Assessing the Quality of Bovine Embryos Produced In Vitro Through the Inner Cell Mass and Trophectoderm Ratio
Excel Rio S. Maylem1, Ma. Elizabeth DC. Leoveras2, Edwin C. Atabay1, and Eufrocina P. Atabay1
1Reproductive Biotechnology Unit, Philippine Carabao Center National Headquarters,
Science City of Muñoz, Nueva Ecija, Philippines
2Department of Biological Sciences, Central Luzon State University,
Science City of Muñoz, Nueva Ecija
*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Embryo quality and implantation potential are the most important factors influencing the rate of successful pregnancies. These two are related to the occurrence of the three morphogenetic process (i.e., compaction, blastulation, and hatching) and the allocation of embryonic cells to the inner cell mass (ICM) and trophectoderm (TE) in response to proper timing of embryonic development. This research was conducted to determine the allocation of ICM and TE of bovine embryos in vitro in relation to its developmental stage and age. The account of this event can be used as benchmark for comparison of good quality embryos for transfer. Using a defined medium – modified synthetic oviductal fluid for IVC – 85 bovine embryos derived from the slaughter house were assessed for cell number and ICM and TE ratio using the Hoechst 33342-propidium iodide differential staining method. Embryos collected on days 7, 8, and 9 were stained, viewed, and examined using fluorescence microscope and Nikon Imaging Software - Basic Research. The results revealed that in terms of total cell number (mean ± SD), the expanded blastocyst on the 7th day (109.29 ± 41.09) and hatched blastocyst on the 8th day (139.5 ± 43.13) yielded the highest total cell number. From these two stages, chi square test determined that the 7th day expanded blastocyst with an ICM:TE count (ratio) of [34.4 ± 15.4]:[73.2 ± 34.9] (0.47) fits to the 1:3 ratio given for a good quality embryo. The results of the present study indicate that the 7th day expanded bovine blastocyst developmental stage and age has the highest potential for pregnancy when transferred owing to its being able to achieve the desired cell number and ICM and TE.
INTRODUCTION
Improving the in vitro production and embryo transfer technologies (IVEP-ET) has been done to continuously meet the goal of genetic improvement in livestock. The IVEP-ET has been recognized to be the most efficient method in producing large number of superior animals (Selokar et al. 2012). In order to achieve this, a good maternal and paternal linkage will allow the development of quality embryos to be transferred and produce offspring that will satisfy the need for large scale production of milk and meat. . . . . read more
REFERENCES
AHLSTRM A, WESTIN C, REISMER E, WIKLAND M, HARDARSON T. 2011. Trophectoderm Morphology: An Important Parameter for Predicting Live Birth after Single Blastocyst Transfer. Human Reproduction 26(12):3289-96.
ATABAY EC, ATABAY EP, DURAN DH, DE VERA RV, MAMUAD FV, CRUZ LC. 2007. Comparison of in-vitro fertilization and nuclear transfer techniques in the production of buffalo pre-implantation embryos. Phil. J. Vet. Anim. Sci. 33(1):29-37.
DELA FUENTE R, KING WA. 1997. Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos. Cambridge Journals Online 5(4):309-320.
GRISART, B, MASSIP A, DESSY F. 1994. Cinematographic Analysis of Bovine Embryo Development in Serum-Free Oviduct-Conditioned Medium. J. Reprod. Fertil. 101:257-64.
HANSEN PJ. 2006. Realizing the promise the IVF in cattle – an overview. Theriogenology 65:119-125.
HARDY KH, HANDYSIDE AH, WINSTON RML. 1989. The human blastocyst: cell number, death and allocation during late pre implantation development. J. of Development 107:597-604.
HOLM P, CALLESEN H. 1998. In Vivo versus in Vitro Produced Bovine Ova: Similarities and Differences Relevant for Practical Application. Reproduction, Nutrition and Deveopment 38:579-94.
IWASAKI S, YOSHIBA N, USHIJIMA H, WATANABE S, AND NAKAHARA T. 1990. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in in vitro and in vivo. J. Reprod. Fert 90:279-284.
MACHA´TY Z, DAY BN, PRATHER RS. 1998. Development of early Porcine embryos in vitro and in vivo. Biology of Reproduction 59:451-455.
MORI M, OTOI T, SUZUKI T. 2002. Correlation between the Cell Number and Diameter in Bovine Embryos Produced in Vitro. Reprod.Domest.Anim 37(3):181-84.
MUENTHAISONG S, PARNPAI R. 2005. Development of cloned swamp buffalo embryos reconstructed with vitrified oocytes. [Master Dissertation]. Phuket, Thailand. Suranee University of Technology. ISBN 974-533-473-1.
NARULA A, TANEJA M, TOTEY SM. 1996. Morphological development, cell number, and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: the effect of IGF-I. MolReprodDev. Jul 44(3):343-51.
Nikon Imaging Software - Basic Research [Computer software]. Retrieved from https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research
PAPAIOANNOU VE, EBERT KM. 1986. Comparative aspects of embryo manipulation in mammals. In Experimental Approaches to Mammalian Embryonic Development. (Ed., Roger Pedersen and Janet Rossant).
RIVERA RM, YOUNGS CR, FORD SP. 1996. A comparison of the number of inner cell mass and trophectoderm cells of pre implantation Meishan and Yorkshire pig embryos at similar developmental stages. Journal of Reproduction and Fertility 106:111-116.
SEIDEL E, SEIDEL SM. 1991. Training Manual for Embryo Transfer in Cattle. 162p.
SELOKAR NL, SAHA AP, SAINI M, MUZAFFAR M, CHAUAHAN MS, MANIK RS, PALTA P, SINGLA SK. 2012. A protocol for differential staining of inner cell mass and trophectoderm of embryos for evaluation of health status. Current Science 102(9):1256-7.
SRIPUNYA N, SOMFAI T, INABA Y, NAGAI T, IMAI K, AND PARNPAI R. 2010. A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes. J Reprod Dev. 56(1):17.
THOUAS GA, KORFIATIS NA, FRENCH AJ, JONES GM, TROUNSON AO. 2001. Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Journal: Reproductive Biomedicine Online – Reprod Biomed Online 3(1):25-29.
VAN SOOM A, BOERJAN ML, BOLS PE, VANROOSE G, LEIN A, CORYN M, DE KRUIF A. 1997. Timing of Compaction and Inner Cell Allocation in Bovine Embryos Produced in Vivo after Superovulation. Biology of Reproduction 57(5):1041-49.
