FERTILIZATION RATE OF CRYOPRESERVED OOCYTES

FERTILIZATION RATE OF CRYOPRESERVED OOCYTES
OF MADURA CATTLE

Sri Wahjuningsih
Faculty of Animal Husbandry, Brawijaya University, Malang

ABSTRACT

The Madura cattle is one of important local cattle, but there no enough available research on oocyte preservation. The aim of this research was to developed oocyte cryopreservation method of Madura cattle using vitrification methods. Medium of vitrification tested was: 40% EG (Ethylene Glycol) + 0.5 M Sucrosa (VS1), 40% GLY (glycerol) + 0.5 M Sucrosa (VS2), 40% DMSO (Dimethylsulfoxide) + 0.5 M Sucrosa (VS3). The viability and the rate of oocyte fertilization after vitrification at in VS1solution) was better (P<0.05) than those were in VS2 and VS3. The vitrification solution of 40% EG + 0.5 M could minimize the damage resulted in vitrification process so that its viability and the fertilization’s rate could be maintained.

Keywords: Fertilization,Oocyte,Madura cattle, Cryopreservation.


INTRODUCTION
Madura cattle is an Indonesian indigenous animal, which their population is getting decreasing nowadays, so that their existence has to be preserved. Until now the studies of Madura cattle are only focused on their characteristics, have never been conducted a study to preserve this indigenous animal using oocyte cryopreservation. Therefore, preserving the indigenous animal will be very interesting to hold the study on Madura cattle oocyte cryopreservation since there is limited report about it. Based on physical phenomena, there are two methods of cryopreservation, those are conventional and vitrification methods. Vitrification method is simple, unexpensive and unnecessary to special equipment in decreasing the temperature step by step, so that it could be applied in place where liquid-nitrogen container available.
The purpose of the research was to develop method for long-term cryopreservation bovine oocyte through improving method of cryopreservation using vitrification method that will be simpler, cheaper, and may be applied especially in under limited facilities in developing country

MATERIAL AND METHODS
Oocyte preparation.
Ovaries were collected from cows at local abattoir and were brought to the laboratory in physiological saline 0.9 % (w/v) at 25 - 30 o C within 3h. Oocytes were aspirated from 2-5 mm follicles with an 18-G needle attached syringe containing PBS (Phosphate Buffered Saline) supplemented with 5 % (v/v) Fetal Calf Serum (FCS) and antibiotics 100 ug/ml streptomycin and 100 IU/ml penicillin G. The oocytes were put into the maturation medium TCM 199 + PMSG 10 IU + hCG 10 IU, cultured for 24 h in 5% CO2 in air at 38.5 C.

Vitrification oocyte
Oocyte were dehydrated by 0.25 M and 0,50 M of sucrose, each of them are 5 minutes, then they are being exposure into vitrification solution for 60 second, then put into 0,25 ml of French straw, then. exposure in nitrogen stem for 10 seconds, straw being put into liquid of nitrogen at -1960 C and saved for 4 weeks for further analysis. Medium of vitrification tested were : (1) 40% EG (Ethylene Glycol) + 0.5 M Sucrosa, (2) 40% GLY (glycerol) + 0.5 M Sucrosa, (3) 40% DMSO (Dimethylsulfoxide ) + 0.5 M Sucrosa.
Statistical analysis
Data was be analyzed using analysis of variance. If there were differences among groups of treatment then they were be analyzed by Duncan`s test for further analysis.

RESULT AND DISCUSSION

Effect of vitrification solution to oocyte viability
The vitrification of oocyte within a solution with 40% of Etylen-Glicol (VS1) showed higher viability (p < 0.05) than another of two solution of vitrification. This result showed that the kinds of cryoprotectant given an effect toward oocyte viability at the all of maturation stages. The rate of oocyte viability after vitrification could be seen on the Table 1.

Table 1. Oocyte viability base on nuclear maturation stages after vitrification in three vitrification solution
Treatment Oocyte Viability in nucleus maturation stages (%)
GV GVBD MT-I MT-II
Control 49/55(89,09)a 40/45(88,89)a 54/68(85,29)a 57/63(90,48)a
VS1 23/68(33,82)b 52/68(76,47)b 54/68(79,42)b 57/68(83,82)b
VS2 18/67(26,86)c 48/68(70,59)c 55/69(79,71)b 48/66(72,73)c
VS3 15/65(23,08)d 48/69(69,57)c 48/68(70,59)c 48/68(70,59)c
The value different superscripts were significantly different (P<0, 05)

There are some factors could influence on the decreasing of oocyte viability, those concentration and kinds of cryoprotectant, oocyte characteristics and temperature. A positive effect of ethylen-glycol had been the best proof in freezing system, conventional or vitrification (Wahjuningsih et al. 2002). After vitrification process of VS1, VS2, or VS3, GV-oocyte showed a lower viability (P<0.05) than GVBD, metaphase I (Mt I) and metaphase II (Mt II). Compared with control-oocyte, the vitrification process of GV-oocyte to VS1, VS2 or VS3 was able to decrease it’s viability (P < 0.05) significantly; and then the oocyte viability at GVBD, MT I and MT II was decreasing after vitrification process to VS2 and VS3.
This result showed that GV-oocyte was more sensitive to vitrification solution than it was at GVBD, MT I and MT II because GV-oocyte has lower permeability than it is at MT I and MT II (Agca et al , 1997). The nature of membrane showed the differences between stages of Oocytes (Agca, et al., 1997). GV and GVBD oocyte had been assessed having lower permeability than Mt-I and Mt-II oocyte and the presence of compacted cumulus could inhibit cryoprotectant enter to the oocyte so they were not enough to protect oocyte toward vitrification process. The lower viability rate after vitrification may be resulted by vitrification and warming process. Based on this research, it was concluded that the rate of viability of Mt-I and Mt-II Oocyte after vitrification were higher than GV and GVBD.
2. The fertilization rate of MT II-oocyte after vitrification process
The vitrification process of MT II oocyte in VS1 (40 % etilen glikol) was showing different significantly (P<0,05) by control oocyte to viability of oocyte rate and fertilization rate. The in vitro fertilization rate of MT-II oocyte after vitrification in VS1(81.25%) was higher than VS2 (68.73%) and VS3 (66.25%)

Table 2. Fertilization rate (%) after vitrification process in VS1, VS2 and VS3
Vitrification The development of pronuclear (%) Viability of
Solution 2PN (%) >2PN (%) Fertilized (%) Unfertilized (%) Oocyte2 (%)
Control 62/80(77,5)a 8/10(10)a 70/80(87,5)a 10/80(12,5)a 59/80(73,75)a
VS1 60/80(75)a 5/80(6,25)b 65/80(81,25)b 15/80(18,75)b 54/80(66,25)b
VS2 40/80(50)b 15/80(18,75)c 55/80(68,75)c 25/80(31,25)c 42/80(52,5)c
VS3 39/80(48,75)c 14/80(17,5)c 53/80(66,25)d 27/80(33,75)d 41/80(51,25)c
Value in one column with different superscripts were significantly different (P<0,05)

The oocyte viability after 18th hours of in vitro insemination was different significantly (P < 0,05) among three of above treatments include 66.25%, 52.5% and 51.25%, each of them at VS1, VS2 and VS3, and significantly lower (p<0.05)than control ones (73.75%). These shown that vitrification process could decrease the ability of Mt-II oocyte in supporting fertilization. Vitrification and warming process caused reformation cell biologically so that they caused decreasing the fertilization rate and further embryonic development (Hyttel, et al., 2000).

CONCLUSION
The viability and fertilization rate after vitrification using 40 % Ethylen Glycol was better than those were Glycerol and Dimethyl Sulfoxide. The vitrification solution of 40% EG + 0.5 M could keep the oocyte’s structure to minimize the damage resulted in vitrification process so that its viability and the fertilization’s rate could be maintained.

ACKNOWLEDGEMENT
This research was founded by Indonesia Toray Science Foundation (ITSF) through The Science and Technology Research Grant. The support is deeply appreciated.

REFERENCES :
Agca, Y., J. Liu, A.T. Peter, E.S. Critser. 1997. Cryoprotectant and water permeability of immature and in vitro matured bovine oocytes. Theriogenology 47:340.
Hochi,S., Kimura,K., Ito,K and Hirabayassi,M., 1996. Effect of nuclear stages during in vitro maturation on the survival of bovine ooytes following vitrification. Theriogenology 46:345. Abstr.
Hyttel, P, G. Vajta and H. Callasen. 2000. Vitrification of bovine oocytes with the open pulled straw method : Ultra structural consequences. Mol. Reprod. and Dev. 56:80-88.
Shaw, JM., A. Oranratnachai and AO. Trounson. 2000. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Therigenology 53:59-72.
Wahjuningsih S., G.Ciptadi, N.Isnaini, Suyadi. 2002. Development of alternative technology to produce bovine embryo using frozen oocytes. Research institution of Brawijaya University. Malang.
Zhu, S., M. Yoshizawa and S. Muramatsu. 1998. Analysis of fertilizability of bovine oocytes cryopreservation in various cryoprotectants after in vitro-maturation and their chromosomes at the first cleavage division. J. Mamm. Ova Research 15:37:42.

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