The study aims to find out maturity dan apoptosisrate of cumulus -oocyte complex in aceh cattle 

after in vitro maturation (IVF). The oocytes used in this  study  amounted  to  95  oocytes from ovary waste  taken  from  slaughterhouse.  The  oocytes  were  collected  by  aspiration  method.  All oocytes were maturated  in DMEM medium at 38°C 5% CO2 for 20 hours. After maturation, maturity   and apoptosis rate of cumulus 

- oocyte complexwas calculated.  

The  results  showed  that    the  maturity  rate  of  aceh  cattle  oocytes  reached  65.71%  and  the apoptosis  rate  reached  13.3%.  Based  on  the  results  of  this  study  it  can  be  concluded  that  the oocytes of aceh catt

le  have good maturity  rate   and  low apoptosis rate ofcumulus - oocyte complex 

after in vitro maturatio

Aulanni'am

Hamny

Panjaitan, Budianto

Widjiati

Universitas Airlangga Repository

                                               Journal of International Dental and Medical Research ISSN 1309-100X                           Cumulus - Oocyte Complex in Aceh Cattle  http://www.jidmr.com                                                                                                                                            Hamny Hamny, and et al        Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017    Page 1066   Maturity and  Apoptosis Rate of Cumulus - Oocyte Complex in Aceh Cattle  after in Vitro Maturation     Hamny Hamny1*, Widjiati Widjiati2, Aulanni’am Aulanni’am3, Budianto Panjaitan1    1. Veterinary Medicine Faculty of  Syiah Kuala University.  2. Veterinary Medicine Faculty of  Airlangga University.  3. Mathematics and Science Faculty of Brawijaya University.    Abstract        The study aims to find out maturity dan apoptosisrate of cumulus -oocyte complex in aceh cattle after in vitro maturation (IVF). The oocytes  used in this study amounted to 95 oocytes from ovary waste taken from slaughterhouse. The oocytes were collected by aspiration method. All  oocytes were maturated  in DMEM medium at 38°C 5% CO2 for 20 hours. After maturation, maturity   and apoptosis rate of cumulus -oocyte complexwas calculated.          The results showed that  the maturity rate of aceh cattle oocytes reached 65.71% and the apoptosis rate reached 13.3%. Based on the results of this study it can be concluded that the oocytes of aceh cattle have good maturity rate  and low apoptosis rate ofcumulus - oocytecomplex after in vitro maturation.  Experimental article (J Int Dent Med Res 2017; 10(3): pp. 1066-1069)       Keywords: Aceh cattle, oocyte, in vitro maturation, apoptosis.        Received date: 30 August 2017                                                Accept date: 16 October 2017          Introduction    Development of aceh cattle as one of genetic resources of beef cattle has been attempted. Various introduction on reproductive technology on aceh cattle keeps being applied in order to obtain maximum productivity and good reproduction. Aceh cattle is one of genetic resources with its population kept and maintained to increase food availability in Indonesia particularly in Aceh Province. Some researchers have applied reproductive technology on aceh cattle, however, the result is not satisfactory1. Reproductive technology  has been much applied on livestock such as cattle. The technology covers in vitro fertilization, in vitro embryo production, embryo transfer, ICSI  (intracytoplasmic sperm injection), and cloning. However, the practice to help aceh cattle development is not maximum yet.     Development  of  rapid  reproductive technology shows that processes that take place inside animal body is able to be done outside animal body which is known as in vitro. In vitro oocyte maturation is conducted in order that primary oocyte is able to complete meiosis process so that it will develop into secondary oocyte which is haploid and has ability to be successfully fertilized by spermatozoa and is able to support next embryo development. During ovulation, oocyte is at break stage of metaphase II until oocyte activation takes place to continue development2,3. Maternal mRNA accumulation and molecular and structural change such as increase of number and size of organelle take place during cytoplasm maturity. Change on the cell organelle is able to make oocyte have ability to support fertilization process and embryo development4. Until now, research on in vitro oocyte maturation process and evaluation on oocyte apoptosis after in vitro maturation on aceh cattle have not been conducted yet.    The study aims to find out aceh cattle oocyte rate after in vitro maturation and evaluate apoptosis of maturatedoocyte. The result of the study is hoped to be able to make use of reproductive technology to help development of aceh cattle population particularly in Aceh Province.    *Corresponding author:  Hamny Hamny   Veterinary Medicine Faculty of  Syiah Kuala University.  E-mail: widjiati1962@gmail.com; initiald.1808da@gmail.com       Journal of International Dental and Medical Research ISSN 1309-100X                           Cumulus - Oocyte Complex in Aceh Cattle  http://www.jidmr.com                                                                                                                                            Hamny Hamny, and et al        Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017    Page 1067   Materials and methods    Aceh cattle oocyte collection   The study used six pairs of ovaries of grown aceh cattle. The ovaries were obtained from slaughterhouse. The ovaries were washed with physiological NaCl  0.95% supplemented  with  gentamycin  0.5 ml and oocyte collection process was then carried out. Oocyte collection was done by aspiration using spuit with needle 18G containing solution of phosphate buffer saline(PBS) 5 ml. Collected Oocytes were put in sterile petri dish and then continued with observation under microscope to observe oocytes used. Oocytes used had normal morphology with characteristics of homogeneous cytoplasm, whole membrane, and surrounded by cumulus cells.   There were eight aceh cattle oocytes successfully collected. Collected oocytes were washed three times in solution of  PBS and then washed using maturation medium of DMEM  (Dulbecco’s modified Eagle’s medium) three times.     In vitro oocyte maturation   Cleaned oocytes were moved to DMEM medium as maturation medium for maturation process which was covered with mineral oil (Sigma, USA). Maturation process was done in  CO2incubator5%  at the temperature of  38ºC for twenty hours until cumulus cells expansion took place.    Examination of cumulus oocyte complex maturity rate    Examination of cumulus oocyte complex maturity rate was conducted microscopically by counting number of mature oocytes based on expansion rate of cumulus cells. Mature oocytes were marked by loose cumulus cells and hyaluronic acid secretion that seems to have expansion. Data obtained were tabulated and percentage was counted.     Examination of cumulus oocyte complex apoptosisusing immunocytochemistry   Examination on apoptosis was conducted using immunocytochemistry method by using antibody to cells that experiencedapoptosis. Mature oocytes from in vitro process were put in object glass and fixated using fixation solution of  mixed metanol andacetic acid glacial (1:3). Fixated oocytes were destained using tunnel method.         Results    Aceh cattle oocytes which were successfully collected amounted to 95 oocytes which met requirement for in vitro maturation. Collected oocytes were then maturated in DMEM medium. Result on in vitro maturation of aceh cattle oocytes is shown in Table 1, Figure 1 and Figure 2.        Table 1. Maturity rate of aceh cattle oocytes after 20 hour in vitro maturation.     Figure 1. Percentage of aceh cattle oocytes after in vitro maturation for 22 hours.       Journal of International Dental and Medical Research ISSN 1309-100X                           Cumulus - Oocyte Complex in Aceh Cattle  http://www.jidmr.com                                                                                                                                            Hamny Hamny, and et al        Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017    Page 1068     Figure 2. Aceh cattle oocytes after in vitro maturation for 20 hours. The figure shows that cumulus cells spread equally to surround oocytes.  A : oocytes, B : cumulus cells.    Based on Table 1 and Figure 1, it is known that aceh cattle oocytes were able to experience in vitro maturation with the percentage of 65.71%. Oocytes after maturation were marked by spread of loose cumulus oophorus which surrounded oocytes and had light color (Figure 2).     Table 2. Apoptosis examination on cumulus oocyte complex after in vitro maturation.      Figure3. Percentage of apoptosis on aceh cattle cumulus oocyte complex after in vitro maturation.       Figure 4. Result of apoptosis examination using immunocytochemistry method. A. Granulosa cell that experiences apoptosis shown by expression of brown color B. Oocyte that experiences apoptosis. C. Cumulus oocyte complex that does not experience apoptosis.    Apoptosis on  granulosa cell and cumulus oocyte complex of aceh cattle after in vitro maturation was expressed by brown color on both structures that indicated bond of antigen and antibody  visualized by chromogenic.      Discussion    Number of oocytes which was successfully collected was relatively low. It was caused by ovary samples taken were waste from slaughterhouse. Regulation that stated that productive female cattle was forbidden to be slaughtered caused relatively low number of ovary waste. Besides, reproductive status of cattle whose ovary was taken was not surely known. There was possibility that ovary was in luteal phase where follicle growth was not as much as active ovary in follicular phase.   Maturity rate of aceh cattle oocytes which reached only 65.71% was also influenced by another factor such as unfresh ovary due to far distance of slaughterhouse and laboratory where oocyte collection was conducted. In addition, Gordon (1994) stated that a factor which influenced the success of oocyte maturity was how   Journal of International Dental and Medical Research ISSN 1309-100X                           Cumulus - Oocyte Complex in Aceh Cattle  http://www.jidmr.com                                                                                                                                            Hamny Hamny, and et al        Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017    Page 1069   to take or collect oocyte from ovary. Technique of taking oocyte influenced oocyte quality5. Proper technique of taking oocyte will not damage cumulus and oocyte. Besides, culture medium used in the study is also able to influence. The study used DMEM as culture medium. It stated that medium type for oocyte and embryo culture highly influenced every stage of oocyte and embryo growth6. In sheep, combined TCM-199 medium was good for oocyte maturation and CR1aa was good for fertilization medium and embryo culture6. In some studies, there are several types of medium culture that are able to be used for in vitro maturation besides DMEM, such as KSOM, Tissue culture medium (TCM)-199, dan Charles Rosenkrans  (CR)1aa TCM-199 medium used for in vitro embryo production of cattle7 and culture cattle embryo on CR1aa medium8,9.    In addition to maturation data, examination on apoptosis that took place in granulosa cell and cumulus oocyte complex of aceh cattle after in vitro maturation was also conducted. Result of examination on apoptosis is presented at Table 2, Figure 3, and Figure 4. Based on the result of the study, out of 30 cumulus oocyte complexes after in vitro maturation, only 13.33% of them experienced apoptosis whereas 86.67% of them did not experience apoptosis.   Brown color showed granulosa cell and cumulus oocyte complex cell that experienced apoptosis so that it would influence nutritional supply from cumulus to oocyte. It led to oocyte disability to have perfect maturity.  Whereas, aceh cattle cumulus oocyte complex that did not experience apoptosis was expressed by staining counter color that is methil green. Cumulus oocyte complex that did not experience apoptosis caused oocyte ability to reach perfect maturity that is metaphase II stage.   Many factors influenced apoptosis to take place and accelerate the process. One of them was medium type used for culture. Medium type was able to influence apoptosis on cumulus cells and was able to reduce oocyte ability to develop to next stage10. Based on kinetic research on oocyte maturation it was known that apoptosis was getting higher as oocyte reached metaphase II which11 likely was caused by reactive oxygen species (ROS)which was increasing too on cumulus cells12 and oocyte13. High Reactive oxygen species made cumulus cells and oocyte become toxic and finally experienced death (apoptosis).    Conclusions     Based on the result of the study, it is concluded that aceh cattle oocyte has ability for in vitro maturation in DMEM medium with low apoptosis rate. Aceh cattle oocyte from slaughterhouse waste has potency to produce mature oocyte by applying in vitro maturation technology.    Acknowledgements     The authors acknowledged the Hibah  Penelitian Antar Perguruan Tinggi (PEKERTI)  2016-2017 for financial aid for the grant and also drh. Dewit for helping the authors during research.      Declaration of Interest      The authors report no conflict of interest.       References    1. Siregar TN, Ruby N,Riady G, Amiruddin. Efek pemberian PMSG terhadap efek respon ovarium dan kualitas oosit kambing lokal prepuber. J. Media Kedokteran. 2004;20(3).  2. Whitaker M. Control of meiotic arrests. Rev. Reprod. 1996; 1:127-35.  3. Crozet, N. M. Dahriel, L. Gall. Meiotic competence of in vitro grown goat oocytes. J.Reprod. Fertil. 2000;118:367-73.    4. Ebner, T., M. Moser, M. Sommergruber, M. Puchner, R. Wiesinger, G. Tews. Developmental competence of oocytes showing increased cytoplasmic viscosity. Hum. Reprod. 2003;18:1294-8.  5. Gordon, I. Laboratory production of cattle embryos. CAB International, Dublin 1994.  6. Yulnawati. Optimalisasi produksi embrio domba secara in vitro : penggunaan medium CR1aa dan pengaruh status reproduksi ovarium. Tesis. Program Pascasarjana Institut Pertanian Bogor, Bogor. 2006.  7. Gandhi AP, Lane M, Gardner DK, Krisher I. A single medium supports development of bovine embryos throughout maturation, fertilization, and culture. Hum. Reprod. 2000;15:395-01.    Journal of International Dental and Medical Research ISSN 1309-100X                           Cumulus - Oocyte Complex in Aceh Cattle  http://www.jidmr.com                                                                                                                                            Hamny Hamny, and et al        Volume ∙ 10 ∙ Number ∙ 3 ∙ 2017    Page 1070   8. Rokentrans CF, First NL. Effects of free amino acids and vitamins on cleavage and development rate of bovine zygotes in vitro. J. Anim. Sci. 1994;72:434-7.  9. Rokentrans CF, Zeng GQ, McNamara GT, Schoff PK, First NL. Development of bovine embryos in vitro as effected by energy substrate. Biol. Reprod. 1993; 49:459-62.  10. Salhab M, Tosca L, Cabau C et al. Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion. Theriogenology. 2011;75:90–104.  11. Ikeda S, Imai H, Yamada M. Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes. Reproduction. 2003;25:369 –76.  12. Cetica PD, Pintos LN, Dalvit GC, Beconi MT. Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life 2001;51:57– 64..  13. Morado SA, Cetica PD, Beconi MT, Dalvit GC. Reactive oxygen species in bovine oocyte maturation in vitro. Reprod Fertil Dev.  2009;21:608 –14.    

Maturity and Apoptosis Rate of Cumulus - Oocyte Complex in Aceh Cattle After in Vitro Maturation

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Maturity and Apoptosis Rate of Cumulus - Oocyte Complex in Aceh Cattle After in Vitro Maturation

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