AIP Conference Proceedings 2168, 020077 (2019); https://doi.org/10.1063/1.5132504 2168, 020077
© 2019 Author(s).
The combination effect of ethylene glycol and palm dates juice on ovarian tissue
of female Sprague-Dawley rats (Rattus norvegicus L.) post-vitrification
Cite as: AIP Conference Proceedings 2168, 020077 (2019); https://doi.org/10.1063/1.5132504 Published Online: 04 November 2019
M. A. Rosvita, N. Anita, A. Abinawanto, et al.
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The Combination Effect of Ethylene Glycol and Palm Dates Juice on Ovarian Tissue of Female Sprague-Dawley Rats
(Rattus norvegicus L.) Post-vitrification
M. A. Rosvita
1, N. Anita
1, a), A. Abinawanto
1, A. A. Jusuf
2, A. Awanis
1, I. Muhiardi
1and Y. Q. A’yun
11Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA), Universitas Indonesia, Depok 16424, Indonesia
2Department of Histology, Faculty of Medicine Universitas Indonesia, Jakarta 10430, Indonesia
a)Corresponding author: [email protected]
Abstract. Preservation of animals and human ovarian tissue by vitrification has been developed to find the efficient methods to overcome the damaging effect from cold shock. The aim of this study is to evaluate the effect of combination of ethylene glycol (EG) as intracellular cryoprotectant and palm dates juice as extracellular cryoprotectant at concentration 3.75 %, 7.5 % and 15 %, respectively with ratio 1:1 in preserving the ovarian tissue after 48 hours vitrification, based on ovarian weight. The ovaries of 21 mature Sprague-Dawley rats in the proestrus stage were isolated and weighed. Ovaries of normal control group (n = 3) were not vitrified, control treatment group (n = 9) was submerged in ethylene glycol (EG) solution and treatment group (n = 9) was submerged in combination of ethylene glycol and palm dates juice solution with three different concentrations inside the cryotube and were continued with vitrification or stored in liquid nitrogen (–196 ºC) for 48 hours, then thawed and re-weighed. The result showed that there was no significant difference between experimental groups on pre-vitrification and post-vitrification ovarian weight (P > 0.05). In conclusion, combination of ethylene glycol and palm dates juice with three different concentrations does not cause significant effect on the ovarian tissue post-vitrification.
Keywords: Palm dates juice, ethylene glycol, ovarian tissue, rats, vitrification
INTRODUCTION
Ovarian tissue preservation in animals and humans has been developed for two decades to realize the ultimate goals of the Assisted Reproductive Technology (ART) [1]. This technique can increase the rate of reproduction in the endangered animals which hard to have offspring in their habitat for conservation purpose, also in livestock [2].
Likewise, in human, the preservation of an ovary is helpful for women who are planned to undergo cancer therapy [3]. The viable ovary can produce an embryo in vitro by in vitro fertilization (IVF) [4]. However, ovarian tissue preservation is quite challenging to get the effective methods with less damaging effect and still maintaining the viability of the ovary. Based on several studies, vitrification or tissue preservation at –196 ºC with rapid freezing is one of the most simple and efficient methods for ovarian tissue preservation [5, 6].
The successful vitrification can be indicated by the minimum damage of cell due to cold shock. Vitrification still has damaging potential on the ovarian tissue by freezing and thawing procedures which can cause an extreme osmotic change in cell, although it is less damaging than conventional cryopreservation [6]. The damaged ovarian tissue can decrease the number of viable follicles, so the follicles cannot mature, and fertilization would be unsuccessful [7].
However, the damaging potential can be overcome by the optimal methods using an appropriate type and concentration of cryoprotectant [8]. Cryoprotectants are essential substances used in protecting and maintaining the cells and tissues from the cold shock effects which needed during vitrification [9]. There are two types of cryoprotectants based on physiochemical properties and membrane permeability; intracellular and extracellular cryoprotectant, respectively.
Application of one type cryopreservation is not enough to protect the intracellular and extracellular of the cells during freezing and thawing procedures. Therefore, the best option to optimize the cell protection is the combination of the two types of cryoprotectants [8].
Ethylene glycol is one of the intracellular cryoprotectants that have been reported to perform better result on ovarian tissue than other cryoprotectants. According to previous studies, ethylene glycol can maintain most of the developing follicles especially primordial follicles in mice and rats ovarian tissue cryopreservation [10-12]. Fathi et al. showed that rat ovarian tissue vitrification with combination of 7.5 % ethylene glycol, 7.5 % DMSO, and 0.25 M sucrose is optimal in preserving the follicles development than other combination cryoprotectant and without sucrose treatment groups [12]. Generally, intracellular cryoprotectant combined with large molecules like synthetic sugar can protect intracellular and extracellular of the cells and tissues, yet chemical toxicity of synthetic cryoprotectants can affect the cells [8, 13]. Natural extracellular cryoprotectant can substitute synthetic sugar causes a less toxic and easily obtained [14]. Many natural extracellular cryoprotectants have been studied, but palm dates can be used as an alternative to increase cell protection effectiveness, because it contains of 72–88 % natural sugar especially sucrose and many essential minerals which can help to stabilize the membrane during vitrification process. Palm dates juice has been reported to be able in maintaining the membrane integrity and motility of bull spermatozoa during freezing [15].
Ovarian tissue vitrification methods have been developed in recent years and always been observed from microscopic evaluation, such as the number of follicles and apoptotic levels [16, 17], but rarely observed through macroscopic evaluation. Previous studies have shown that the ovarian tissue weight is associated with the follicular number [18]. However, the effect of palm dates juice as natural extracellular cryoprotectant on rat ovarian tissue and the effect on the weight of ovary after vitrification has not been reported yet. Accordingly, the objective of the present study is to evaluate the effect of combination of ethylene glycol (EG) as intracellular cryoprotectant and palm dates juice as extracellular cryoprotectant at concentration of 3.75 %, 7.5 % and 15 %, respectively with ratio 1:1 on ovarian tissue weight after 48 hours vitrification, based on Fathi et al. study [12]. Concentration of 3.75 % and 15 % is added to evaluate the effect with minimum and maximum concentration.
MATERIALS AND METHOD Animals and Experimental Group
Twenty-one female Sprague-Dawley rats aged 12 weeks with average body weight 145–231 g, were studied in Department of Biology, Faculty of Mathematics and Sciences, Universitas Indonesia, Depok. The animals were derived from Indonesian Ministry of Health, Jakarta and kept since pre-matured aged (8 weeks) in cages consisted of three rats under controlled condition; room temperature (26–28 °C) and the air circulated by exhaust fan; with a 12 hour light/dark cycle (lighting from 06:00 to 18:00; dark, 18:00 to 06:00). They were fed with pelleted food (CV PD Kasman) and given drink of water ad libitum.
The experiment was designed with seven treatment groups, which each group consisted of three ovaries to compare the ovarian weight before vitrification and after different vitrification treatments. The following are the experimental groups: normal control groups were fresh ovaries (not vitrified); control treatment group 1 was vitrified with 3.75 % ethylene glycol solution; control treatment group 2 was vitrified with 7.5 % ethylene glycol solution; control treatment group 3 was vitrified with 15 % ethylene glycol solution; treatment group 1 was vitrified with combination solution of 3.75 % ethylene glycol and 3.75 % palm dates juice; treatment group 2 was vitrified with combination solution of 7.5 % ethylene glycol and 7.5 % palm dates juice; treatment group 3 was vitrified with combination solution of 15 % ethylene glycol and 15 % palm dates juice. This experiment was approved by The Ethics Committee of Faculty of Medicine, Universitas Indonesia on February 12th, 2018 with the registered number of 18-02-0148.
Estrous Cycle Determination
Animals in aged 10 weeks were checked for estrous cyclicity by vaginal cytology, every morning between 08:00 and 09.00 a.m. to get a constant cycle for two weeks. The vaginal fluid of each animal cage was collected using micropipette filled with 10 µL of aquabidest, by inserting the tips into the rat vagina at depth approximately 1 cm, then aquabidest flushed into the vagina and back out with the vaginal fluid. Three drops vaginal fluid was placed evenly on the glass slide. The slides were fixated in 80 % methanol and stained by 10 % Giemsa stain solution. Stained slides were observed under a light microscope and documented by imaging software to evaluate and determine the estrous cycle. Animals in proestrus stage (dominated by small nucleated epithelial cells) were selected to continue the next procedures [19-21].
Ovarian Tissue Preparation
Animals in proestrus stage were euthanized by ketamine in anesthetized dose which administered intraperitoneally (IP) and continued with cervical dislocation. The animal body was opened from urethra to diaphragm and the ovaries were dissected free from other tissues, fat and mesentery [22]. All ovaries were given treatment according to the experimental groups, however only the right ovaries were studied in this experiment.
Vitrification Solution
Vitrification solution in this experiment was a modification according to Fathi et al. and Milenkovic et al. [12, 23], contains of NaCl 0.9 % as physiological solution or solvent and one or two types of cryoprotectant. Ethylene glycol as intercellular cryoprotectant and palm dates juice that main content is carbohydrates especially in the form sucrose, fructose and glucose, also contains some essential minerals (CV AMS, Bogor) as extracellular cryoprotectant were diluted with NaCl 0.9 % in a bottle using micropipette according the treatment vitrification or concentration of cryoprotectants. Total volume of vitrification solution for each ovary was in 1:20 ratio. Volume of ovaries was ± 0.1 mL, then 2 mL vitrification solution was needed for one ovary. Vitrification solution was not needed in normal control group ovaries; treatment control group contained intracellular cryoprotectant (ethylene glycol); and treatment group contained combination of intracellular and extracellular cryoprotectant. The ratio of vitrification solution for treatment group ovaries was 1:1. Table 1 shows composition of vitrification solution for each experimental group.
Vitrification and Thawing Procedures
The ovarian vitrification method was a modification according to Milenkovic, Mohamad et al. and Rosadi et al.
[23-25]. Treatment control group and treatment group ovaries submerged in vitrification solution inside the cryotube according to the treatment. The cryotubes were exposed with 10 seconds nitrogen vapor for adjustment (equilibration).
Then, immediately plunged into liquid nitrogen at –196 °C and stored for 48 hours until thawing. For thawing,
TABLE 1. Composition of vitrification solution for each experimental group.
Experimental Group Ethylene Glycol (EG) (µ)
NaCl 0.9 % (µ)
Palm Dates Juice (PDJ) (µ)
NaCl 0.9 % (µ)
Normal Control Group (without vitrification) - - - -
Treatment Control Group 1 (EG 3.75 %) 75 1925 - -
Treatment Control Group 2 (EG 7.5 %) 150 1850 - -
Treatment Control Group 3 (EG 15 %) 300 1700 - -
Treatment Group 1 (EG 3.75 % + PDJ 3.75 %) 37.5 962.5 37.5 962.5
Treatment Group 2 (EG 7.5 % + PDJ 7.5 %) 75 925 75 925
Treatment Group 3 (EG 15 % + PDJ 15 %) 150 850 150 850
cryotubes were removed from liquid nitrogen and directly incubated in water bath at 37 °C for 3 minutes or until completely thawed; continued with re-weighing the ovaries.
Ovarian Weights Measurement
All right ovaries from animals were weighed on a digital analytical scale before vitrification procedures. All vitrified (treatment control group and treatment group) ovaries were weighed again after two days stored [26, 27].
Statistical Analysis
Statistical analysis was performed using the program of Statistical Product and Service Solution (SPPS) for Windows version 23. Normality and homogeneity of the data was analyzed by Shapiro-Wilk test and Levene test (α > 0.05). The one-way of analysis of variance (ANOVA) test and Post Hoc Least Significant Difference (LSD) was used to analyse the differences on ovaries weight between the experimental group (P ≤ 0.05).
RESULTS AND DISCUSSION
Twenty-one rat ovaries in the proestrus stage were used in this study. The weight of the ovaries was observed from seven experimental groups with three repetitions and the results are shown in Fig. 1 and Table 2. The weight of pre- vitrification ovaries are ranged from 0.0478 ± 0.005 to 0.0705 ± 0.012 g with the mean of 21 ovaries of 0.0581 ± 0.015 g (P = 0.354). After vitrification, the weight was slightly decreased with total mean of 0.0559 ± 0.015 g; ranged from 0.0469 ± 0.004 g to 0.0674 ± 0.015 g (P = 0.363). Statistically, the samples were normally distributed and homogeneous, also there were no significant differences between the experimental group on pre-vitrification and post-vitrification ovarian weight based on one-way ANOVA (P > 0.05) and LSD test was not performed due to the ANOVA value.
According to Murasawa et al. [18], there is a relationship between ovarian weight and follicular population that founds inside the ovarian tissue. The study stated that the weight of follicles itself contributes to the total ovarian weight, so the weight of an ovary can reflect the potential of the ovary to produce the mature follicles [18]. This report can be fundamental for this study to evaluate the possibility for pre-estimating the follicles inside ovarian tissue due to changes in ovarian tissue post-vitrification using several variations of cryoprotectant’s concentration.
FIGURE 1. Pre-vitrification and post-vitrification of 21 rat ovarian tissue weights. The blue graphics are vitrified excepts for normal control group (NC). The same letters indicate no significant differences between experimental group
(one-way ANOVA, P > 0.05).
TABLE 2. Pre-vitrification and post-vitrification of 21 rats ovarian tissue weights. Values are mean ± SD of three repetitions.
Experimental Groups (g) Total Mean
of All Groups (g)
NC TC1 TC2 TC3 T1 T2 T3
Pre-vitrification 0.0610 ± 0.022
0.0478 ± 0.005
0.0520 ± 0.018
0.0498 ± 0.008
0.0569 ± 0.005
0.0705 ± 0.012
0.0687 ± 0.018
0.0581 ± 0.015 Post-vitrification 0.0610 ±
0.022
0.0469 ± 0.004
0.0498 ± 0.016
0.0454 ± 0.011
0.0544 ± 0.007
0.0676 ± 0.015
0.0661 ± 0.018
0.0559 ± 0.015 NC : Normal control group (without vitrification)
TC1 : Treatment control group 1 (vitrification with EG 3.75 %) TC2 : Treatment control group 2 (vitrification with EG 7.5 %) TC3 : Treatment control group 3 (vitrification with EG 15 %)
T1 : Treatment group 1 (vitrification with combination EG 3.75 % and palm dates juice 3.75 %) T2 : Treatment group 2 (vitrification with combination EG 7.5 % and palm dates juice 7.5 %) T3 : Treatment group 3 (vitrification with combination EG 15 % and palm dates juice 15 %)
The mean of the ovarian weight of normal control group (NC) or not vitrified ovaries was 0.0653 ± 0.019 g. The weight of NC group ovaries is used to compare with other experimental group to evaluate the effect of cryoprotectant also the weight of treatment control (TC1, TC2 and TC3) and treatment group (T1, T2 and T3) ovaries after treatment are expected to be not much different with the initial weight and NC. However, post-vitrification ovarian weight in TC and T groups decreased from the initial weight. The decrease of ovarian weight may affect the viable follicles inside the ovarian tissue.
Previous studies reported that vitrification success can be affected by the concentration and types of cryoprotectant use for maintaining the cells almost the same as the initial state [8]. Ethylene glycol is known as one of the best cryoprotectant to protect the intercellular of ovarian tissue during vitrification procedures, but it has a potential toxicity and could be harmful to the follicles. This issue can be overcome by combining it with natural extracellular cryoprotectants like palm dates juice which have natural sugar and many minerals to protect the extracellular of cells and tissue during freezing and thawing. The sugar can bind covalently with protein and lipid to stabilize the membrane and the mineral can maintain the stability of osmotic pressure on cell [15]. Nevertheless, the ovarian tissue vitrification study with a combination of ethylene glycol and palm dates juice is still limited to animal test and there is no data about post-vitrification ovarian weight evaluation.
In this study, the result of pre-vitrification and post-vitrification ovarian weight show there are no significant differences between normal control (NC) groups and other experimental groups; between three different treatments on treatment control (TC) group (TC1, TC2 and TC3); and between three different treatments on treatment groups (T1, T2 and T3) (Fig. 1). No difference between the NC group and TC groups also T groups indicates there is no change in the ovarian tissue, and it may not affect much to the follicles. The post-vitrification means of ovarian weight on TC1, TC2 and TC3 respectively, are 0.0459 ± 0.051 g, 0.0498 ± 0.016 g and 0.0511 ± 0.009 that slightly decreased from the pre-vitrification ovarian weight with percentages of decrease are 2.02 %, 4.29 % and 8.83 %. It caused by the increase concentration of ethylene glycol (EG) that used to protect the ovarian tissue from cold shock effect, but it is not clearly proven with this parameter. The decrease of post-vitrification ovarian weight is also occurred in T1 (0.0545 ± 0.007 g), T2 (0.0674 ± 0.014 g) and T3 (0.0661 ± 0.018 g) with percentage of decrease are 4.39 %, 4.12 % and 3.79 %, may be caused by the combination of EG and palm dates juice. The previous studies reported that a combination of intracellular and extracellular cryoprotectants is better than using one type of cryoprotectant to prevent the ice crystals formation by means of ethylene glycol penetrates into cells to maintain the inner cells properties and palm dates juice stayed in outer cells to stabilize the membrane from rapid changes in osmotic pressure [8]. However, between post-vitrification ovaries of TC1, TC2, and TC3; post-vitrification of T1, T2, T3, there is no significant differences (P > 0.05) which indicates there is no significant effect on the change of ovarian weight that correlates with follicles number inside the ovarian tissue. It makes difficult to draw a conclusion about the most optimal cryoprotectant treatment for ovarian tissue vitrification.
CONCLUSION
This study showed that vitrification with combination of ethylene glycol and palm dates juice does not affect the rat ovarian tissue weight. However, the most optimal cryoprotectant treatment for ovarian tissue vitrification cannot be concluded from this study, so further investigation on microscopic evaluation is necessary with the same concentration and combination of cryoprotectant.
ACKNOWLEDGMENTS
This work was financially supported by Universitas Indonesia under research grant PITTA 2018 (Publikasi Internasional Terindeks untuk Tugas Akhir Mahasiswa Universitas Indonesia 2018).
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