NUSANTARA BIOSCIENCE ISSN: 2087-3948 Vol. 9, No. 2, pp. 202-208 E-ISSN: 2087-3956 May 2017 DOI: 10.13057/nusbiosci/n090216 Fertilization and development of mice (Mus musculus) embryo in vitro after supplementing the extract of Pandanus conoideus KARTINI ERIANI 1,♥ , PUTRI LAILAN TIFANI 1 , SYAHRUDDIN SAID 2 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. Jl. Syeh Abdurrauf No. 3, Banda Aceh 23111, Aceh, Indonesia. Tel. +62-85277060751, ♥ email: kartini_eriani@unsyiah.ac.id 2 Research Center for Biotechnology, Indonesian Institute of Sciences, Jl.Raya Bogor Km 46 Cibinong, Bogor 16911, West Java, Indonesia Manuscript received: 31 August 2016. Revision accepted: 20 April 2017. Abstract. Eriani K, Tifani PL, Said S. 2017. Fertilization and development of mice (Mus musculus) embryo in vitro after supplementing the extract of Pandanus conoideus. Nusantara Bioscience 9: 202-208. An extract of red fruit (ERF/Pandanus conoideus Lam.) contains beta-carotene and alpha-tocopherol, i.e. antioxidant compounds group which can stop the formation of free radicals during in vitro fertilization. Reactive oxygen species (ROS) is a free radical which resulted in an oxygen derivate. This research aimed to determine the effect of ERF supplementation on fertilization rate and embryo in vitro development of mice. The study used Completely Randomized Design (CRD) with three group of treatments (ERF dosage supplement: 0 mL, 0.05 mL, and 0.1 mL) and three replications. Each dosage was added to five mice in 7 days. In total, 45 samples were used. Research procedure included extraction of P. conoideus Lam, treatment on the sample, superovulation, preparation of sperm, oocyte collection, in vitro fertilization, and in vitro culture. Parameters observed were the quality of oocyte, the fertilization rate of mice and the development stage of in vitro pre-implantation mice embryo. Acquired data analyzed quantitatively using One Way ANAVA and Duncan’s Multiple Range Test (DMRT) at 5% significance level. The result showed that all treatments did not affect significantly (p>0.05) on the quality of oocyte, in vitro fertilization of oocyte and in vitro early development of embryo. Suggested for further research about the improvement technique of in vitro fertilization and early embryo development. Keywords: Extract Pandanus conoideus, in vitro fertilization, early embryo development, in vitro culture INTRODUCTION The development of biotechnology nowadays plays a vital role in many areas such as agriculture, farming, fishery, medicine, and health. Gordon (1994) stated that one of the advantages of biotechnology in farming is increasing farming product including reproductive technology such as artificial insemination, embryo transfer, cryopreservation, in vitro fertilization (IVF), spermatozoa sexing, embryo cloning, genetic engineering, and other forms of biotechnology in veterinary area. One of the present reproductive technology that has been explored widely is in vitro embryo production (IVEP). In vitro embryo production is a form of assisted reproductive technology (ART) consisted of in vitro maturation (IVM), in vitro fertilization and in vitro culture (IVC). Rabbit, mice, human, pig, cow and sheep embryos have been successfully produced (Hafez and Hafez 2000). The success of in vitro fertilization depends a lot on the quality of oocytes. Oocytes found in female reproductive duct will have good qualities if it is free of free radicals. Female reproductive duct naturally produces free radicals through metabolism process. Under certain conditions, in the body, free radicals are formed a lot more than antioxidant which works as free-radical neutralizer by binding the free-radicals. Reactive oxygen species (ROS) is free radicals in the form of oxygen derivative (Agarwal et al. 2003). Reactive oxygen species can cause damage to cell structure like mitochondria and microtubule. It also causes the disruption of cell function when the critical concentration of ROS is overwhelming (Roushandeh et al. 2008). Thus, influence the qualities of oocyte, spermatozoa, embryo and also the media culture (Agarwal et al. 2005). Cell physiological functions must be kept normal by constant inactivation of ROS or by keeping free radicals at the low number by giving antioxidants such as tocopherol (vitamin E) and beta-carotene (vitamin A) (Sarungallo et al.2015). Red fruit (Pandanus conoideus Lam) is an endemic fruit from Papua and has been used by local people as a natural antioxidant. According to Sarungallo et al (2015) P. conoideus contains a natural antioxidant indicated by the high level of α-tocopherol and β-carotene. Alpha- tocopherol and β-carotene are antioxidant compounds that can bind to free radicals (Wojcik et al. 2010). Alpha- tocopherol (vitamin E) is known as a secondary antioxidant that prevents the forming of free radicals, inactivate oxygen singlet and prevents lipid peroxidation on the plasma membrane (Rajalakshmi and Narasimhan 1996). β-carotene is a pigment isoprenoid in plants that can destruct and inactivate ROS and protects cells from oxidative damage (Bendich and Saphiro 1986). The study conducted by Chen et al (2011) reported that P. conoideus contains both antioxidant compounds, each 700 ppm of α-tocopherol and 500 ppm of β-carotene (Schierle et al. 2003). The antioxidant compounds in P. conoideus are expected to bind to ROS found in the female reproductive system.