The haching capacity of bovine embryos obtained from diffeerent quality embryos, diffeerent sources of oocytes and cultured after thawing out frozen

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2008, Vol. 53, N ◦ . 5, pp. 144-148 THE HACHING CAPACITY OF BOVINE EMBRYOS OBTAINED FROM DIFFEERENT QUALITY EMBRYOS, DIFFEERENT SOURCES OF OOCYTES AND CULTURED AFTER THAWING OUT FROZEN Le Ngoc Hoan and Van Le Hang Hanoi National University of Education Abstract. In this research, we have compared the growing capacity after thawing out frozen embryos which had different qualities and were formed from different source of oocytes. The results show that, the embryos which have standard forms, embryonic cells connected closely, without or few seper- ated cells or crushed pieces outside would have high growing capacity of frozen embryos after thawing out. Oocytes collected from live cows were able to form embryos which had higher specific vital force after thawing than ones formed from oocytes collected from slaughtered ovums. 1. Introduction Embryo freezing is a process to transfer from ordinary physiological statement to anabiosis to preserve and keep in low tempratures of -196 o C (in liquid nitrogen). Frozen embryos will still live and grow after thawing out and they can be used to culture on accepted, co-phase in rut cows or used in other studies. Embryo freezing technology has brought many advantages about domestic an- imal variety tasks. Frozen enbryos can be tranported easily from country to country rather than transporting live domestic animals. Transporting frozen embryos avoids unexpectedly disadvantage that changes to animals and avoids infecting epidemic diseases among regions. Moreover, embryo freezing technology is the basic to main- tain a bank of frozen embryos of valuable and rare animals. In this paper, we studied the impact of the embryos' quality on their grow- ing capacity after thawing out to find out the appropriate embryos to preserve and rejecting the decayed ones that have a part in economizing materials, chemical substances and time of embryo forming in vitro. In this research, we have also com- pared the developing capacity of embryos which formed from two different oocyte resources: one group was formed from slaughter-house collected oocytes; the other group was formed from live cows' oocytes collected by ultrasound technology called Ovum Pick Up (OPU). Consequently, we can see that how the source of oocyte 144 The haching capacity of bovine embryos obtained from different quality embryos... impacts on embryo freezing results As a result, it can help us to select the appropri- ate oocyte resource right at the beginning of forming embryos in vitro and embryo preservation. 2. Content 2.1. The impact of embryos'quality on growing capacity of em- bryos after thawing out In this research, we have collected 983 oocytes from slaughtered ovums. The oocytes which were fertilized and grown in vitro until the seventh day and then collected 97 morulas and 53 blastulas. Subsquently, embryos were classified by the method of Lindner, Wright [1]. Table 1. Classification of the quality of embryos by the methodology of Lindner Wright, 1983 The quality of embryos Targets Type A Have standard form, reflect exact embryonic growing stages, membrane is transparent, round; block of embryonic cells is dark, divide equally, clearly; embryonic cells connect closely, without seperated cells or crushed pieces outside. Type B Have standard form, reflect exactly embryonic growing stages; membrane is transparent, round; but block of em- bryonic cells is not dark and equal as embryos of type A; maybe have seperated cells and some crushed pieces. Type C Colour is not identical; embryonic cells connect loosely, have many seperated cells and some crushed pieces that inter- posed or build up small masses. The result of embryos classification before freezing was presented in Figure 1, Figure 2 and Table 2. Figure 1. Photograph of morula classification (magnification 240 times) 145 Le Ngoc Hoan and Van Le Hang Figure 2. Photograph of blastula classification (magnification 240 times) Table 2. Result of embryos classification before freezing The quality of embryos Morula Blastula Number Rate (%) Number Rate (%) Type A 75 77.32 27 50.94 Type B 18 18.56 23 43.40 Type C 4 4.12 3 5.66 Total 97 100 53 100 The Table 2 shows that, in the total number of 97 collected morulas, type A made up a higher ratio than type B (77.52% against 18.56%). Morulas of type C made up the lowest ratio (4.12%). In the total number of 53 formed blastocysts, the ratio of type A is not more differential than type B (50.94% against 43.40%). The collected morulas of type C ratio is the lowest (5.66%). This result would affirm that the developing of embryos in the test-tube growing environment is very good. Figure 3. Absorb embryos into blades of grass to freeze All of 150 collected embryos were frozen in every group of the quality of em- bryos. The frozen process was implemented by one step of frozen method by ethylene glycol of Saitoh et al. [2]. These embryos were absorbed into blades of grass (Figure 3) which contain Ethylene Glycol environ- ment and moved to preservation in liquid nitrogen (-196 o C). After one-week-preservation in a temperature of -196 o C, embryos would be thawed and grown in 48 hours to evaluate the developing of embryos. The result was written in Table 3 and Table 4. 146 The haching capacity of bovine embryos obtained from different quality embryos... Table 3. The development of embryos after thrawing out The quality of embryos Number The number of thawed embryos Membrane-escaped embryos Number Rate (%) Type A 102 102 85 83.33 Type B 41 41 17 41.46 Type C 7 7 0 0.00 Figure 4. Stage of blastula escaping membrane (magnification 240 times) In the Table 3, we have seen that after thawing out from frozen, the ratio of embryos which can grow up to membrane-escaped stage of blastula was a clear difference among different types of embryos. Embryos of type A made up the highest ratio of embryos de- velopment after thawing, achieving 83.33%. Embryos of type B made up much lower developing ratio, only achieved 41.46%. Especially, embryos of type C have not got continued development capacity after thawing out. Therefore, it is seen that there are only embryos of type A and type B which are appropriate for the embryo preservation process. 2.2. The impact of the quality of oocytes on the developing capacity of embryos after thawing out On experiments from cows which were fed in the Camp of Domestic Animal Food Test - within the Vietnamese National Institute of Animal Husbandry (NIAH), we have collected 206 oocytes after 7 ultrasound courses (OPU). These oocytes which were grown familiarly would be kept with sperm to fertilize and continue being grown after 7 days, we have collected 41 appropriate qualitaty embryos (embryos of type A and type B). All of these 41 embryos were frozen and preserved in liquid nitrogen. After one week, embryos were thawed out and grown in 48 hours to appraise their devel- opments. The result were written in the Table 4. Table 4. The development of embryos after thawing out frozen Targets Number Rate (%) Number of thrawed embryos 41 100 Membrane-escaped blastulas 31 75.61 The Table 4 shows that in the total number of 41 thawed-embryos, there were 31 embryos which continued growing up to membrane-escaped stage of blastula, achieving 75.61%. 147 Le Ngoc Hoan and Van Le Hang Figure 5. A graph on comparison of developing capacity of embryos formed from two different sources of frozen oocytes after thawing out In comparison with the result of thawing embryos which were formed from oocytes collected from slaughtered- ovums in our research, the ratio of embryos which were grown up to membrane-escaped stage blastulas col- lected by OPU technology were obvi- ously higher (P < 0.05) (75.61% against with 102/143 = 71.33%) (Figure 5). It is said to be collected oocytes with higher quality by OPU technology, namely: the oocytes were collected from selected cows, with clear origins, and looked after carefully; term of oocytes collecting was short, as usual after absorbing out of cows in 15 - 20 minutes, these oocytes were transmitted to the laboratory. Whereas oocytes collected from slaughtered cows had not a clear source of cows and were not fed carefully; term of oocytes collected approximately at 0 to 4 a.m, then transmitted to the laboratory. Due to a higher quality of oocytes, the embryos which were formed from OPU had a higher quality and the vital force of thawed embryos were specificly higher than the others formed from slaughtered collected oocytes. 3. Conclusion As the comparisons given above, it is said that the quality of embryos af- fected evidently on the developing capacity of embryos after thawing out. There were only embryos of type A and type B having good qualitty appropriate to freeze and preserve embryos. Whereas embryos of type C had not a developing capacity after thawing out. The source of oocytes affected manifestly on the developing capacity of thawed- based embryos. Oocytes collected by OPU which could form numerous high embryos made up a much higher ratio of developing embryos after thawing out than ones formed from slaughtered collected oocytes For this reason, ultilizing sources of oocytes collected by OPU and classifying embryos before freezing to collect the appropriate qualitative embryos (type A, type B) can bring many advantages, shorten time, economize materials and also make up high ratio of developing embryos after thawing out. REFERENCES [1]. Lindner G.M. and Wright R.W., 1983. Bovine embryo morphology and evaluation. Theriogenology, Vol. 20, pp. 407-416. [2]. Saitoh N., Kanagawa H. and Shimihira I, 1995. Manual of bovine embryo transfer. Japan Livestock Technology Association, pp. 87-116. 148