was significantly effective on in vitro bovine embryo development especially ratio to reach blastocyst. The cells for both ICM and TE decreased by the effect of freezing in all treatment groups in the Experiment 2 compared with Experiment 1. Interestingly, the LIF treatment showed fewest variations. In addition to this, for average number of ICM and TE cells, LIF treatment showed fewest variation compared with other treatments (ICM: 23.5 vs 19.5, TE: 53.6 vs 51). These results are the first to demonstrate that the addition of IGF-I along with LIF to the culture medium was found to be beneficial for bovine embryonic development based on cellular cryotolerance after vitrification. Ó 2015 Elsevier Inc. All rights reserved.
Introduction The improvement of in vitro culture systems are important for production of embryos with high developmental competence that are used in embryo transfer programs and biomedical researches [17]. Although with the advancements of culture procedures, in vitro produced (IVP) embryos it usually shows low quality and viability than developed in vivo counterparts. Besides, IVP embryos are more sensitive to cryoinjury than in vivo-derived embryos, such as shown by lower cell numbers, less compaction and a lower number of tight-junctions compared to embryos in vivo [11,37,38,42]. Tight-junction formation is a prerequisite for blastocoel formation, expansion of the blastocyst and final differentiation of two distinct cell populations: the trophectoderm (TE) and the inner cell mass (ICM). It is well known that the ratio and allocation of these cells is considered to be a potential indicator on embryo
q Statement of funding: This work was funded by the Research Foundation of Ondokuz Mayis University, Samsun/TURKEY. This article was produced by the same name PhD thesis study. Project ID: PYO.VET.1904.11.011. ⇑ Corresponding author. E-mail address: [email protected] (A. Kocyigit).
quality and crucial for implantation [40,44]. Therefore, developing an optimal culture system is essential to improve quality of embryos produced in vitro. There are several ways to improve blastocysts quality and cryotolerance during in vitro culture. One way to improve blastocysts quality is supplementation of culture media by growth factors and cytokines. Insulin-like growth factor I (IGF-I) have been reported to be mitogens inducing a positive effect on development of embryos [41]. Moreover, growth factors are involved in the compaction and formation of the blastocyst, activation of transport systems responsible for the uptake of glucose, inhibition of apoptosis, enhancement of endocytosis, probably protein turnover [14,27]. One of the cytokines is leukemia inhibitory factor (LIF), a pleiotropic member of the interleukin-6 (IL-6) family. Leukemia inhibitory factor receptors (LIFR) have been detected in embryos of different species that is considered essential for blastocyst development such as hatching rate, cell count and cryotolerance [2,4,16,26,45]. LIF was originally implicated in regulating the early development of the embryo and promoted the development of bovine embryos and increased the viability and cryotolerance of bovine blastocysts. Cryotolerance may be a useful indicator of blastocyst quality [31,36]. As a functional criterion for the evaluation of blastocysts
http://dx.doi.org/10.1016/j.cryobiol.2015.05.068 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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cryotolerance after vitrification, a post-thaw re-expansion capacity and blastocysts cell allocation have been used [24]. Numerous studies, with contrary views, reported that IGF-I combination with cytokines and added to SOF medium has improved development and cryotolerance of bovine blastocysts [16,32,39]. The objectives of the present study were: (1) to examine the effects IGF-I and LIF on bovine embryo development potential and (2) to investigate the cryotolerance and survivability of vitrified blastocysts obtained from embryos cultured in a defined media.
(10 ll/ml), penicillin (100 U/ml) and streptomycin (100 lg/ml) on the day of use a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 °C. In the current study, LIF was added to the culture medium at Day 4 post insemination (p.i.), while IGF-1 was supplemented in the culture medium at Day 0 p.i (fertilization day). A concentration of 100 ng/ml was unprompted selected for addition to culture media following our study of previous reports.
Materials and methods
In Experiment 1, effect of IGF-I and LIF on the bovine embryo development competence rate studied. Presumptive zygotes were cultured in four different treatments (six replicates). Growth factor and cytokine added to the culture medium were as follows; treatment I: SOF alone, treatment II: SOF plus 100 ng/ml LIF, treatment III: SOF plus 100 ng/ml IGF-I, treatment IV: SOF plus 100 ng/ml IGF-I and 100 ng/ml LIF. The growth factor and cytokine used in our study were human recombinants. Zygotes were cultured to the blastocyst stage and assessed for morphology approximately 168 h (day 7) post insemination, immediately prior to differential staining.
All chemicals used in this study were purchased from Sigma– Aldrich (Istanbul, TURKEY), except where otherwise indicated. Collection and in vitro maturation of oocytes Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory at approximately 34 ± 2.0 °C in physiological saline solution supplemented with gentamycin sulfate (0.1 ll/ml). Cumulus oocyte complexes (COCs) were recovered from follicles 2–8 mm in diameter by aspiration. The COCs were collected in 3–4 ml Hepes-buffered Medium-199 containing Earle’s salts and supplemented with 1% v/v antibiotic–antimycotic solution. Before in vitro maturation, the COCs were assessed morphologically and only oocytes with compact, non-atretic cumulus investment and evenly granulated cytoplasm were selected for maturation. Maturation medium was sodium bicarbonate-buffered Medium-199 with sodium pyruvate (5.5 lg/ml), antibiotic–antimycotic solution (1% v/v), heat-inactivated fetal calf serum (FCS, 10% v/v). The COCs were matured for 22 h in a humidified atmosphere of 5% CO2 in air at 38.5 °C. Spermatozoa preparation and in vitro fertilization After a 22 h maturation period, oocytes were transferred into 44 ll fertilization drops. The fertilization medium was glucose-free modified Tyrode’s albumin lactate pyruvate (TALP) supplemented with bicarbonate (25 mM), Na-lactate (22 mM), Na-pyruvate (1 mM), fatty acid-free bovine serum albumin (BSA) (6 mg/ml), and heparin-sodium salt (184 units/mg heparin, 10 mg/ml) and antibiotic–antimycotic solution (0.5 ll/ml) (pH 7.4 and 280–300 mOsm/kg). Frozen–thawed semen was used for the fertilization of oocytes. Percoll density gradient system was used for the separation of the motile fraction of the frozen–thawed semen [34]. Sperm were then diluted to 50 106 spermatozoon/ml in TL-HEPES, including the 2 106 spermatozoa/ml as final concentration. The fertilization procedure was completed by adding 2 ll of diluted sperm, 2 ll heparin (5 lg/ml) and 2 ll of PHE solution (20 lM penicillamine, 10 lM hypotaurine, and 1 lM epinephrine in final concentration) into the fertilization drops containing oocytes. The oocytes were fertilized with 2 ll diluted semen per fertilization drops for 22 h in a humidified atmosphere of 5% CO2 in air at 38.5 °C. In vitro culture Cumulus cells surrounding the oocytes were removed from presumptive zygotes at approximately 22 h post-insemination by vortexing 3 min. The zygotes were transferred in groups of 20–30 for the culture droplets. Embryos were cultured in the synthetic oviduct fluid (SOF) medium supplemented with pyruvate (0.4 mM), fatty acid free (BSA-FAF) (8 mg/ml), 100 minimum essential medium (MEM) (20 ll/ml), 50 basal medium eagle (BME)
Experiment 1
Experiment 2 This part of the study, the effect of IGF-I and LIF on the subsequent development of bovine blastocysts after freezing and thawing was examined. Presumptive zygotes maturated and fertilized using standard procedures were cultured in four treatments as in Experiment 1 (four replicates). Early blastocysts were harvested on day 6 or day 7 of in vitro culture. After two initial washes in SOF, blastocysts were placed in a 10% glycerol solution for 1 min. Embryos then transferred to 20% ethylene glycol (EG) + 10% glycerol solution for 1 min. Finally embryos then transferred to %25 EG + %25 glycerol for 25 s. The blastocysts were immediately drawn by capillary action into sterile straws, along with a maximum of 1–2 ml medium. Blastocysts were vitrified using the simple method of directly submerging embryos into liquid nitrogen after brief exposure to a cryoprotectant solution. In the thawing procedure (one day after cryopreservation), straws were thawed in a water bath at 35 °C for 15 s, and their contents were released into 0.5 M sucrose solution. Blastocysts were recovered and equilibrated in respectively 0.5 M and 0.25 M sucrose for 5 min, then washed once in SOF medium containing BSA-FAF (8 mg/ml) and cultured in SOF drops at 24 h. The blastocysts were considered viable if they re-expanded or hatched from the zona pellucidae. Determination of the numbers of ICM and TE cells Finally expanded blastocysts from the non-vitrified in Experiment 1 and vitrified in Experiment 2 to determine the differential count of cells in the ICM and TE. Expanding blastocysts characterized by zona pellucidae showing signs of thinning and with slightly increased embryo diameters by day 7 or 8 was stained as described by Van Soom et al. [40]. Briefly, zona intact blastocysts were incubated with 10 mM picrylsulphonic acid (Sigma, in cold Ca2+-free PBS) for 5 min in the refrigerator (4 °C). Then they were washed and incubated for 30 min at 39 °C in anti-dinitrophenyl antibody (Sigma) diluted to 30% (v/v) with Ca+2 free PBS. Embryos were then transferred into guinea pig complement (55852, ICN biochemicals, Irvine, CA, USA) diluted to 20% (v/v) in Ca2+-free PBS containing 50 lg/ml propidium iodide, 12.5 lg/ml bisbenzimide, and 50 lg/ml RNAse a for 30 min at 39 °C. Finally, embryos were fixed in 2% paraformaldehyde in PBS for 1–2 min at room temperature. Before mounting on slides with a 10 ll drop of 0.2 M 1,4 diazabicyclo-octane in 50% glycerol (v/v) in Ca2+-free
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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PBS as an anti-fading solution. Embryos were examined in whole mount under a fluorescence microscope (Nikon Invert Microscope Eclipse Ti-FL, 340–380 nm excitations and 430 nm suppression). Thus, ICM nuclei labeled with bisbenzimide appeared blue and TE nuclei labeled with both bisbenzimide and propidium iodide appeared pink to red. Numbers of ICM and TE nuclei were counted directly under the fluorescence microscope.
and embryos consisted average of 104.9 cells in total, with 31 cells in the ICM and 73.8 cells in the TE. In treatment III, 53 embryos counted and embryos consisted average of 77 cells in total with 23.5 cells in the ICM and 53.6 cells in the TE. In treatment IV, 45 embryos counted and embryos consisted of an average of 99.8 cells in total with 29.2 cells in the ICM and 70.6 cells in the TE. For ICM/total cell ratio yield, cell ratio increased in favor of the ICM in treatment IGF-LIF, whereas decreased in the other treatments (0.37–0.32, 0.29–0.26, 0.30–0.26 and 0.29–0.31, respectively). As shown in Table 2, their allocation to TE and ICM cell numbers and ICM/Total cell ratio in stained blastocysts. For total cell number yield, treatment BSA showed the highest variation (96.4 vs 72.2). The LIF alone treatment showed fewest variation (77.03 vs 70.56) (Fig. 1). In Experiment 2, 40 blastocysts counted in treatment I and blastocysts consisted average of 72.2 cells in total, with 23.35 cells in the ICM and 48.92 cells in the TE. In treatment II, 39 blastocysts counted and blastocysts consisted average of 88.68 cells in total, with 24 cells in the ICM and 64.68 cells in the TE. In treatment III, 46 blastocysts counted and blastocysts consisted average of 70.56 cells in total with 19.5 cells in the ICM and 51.06 cells in the TE. In treatment IV, 38 blastocysts counted and blastocysts consisted of an average of 81.46 cells in total with 22.53 cells in the ICM and 63.14 cells in the TE. In the Experiment 2, both ICM and TE cell number decreased for all treatment groups compared with Experiment 1. Interestingly, the LIF treatment showed fewest variations for all cell types. In addition to this, for average number of ICM and TE cell, LIF treatment showed fewest variation compared with other treatments (ICM: 23.5 vs 19.5, TE: 53.6 vs 51) (Fig. 2).
Statistical analysis Blastocysts were randomly allocated to each treatment group and all experiments were replicated at least four times. Statistical data analysis was performed by using the general linear models (GLM) procedure of statistical analysis system (SAS) [21]. One-way Analysis of Variance (ANOVA) followed by Duncan’s Multiple Comparison Test was used for statistical comparison of the groups. All values reported as least-squares means ± SEM, and statistical differences were considered as significant when P values were less than 0.05. Results Blastocyst development BSA group had significantly lower percentage of morula rate compared to the other groups. For blastocysts rate yield, that of LIF alone (24.1%, n = 295) or combined with IGF-I (24.0%, n = 249) resulted in higher than BSA alone treatment (18.7%, n = 434). Additionally LIF alone or supplemented with IGF-I yielded similar results in terms of development to the blastocyst stage. For the blastocysts/cleavage rate yield, LIF resulted in highest rate in all groups. IGF-I alone or supplemented with LIF yielded similar results (34.6%, n = 335 and 29.6%, respectively) in terms of development to the blastocysts/cleavage rate. Additionally BSA (26.3%) had the significantly lower than that for all other treatments (P < 0.001). Data from Experiment 1 are shown in Table 1.
Discussion The present study examined the developmental capacity and cryotolerance of cultured bovine embryos in defined media (SOF) supplemented with IGF-I and LIF. The results suggested that cleavage, morula and blastocyst yields were significantly higher in embryos cultured in media containing IGF-I and LIF than in media containing alone BSA. Moreover, the addition of growth factor and cytokines to the culture medium was found to be beneficial for re-expansion rates and cellular cryotolerance of bovine blastocysts after vitrification. In recent years, numerous studies have been performed to improve in vitro culture of embryos by supplementing culture medium with components that may be advantageous for embryo development. These include growth factors, energy substrates, amino acids and albumin [5,18,19,39]. It is known that, embryotrophic factors that have been shown to be released by the co-cultured cells, such as growth factors and cytokines are still absent in these new generation defined culture media compositions [3,7,16,39]. To improve bovine blastocyst development, several growth factors and cytokines have been added to embryo culture media as they are known to be involved in preimplantation developments of mammalian embryos.
Blastocyst viability and cell allocation following vitrification In Experiment 2, no differences were observed between the treatment I and III regarding the blastocyst re-expansion rate after 24 h thawing (38.69%, n = 40 and 37.12%, n = 46). Furthermore no differences were observed between the treatment II and IV (45.76%, n = 39 and 46.11%, n = 38). On the contrary, the blastocyst re-expansion rate of the treatment II and IV was higher than that of the treatment both I and III (P < 0.001). A total of 380 expanded blastocysts were allocated to the same groups of treatments than in the previous experiments; 217 out of them were fixed as fresh for differential staining (Experiment 1), while the remainder 163 ones were vitrified/warmed for differential staining (Experiment 2). In Experiment 1, 54 embryos counted in treatment BSA and embryos consisted average of 154 cells in total, with 48.3 cells in the ICM and 60 cells in the TE. In treatment II, 65 embryos counted
Table 1 Effects of IGF-I and LIF on in vitro developmental competence of bovine embryos. Groups BSA IGF-I LIF IGF-I + LIF D DD
Zygotes (n) 434 335 295 249
Cleavage (% ± SEM)D 71.04 ± 1.3 65.61 ± 2.0 66.76 ± 3.9 73.39 ± 1.7
Morula (% ± SEM)DD b
26.90 ± 3.4 45.80 ± 3.5a 38.62 ± 4.8a 47.90 ± 2.1a
Blastocyst (% ± SEM)DD b
18.72 ± 1.2 22.60 ± 0.9ab 24.17 ± 2.1a 24.03 ± 2.0a
Blastocyst/cleavage (% ± SEM)DD 26.38 ± 1.6b 34.65 ± 1.7ab 37.22 ± 3.5a 29.60 ± 3.1ab
There were no significant differences among any groups (P > 0.01). Values within a column with no common superscripts are significantly different (P < 0.001)
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Table 2 Effect of LIF and IGF-I on the blastocysts cell allocation following vitrification. Groups
Blastocyst cell number (n ± SEM)D Total cell
BSA IGF-I LIF IGF-I + LIF D
TE
ICM
Non-vitrified
Vitrified
Non-vitrified
Vitrified
Non-vitrified
Vitrified
96.44 ± 2.3b 104.93 ± 1.9a 77.03 ± 1.3c 99.82 ± 2.3ab
72.28 ± 2.1c 88.68 ± 3.2a 70.56 ± 0.7c 81.46 ± 4.4b
60.07 ± 1.6b 73.87 ± 1.3a 53.64 ± 1.1c 70.64 ± 1.0a
48.92 ± 1.5b 64.68 ± 2.3a 51.06 ± 0.8b 63.14 ± 1.3a
36.37 ± 1.3a 31.06 ± 0.8b 23.54 ± 0.7c 29.20 ± 0.6b
23.35 ± 1.3a 24.00 ± 1.3a 19.50 ± 0.6b 22.53 ± 0.5a
Values within a column with no common superscripts are significantly different (P < 0.05).
Fig. 1. Average number of total cells in blastocysts treated with Experiment 1 (non-vitrification) and Experiment 2 (vitrification) at after 24 h warming.
Fig. 2. Average number of TE and ICM cell in blastocysts treated with Experiment 1 (non-vitrification) and Experiment 2 (vitrification) at after 24 h warming.
One of these factors, leukemia inhibitory factor (LIF) plays an important role in early embryonic development and thought to be essential for the maintenance of totipotency of embryonic stem cells and LIF increases the proliferation rate of murine primordial germ cells in vitro [39]. It is known that LIF supplementation of in vitro embryo culture medium enhances embryo development in human [43], murine [29], ovine [35] and bovine [16] models. Furthermore, mLIF or hLIF is able to stimulate implantation in mice [10,29] and to enhance embryo development in vitro in sheep embryos [11] and in frozen/thawed [16], parthenogenetic and in vivo- and in vitro-produced bovine embryos [12]. The role of LIF in bovine embryo culture has been controversial in previous studies [13,26,39] in which the effects of different supplements, such as growth factors, synthetic molecules and BSA were examined. IGF-I is one of the most important members of the growth factor family. Some reports indicate that IGF-I can increase the number of total cells and ICM and decrease the percentage of blastomeres that were apoptotic [19,39,41], especially apoptosis tending to occur predominantly in the ICM [1]. It can be speculated that IGF-I might increase ICM cell numbers by reducing the prevalence of apoptosis within the ICM. Present study results showed that LIF alone or combined with IGF-I was significantly effective on in vitro bovine embryo development especially ratio to reach blastocyst. Our results are in
agreement with the findings of Sirisathien et al. [39] but in contrast to those of Vejlsted et al. [44] reported that the addition of 1000 U/ml LIF to the culture medium was found to be beneficial for bovine in vitro embryonic development. In assessing the viability of the embryos, morphological observations are most widely used as a gross indicator, but blastocyst cell number and the ICM/total cells ratio are valid indicators for the viability of preimplantation embryos, while morphological criteria alone is a poor indicator. Proliferation and differentiation of somatic cells are regulated by autocrine and paracrine secretion of mitogenic growth factors by maternal and embryonic tissues at specific stages of development as evidenced by measurements of total cell numbers and ICM:TE ratios [40,44]. Diaz-Cueto and Gerton [6] reported that IGF-I receptors are only localized in the ICM. Stimulation of cell proliferation is specific to the ICM and the TE is not affected. In consistent with our results, Makarevich and Markkula [24], indicated that the addition of IGF-I to the culture medium increased the total cell number of blastocysts and lowered the apoptotic index by decreasing the number of apoptotic cells per embryo. The ICM/total cell ratio of the bovine blastocysts obtained in the present study were similar to previous studies findings of Choi et al. [5] and Orsi and Leese [33], but lower than studies of Duque et al. [9]. Differences between the present study and others may be related to differences in culture composition such as macromolecule sources, LIF
Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068
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concentration and laboratory conditions. In agreement with previous studies [23,32,39], our results demonstrated that, IGF-I treated expanded blastocysts were found to have higher total cell number and ICM nuclei than treatment LIF and BSA blastocysts. During the last decade, new strategies to produce embryos with improved ability to resist stressing sources (such as vitrification) have been proposed. The quality of the IVP bovine blastocyst has been classically associated with their sensitivity to cryopreservation [22,31,37] and to the amount and proper allocation of the embryonic cells [40]. Blastocyst cell numbers are usually reduced in IVP embryos as compared to in vivo-derived embryos [20,28]. The viability of animal embryos after cryopreservation can affect efficiency of embryo transfer and birth rate. Therefore, many efforts have been undertaken for several decades to improve post-thaw quality and viability of mammalian embryos. One of approaches is to add various substances with growth-stimulating and/or membrane-stabilizing effects either before freezing or post-thawing or during both periods. Culturing bovine IVP embryos in the presence of varying concentrations of LIF has brought unclear findings with regard to embryo developmental kinetics, cryotolerance and cell count [2,12,13,16,26,39]. Generally, increased blastocyst yield may not be correlated with higher developmental potential. Therefore, other criteria for the evaluation of embryo viability after cryopreservation are required. In our study we used invasive parameters of embryo quality, such as blastocyst cell number, ICM and TE cell allocations. As is known, the ICM/total cell ratio is crucial to the developmental competence of the embryo, as it is assumed that a certain minimal number of ICM cells are required to obtain a pregnancy. It is unknown whether specific traits of ICM and TE could lead to differences in survival to vitrification, although a detrimental effect (such as apoptosis) of vitrification on the numbers of cells on the ICM has been observed [15,30]. The probable reason for reduced embryo viability following freezing and thawing is the disruption of the cytoskeleton, as a result of intracellular ice formation [8,25]. In our study results showed that the re-expansion rate of blastocysts were similar for both types of embryos on treatments BSA and LIF, whereas significantly lower than treatments IGF-I and IGF-I plus LIF. This may be due to the average numbers of cells, because total cell nuclei and ICM nuclei were lower in LIF treated blastocysts compared with other treatments. These results are similar to that reported by Vejlsted et al. [44] but in contrast to that Sirisathien et al. [39] and Neira et al. [32]. Furthermore, Han et al. [16] reported that addition of LIF before freezing improved the cryotolerance of IVF-derived bovine early blastocysts after freezing and thawing. In light of our findings, the data observed suggest that when culture media supplemented with IGF-I and LIF before vitrification, cultures system produces more blastocyst and embryos produced had a higher rate of re-expansion after vitrification. In addition to this, for average cell number yield, LIF treatment showed fewest variations compared with other treatments. In conclusion, the addition of IGF-I along with LIF to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts yields, re-expansion rate and cellular cryotolerance after vitrification. The positive effect of LIF on the cellular cryotolerance of bovine blastocysts after vitrification, as reported here, was not observed in earlier work. Moreover, those measures alone are not enough criterions to evaluate blastocysts cryotolerance, so these results should be supported by the findings of pregnancy.
Conflict of interest None.
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Please cite this article in press as: A. Kocyigit, M. Cevik, Effects of leukemia inhibitory factor and insulin-like growth factor-I on the cell allocation and cryotolerance of bovine blastocysts, Cryobiology (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.05.068