AIP Conference Proceedings 2168, 020098 (2019); https://doi.org/10.1063/1.5132525 2168, 020098
© 2019 Author(s).
The effect of different concentrations of
ethylene glycol and skim milk in vitrification of ovarian rat (Rattus norvegicus L.) strain Sprague-Dawley
Cite as: AIP Conference Proceedings 2168, 020098 (2019); https://doi.org/10.1063/1.5132525 Published Online: 04 November 2019
Y. Q. A’yun, N. Anita, A. Abinawanto, et al.
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The Effect of Different Concentrations of Ethylene Glycol and Skim Milk in Vitrification of Ovarian Rat
(Rattus norvegicus L.) Strain Sprague-Dawley
Y. Q. A’yun
1, N. Anita
1, a), A. Abinawanto
1, A. A. Jusuf
2, A. Awanis
1, I. Muhiardi
1and M. A. Rosvita
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. Vitrification as a part of the assisted reproductive technology (ART) has become the main concern of reproductive organ preservation. Vitrification is a development of reproduction biotechnology to overcome reproductive problems. Ethylene glycol (EG) as intracellular cryoprotectant has been commonly used in rat ovary vitrification, while skimmed milk (SM) has been previously used as extracellular cryoprotectant in fish sperm cryopreservation. The research is aimed to find the best concentration among 3.75 %, 7.5 % and 15 % of EG and SM in preventing ovarian tissue 48 h after vitrification. The selected 21 ovaries were used in this study. The samples (n = 21) were divided into 7 groups. Control group (NC 1, NC 2, NC 3, n = 3) includes sample without vitrification. Control treatment group (TC 1, TC 2, TC 3, n = 9) includes sample that vitrificated with EG in concentrations of 3.75 %, 7.5 % and 15 %, respectively. Treatment group (T 1, T 2, and T 3; n = 9) includes samples vitrificated with EG and SM mixture in concentrations of 3.75 %, 7.5% and 15%, respectively. The results showed that the weight of the ovarian tissue pre-vitrification and post-vitrification in TC 1, TC 2, TC 3, T 1, T 2 and T 3 were not significantly different. In conclusion, this research showed that vitrification of rat ovary using ethylene glycol and skim milk mixture may have no effect on weight of ovarian tissue.
Keywords: Vitrification, ethylene glycol, skim milk, Rattus norvegicus, ovary
INTRODUCTION
According to the IUCN (2012), 1.131 mammals were classified as endangered, threatened, or vulnerable [1]. The assisted reproductive technology, therefore, is needed to resolve the species extinction problems. One of ART method that can be applied in preserving the cells or organ in liquid nitrogen is vitrification. Vitrification method is more preferable in preserving organ because it is considerably more effective with a minimum risk of organ damage and has lower technical difficulty. The successful of vitrification can be influenced by cryoprotectant. Cryoprotectant consisted of intracellular and extracellular substances [2]. Vitrification uses the combination of intracellular and extracellular cryoprotectant have been reported. DMSO, EG, GLY, and PROH have been used as the intracellular cryoprotectant, while fetal bovine serum was used as the extracellular cryoprotectant for preserving mice ovary [3].
Egg yolk has been used as the natural extracellular for preserving the human ovary [4].
Vitrification has been applied to several ovarian tissue in mammals, for example Mus musculus [2], Macaca fascicularis [5], Capra aegagrus [6] and Rattus norvegicus [7]. The effect of DMSO to protect the Rattus norvegicus
ovarian in the liquid nitrogen for 24 hours has been reported [7]. Further, the combination of EG, DMSO, FBS, and sucrose were suitable as the cryoprotectant for preserving Rattus norvegicus ovarian at -196 °C [7].
Ethylene glycol as an intracellular cryoprotectant has a low toxic effect and shows a rapid diffusion into cells compared to glycerol, DMSO, and PROH [8-10]. On the other hand, skim milk can also be used as the extracellular cryoprotectant to prevent cell damage due to freezing and thawing during vitrification [11]. In the previous study, it was shown the advantage of skim milk for protecting the spermatozoa of Barbonymus gonionotus (Java Barb) when frozen at -34 °C for 24 hours [12]. However, the combination effect of EG and skim milk for preserving the ovarian rat in liquid nitrogen (-196 °C) has not been reported yet. Accordingly, the present study, therefore, is to evaluate the combination of EG and skim milk on ovarian weight of Rattus norvegicus after vitrification for 48 hours.
MATERIALS AND METHODS Materials
The test material used were ethylene glycol and skim milk. The animals used were 21 tails female Rattus norvegicus L. Sprague-Dawley strain, 12 weeks of age and 144.17 ± 23.98 g of weight. The animals were obtained from Kemenkes RI, Jakarta. The animals were given drinking water and food pellets derived from CV PD Kasman, Sunter Jaya, North Jakarta.
Animal Test’s Maintenance
Twenty-one rats were divided into 9 cages and placed in a plastic tray. Before the treatment begin, the rats were acclimatized for 14 days. The rats were given ad libitum (free-feeding) access to food and water, also weighed daily.
Animal cages were placed in the homes of animal Faculty of Mathematics and Science Universitas Indonesia, Depok.
The air was circulated by exhaust fan with a room temperature about 27 °C. The lamp tube was set up with a 12-hour light and dark cycle (lighting from 06:00 to 18:00; dark from 18:00 to 06:00). Test animal maintenance was approved by Ethical Clearance Committee of Faculty of Medicine Universitas Indonesia.
Experimental Group
The experiment was designed with seven treatment groups, which each group consisted of three ovaries to compare the ovarian weight pre-vitrification and post-vitrification. 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 % skim milk; treatment group 2 was vitrified with combination solution of 7.5 % ethylene glycol and 7.5 % skim milk; treatment group 3 was vitrified with combination solution of 15 % ethylene glycol and 15 % skim milk.
Determination of Estrous Cycle
Animals were checked for estrous cyclicity at 08:00 a.m. for two weeks by vaginal cytology. The vaginal fluid was collected using micropipette with 10 µL of aquabidest and then inserting the tips into the rat vagina [13]. The vaginal fluid was placed on the glass slide and next the slides were fixated in 80 % methanol then stained by 10 % Giemsa stain solution [14]. The slides were observed with microscope (Leica DM500) and then documented by image driving software (LAS EZ v.2.0.0) to determine the estrous cycle of animals [15].
Ovarian Tissue Preparation
Animals in proestrus phase were euthanized by ketamine (anesthetized dose) and then continued with cervical dislocation. The animal was dissected from urethra to diaphragm, and then both ovaries were separated from the body.
Ovary used in this study was only the right part [16].
Vitrification Solution
Vitrification solution in this study contained two, intracellular and extracellular cryoprotectants (Table 1).
Intracellular cryoprotectant consisted of EG and NaCl 0.9 %, meanwhile extracellular cryoprotectant consisted of skim milk and NaCl 0.9 %. EG served as intracellular cryoprotectant, skim milk as extracellular cryoprotectant, and NaCl 0.9 % as a physiological solution. Two millimeters of vitrified solution are needed for one ovary with a volume of 0.1 mL. The ratio of each ovary per vitrification solution for treatment group was 1:1 (intracellular cryoprotectant:
extracellular cryoprotectant) [17, 18].
Equilibration Process
The cryotubes containing the ovaries of the control group and treatment group which was submerged in vitrification solution was labeled, and allowed to equilibrate for 10 seconds in liquid nitrogen vapor (-120 °C) to allow time for the ovary to be exposed to the cryoprotectant before freezing [19, 17].
Freezing Ovary
The cryotubes were then placed in the liquid nitrogen at -196 °C and preserved for 48 hours [19].
Thawing Ovary
After 48 hours, the samples were removed from liquid nitrogen and directly thawed in a water bath at 37 °C for 3 minutes, then the ovaries were re-weighed [17].
Ovarian Weights Measurement
Ovaries from animals were weighed with Precisa XT220 digital analytical balance before vitrification process.
After 48 hours all ovaries were then re-weighed.
TABLE 1. Composition of vitrification solution for each experimental group.
Experimental Group Ethylene
Glycol (EG) (µ)
NaCl 0.9 % (µ)
Skim Milk (SM) (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 % + SM 3.75 %) 37.5 962.5 37.5 962.5
Treatment Group 2 (EG 7.5 % + SM 7.5 %) 75 925 75 925
Treatment Group 3 (EG 15 % + SM 15 %) 150 850 150 850
Statistical Analysis
The data was analyzed statistically using the program of Statistical Product and Service Solution (SPPS) for Windows version 24. The normality and homogeneity of the data were measured by Shapiro-Wilk test and Levene test (P ≤ 0.05). If the data was distributed normally and homogeny then the analysis will be continued with one-way ANOVA and multiple comparison test (LSD) statistic parametric.
RESULTS AND DISCUSSION
The mean ovarian weight post-vitrification in control group (NC) was 0.0610 ± 0.0223 g (Table 2 and Fig. 1).
Despite the invariability, the weight of NC was still in normal range of ovarian weight, so that it can be used as a comparison against the other treatment groups [20].
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 ± 0.0223
0.0478 ± 0.0050
0.0520 ± 0.0183
0.0498 ± 0.0082
0.0899 ± 0.0105
0.0699 ± 0.0121
0.0896 ± 0.0213
0.0657 ± 0.0214 Post-
vitrification 0.0610 ± 0.0223
0.0469 ± 0.0039
0.0498 ± 0.0160
0.0454 ± 0.0107
0.0714 ± 0.0103
0.0625 ± 0.0116
0.0724 ± 0.0238
0.0585 ± 0.0168
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 skim milk 3.75 %) T2 : Treatment group 2 (vitrification with combination EG 7.5 % and skim milk 7.5 %) T3 : Treatment group 3 (vitrification with combination EG 15 % and skim milk 15 %)
FIGURE 1. Pre-vitrification and post-vitrification of 21 rat ovarian tissue weights. The red graphics are vitrified except for natural control group (NC). The same letters indicate no significant differences between experimental group
(one-way ANOVA P > 0.05 and LSD test were not performed due the ANOVA value)
The post-vitrification of ovarian weight on TC1, TC2, and TC3 are 0.0469 ± 0.0039 g; 0.0498 ± 0.0160 g; and 0.0454 ± 0.0107 g, respectively. The decrease of post-vitrification ovarian weight on TC1, TC2, and TC3 are caused by ethylene glycol as intracellular cryoprotectants. However, the results of the statistical tests show that there is no significant difference between post-vitrification weight of ovarian on TC1, TC2, and TC3 against NC, due the results show no significant difference, the multiple comparison test (LSD) was not performed. This condition is allegedly due to the ethylene glycol are high permeability, that it can protect membrane intracellular during vitrification and the least toxic compared to DMSO, PROH, and glycerol [21, 11].
The mean ovarian weight post-vitrification on T1, T2, and T3 are 0.0714 ± 0.0103 g; 0.0625 ± 0.0116 g; and 0.0724 ± 0.0238 g, respectively. The statistical analysis showed there is no significant improvement of ovarian weight on T1, T2, and T3 against NC, TC1, TC2, and TC3. This is due to the use of a combination of ethylene glycol and skim milk as cryoprotectant. Ethylene glycol protects the intracellular membrane and casein in skim milk protects the extracellular membrane. Casein in skim milk affects the viscosity and prevents formation of ice crystals, decreases intracellular cryoprotectant (ethylene glycol) toxicity without compromising vitrification properties. Ethylene glycol can be used as a single cryoprotectant but skim milk cannot be used due the casein has a big molecule, which impossible to penetrate into the intracellular membrane and protect the ovary during vitrification [22, 23]. However, this parameter cannot show the treatment was successful due the results of statistical test show no significant difference between TC1, TC2 and TC3.
CONCLUSION
This study demonstrated that the combination of ethylene glycol and skim milk does not affect the ovarian weight.
The further research is needed to find out which concentration of cryoprotectant is the most optimum to determine the number of normal follicles due the ovarian weight alone cannot explain the follicular condition.
ACKNOWLEDGMENTS
We would like to send our gratitude to Universitas Indonesia and Directorate General for Financial Support by Hibah Publikasi Internasional Terindeks untuk Tugas Akhir (PITTA) UI 2018. We would also to thank the staffs of Laboratory of Animal Physiology of Biology Department, Universitas Indonesia for their courtesy and assistance.
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