Summary of Biology doctoral thesis: Research on oocytes maturation and embryo production from vietnamese Ban native pig by in vitro techniques

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Research is conducted on the Ban Pig model with goals: To determine the potential of Ban Pig's oocyte exploitation; to determine cultivate conditions of the oocyte mature in Ban Pig; success in establishing create Pig’s embryo system through in vitro fertilization (IVF) and cloning (C).

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Nội dung Text: Summary of Biology doctoral thesis: Research on oocytes maturation and embryo production from vietnamese Ban native pig by in vitro techniques

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- NGUYEN THI NHUNG RESEARCH ON OOCYTES MATURATION AND EMBRYOS PRODUCTION FROM VIETNAMESE BAN NATIVE PIG BY IN VITRO TECHNIQUES Majors: Biotechnology Code: 9 42 02 01 SUMMARY OF PH.D THESIS Hanoi, 2021
Thesis was conducted at: 1. Graduate University of science and technology- Vietnam academy of science and technology 2. Insitute of Biotechnology The first instructor: Ph.D. Bui Xuan Nguyen The second instructot: Ph.D. Nguyen Viet Linh PhD dissertation reviewer 1: PhD dissertation reviewer 2: PhD dissertation reviewer 3: The thesis will be presented in Doctoral Thesis Evaluation Committee meeting at Graduate university of science and technology - Vietnam Academy of Science and Technology at ....hr ......., date ....... month.…… 2021. The thesis can be found at: - Library of Graduate university of science and technology - Vietnam National Library - Institute of Biotechnology
AUTHOR’S PUBLICATION RELATED TO THE TOPIC 1. Nguyen Thi Nhung. Nguyen Thi Hong. Nguyen Tien Đat. Nguyen Hoang Thinh. Tamas Somfai. Kazuhiro Kikuchi. Ngo Thi Kim Cuc. Nguyen Thanh Son. Đong Van Quyen. Chu Hoang Ha. Bui Xuan Nguyên. Nguyen Viet Linh. Effects of culture medium on the development of pig embryos obtained by somatics cell nuclear transfer Academia Journal of Biology. 2018. 40(2se):101-105. Doi:10.15625/0866-7160/v40n2se.11610. 2. Nguyen Thi Nhung. Dong Van Quyen. Chu Hoang Ha. Tamas Somfai. Kazuhiro Kikuchi. Ngo Thi Kim Cuc. Nguyen Thi Hong. Bui Xuan Nguyen. Barbara Beck-Woerner. Nguyen Viet Linh. Effect of donor cell type on the development of pig embryos produced by somatic cell nuclear transfer. Journal of Biotechnology 17(2): 1-5. 2019. 3. Nhung Thi Nguyen. Nguyen Xuan Bui. Viet Linh Nguyen. Van Khanh Nguyen. Kazuhiro Kikuchi. Hiep Thi Nguyen. Hong Thi Nguyen. Hoang Thinh Nguyen. Quyen Van Dong. Hoang Ha Chu. Ngo Thi Kim Cuc. Tamas Somfai. Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: the effects of different in vitro oocyte maturation systems. Anim Science Journal. 2020;91:e13412. 4. Nguyen Viet Linh. Thi Hiep Nguyen. Nguyen Thi Nhung. Nguyen Thi Hong. Nguyen Tien Dat. Nguyen Hoang Thinh. Nguyen Khanh Van. Tamas Somfai. Kazuhiro Kikuchi. Optimization of the in vitro fertilization protocol for frozen epididymal sperm with low penetration ability in a native Vietnamese pig. Anim Science Journal. 2018 Aug;89(8):1079-1084.-3. Issue-1. Jan-Feb. 2019 ISSN: 2456-8635. 5. Huynh Thi Huong. Nguyen Thi Nhung. Nguyen Hoang Thinh. Nguyen Viet Linh. The impact of cell lineages gender cultivating in the development of Ban Pig's cloning embryo. The Magazine of Animal Husbandry Association ò Viet Nam. Issue 256 April 2020.
1 INTRODUCTION Development of reproductive biotechnology with the new booming achievements of embryological transfer, in vitro fertilization, cloning, preservation of frozen reproductive and embryonic cells, the interference of reproductive technique and genetic technological therapy on reproductive cells have developed tremendous potential applications in plenty of aspects: improving animal productivity; food security assurance; conservation of biodiversity, the balance of ecosystems and development of modern pharma medical technology. The pigs are one of the most important factors in Biotechnology. They are not only food resources but also are one of the factors in the biological diversity preservation process with giant potential medical applications. Pigs are the big- size animal that has the organ, genome, physiology process, physical and immune system similar to human. So, it is considered that is the most appropriate object for research of transplantation (xenotransplantation). Because of the alive ability of the individual that doesn't carry Porcine Endogenous Retrovirus (PERV), Ban Pig appraised is an important potential object in xenotransplantation technology and the precious genetic genome resources need to be preserved and developed. So we suggest the subject: “ Research on oocytes maturation and embryo production from vietnamese Ban native pig by in vitro techniques”. Goals: Research is conducted on the Ban Pig model with goals: (1). To determine the potential of Ban Pig's oocyte exploitation;(2). To determine cultivate conditions of the oocyte mature in Ban Pig; (3). Success in establishing create Pig’s embryo system through in vitro fertilization (IVF) and cloning (C). Base on researches: (1). Research the structure of ovary and potential of Ban Pig’s oocyte exploitation through the season; (2). Research cultivate oocyte mature by standardized medium method; (3). Research optimization in producing in vitro Ban Pig’s embryos through fertilization medium, condition of fertilization, and embryonic culture; (4). Research produces Ban Pig’s embryos by somatic cell nuclear transfer. The new points in thesis: (1) Thesis provides the new informations about the effects of season, the cultivate medium of oocyte mature, the medium and fertilization regime, cultivate embryos and preservation of frozen in vitro fertilization of embryos, creating cloning embryos by transplantation to establish a successful system of creating Ban Pig's embryos. (2). Being the first research about applying the Piezo method to produce cloning pig’s embryos in Vietnam.
2 CHAPTER 1. RESEARCH OVERVIEW 1.1. Structure of Ovary, oocyte matureness, the fertilization process, and development of in vitro embryos in Pig. 1.1.1. The structure of Ovary and in vivo oocyte mature in Pig The ovary is a part of the female genitalia that has a function in creating oocyte cells (female gamete - oocyte). The ovary cortex area consists of numerous ovarian follicles in the different developed periods, such as classic, primary, secondary, and follicle mature. Ovarian follicles are the basic structural unit of the ovary that has a function in maintaining the oocyte’s development (oogenesis), make sure the ability of fertilization and embryo’s formation. 1.1.2. Fertilization and in vivo embryos development in Pig 1.1.2.1. Stages of fertilization Like many other species, Pig’s fertilization includes many stages such as sperm activation, sperm attaches in zona membrane of the oocyte, acrosome reaction, sperm goes through zona membrane, it gets in oocyte’s cytoplasm and associates with oocyte, the oocyte is activated. 1.1.2.2. Stages of embryo development in Pig After fertilization for 30 hours, the oocyte is cleaved into two cells, quickly keep cleaved become 4,8 cells. The morula is formed on day 3 or 4 after fertilization. It continues to cleaved and form the embryotic follicle and starts nesting in the uterus. 1.1.3. The effect of the season on Pig’s reproductive activities The pig is a multi-pregnancy animal, although Pig’s oocyte exploitation has been through all the season of the year, the season factor has a significant effect on the quantity and quality of the oocyte. 1.2. Research situation in producing in vitro pig’s embryos 1.2.1. Culturing in vitro oocyte mature of Pig, the impact of oocyte’s material resource and oocyte mature cultivate condition. The experiment results in cultivating oocyte mature get from different size follicles shows that the quality and state of follicles have a big impact on oocyte matureness. The medium cultivating oocyte mature usually includes base medium (Hank medium with L-glutamime and Hespes), added the serum (fetal calf serum: FCS) or follicular fluid (FF), and other elements such as gonadotropin, hormone growth factors (LH, FSH), Dibutyryl-cAMP (dbc- AMP).
3 1.2.2. Creation of Pig in vitro fertilization embryos, the impact of sperm quality, fertilization regime, cultivation, and preservation embryos. IVF is controlled by numerous effect factors, not only preservation method and sperm activation but also medium that cultivates oocyte mature, fertilization regime, cultivate embryos medium and preservation method. 1.2.2.1. Preservation and sperm activation In 1988, Nagai and assistances succeed in using frozen Pig’s sperm to create in-vitro embryos. The research of Nagai (1988) and Kikuchi (1998) team show that sperm got from epididymis have cold resistance better than sperm got after emission, maybe frozen and take part in mature oocyte 1.2.2.2. Fertilization, cultivate embryo medium, and the effect factors to fertilization percentage. Two kinds of medium are usually basically used are Tyrode’s albumin lactate pyruvate (TALP) and Fertilization Medium for Porcine oocytes (Pig- FM). Add 10 mg/mL BSA and 2 mmol/L caffeine, ovarian follicular fluid (pFF) into cultivating oocyte and sperm medium, increase the significant percentage of sperm intrusion, cleaved rate, and development of embryos. The medium such as Porcine Zygote Medium-3 (PZM-3) and PZM-5 are considered that have better efficiency in producing embryos, so mostly used in many years recently. The biggest obstacle that affects in vitro Pig’s embryos production efficiency is the polysperm fertilization phenomenon (polyspermic). The percentage of polyspermy embryos fluctuates from 13% to 19%. The cumulus cell plays an important role that affects monosperm fertilization. The highest in vitro efficiency occurs if oocyte cells have an cumulus cell layer and fertilized in concentration 0.5x105 sperm/ml. 1.2.2.3. Preservation of Frozen in vitro Pig’s embryos Researches about frozen in vitro Pig’s embryos have conducted early for a while. Nevertheless, embryo preservation has tremendous struggle because Pig’s embryos are so sensitive to temperature variation. The quickly frozen method with minimum volume (minimum volume cooling: MVC), directly drop embryos into liquid nitrogen method (microdrop), or onto metal surface cooled by liquid nitrogen, new Hollow Fiber Vitrification (HFV) method have used in frozen Pig’s embryos at follicle embryo. 1.2.3. Producing Pig’s embryo by cloning 1.2.3.1. Cloning engineering, methylation, reprogramming in in vitro embryo The technique effects to cloning results.Cloning engineering frequently is a classic technique that uses a microsurgeon microscope to remove and transplant nucleus by microneedle, the remove and transplant technique
4 combines microsurgeon microscope attached to the piezo process and handmade cloning technique. 1.2.3.2. Interspecies somatic cell nuclear transfer Somatic cell nuclear transfer is a technique that produces in vitro embryos by transferring cell nuclear of one species into enucleated oocytes of another species (Interspecies somatic cell nuclear transfer –iSCNT). Interspecies cloning is considered a potential solution to re-create, preserve rare animals that have been in danger of extinction. In 1988, Wells and his assistances announced that they succeed in cloning an individual cow of Enderby Island (only one individual survived from the most valuable poultry in the world). 1.2.3.3. Pig cloning Oocyte quality, season, medium factors such as photoperiod, the temperature can affect oocyte and cloning quality. The research of Korume and his assistant in 2013 show when conducted to transplant cloning embryos, pregnant rates are highest in winter (5.3%) and lowest in summer. Recently, it is announced the highest cloning productivity is 4.1% out of born Pig after transplanting embryos. 1.3. In vitro Ban Pig’s embryos research 1.3.1. Morphological and reproductive features of Ban Pig Ban Pig is considered an ideal object in transplantation technique (xenotransplantation) because it has dimensions, genome features similar to humans. Additionally, Ban Pig is a unique breed only that has low PERV virus copies, so it has powerful potential in medical research and application, especially transplantation. Nowadays, Ban Pig populations have about 30000- 32000 individuals but mostly be hybrid, distribute almost in the high mountain district of Hoa Binh, is there essential concentrate in Da Bac, Cao Phong, Lac Son, Mai Chau, Tan Lac. 1.3.2. Research cultivating mature oocyte and producing embryos of Ban Pig in Vietnam The experiment about cultivating oocytes mature and producing in vitro embryos of Ban Pig deployed the first time in the circle of Science Collaboration Project Vietnam-Japan (JSPS-VAST, 2005-2007). In 2008, Uoc and his assistants announced research of producing interior mini Pig’s embryos by a combination of in vitro fertilization and cloning. The authors also succeed in conducting create embryo testing experiment by transplant Ban Pig’s somatic cells into Landrus Pig’s oocyte cell, with the rate of unity between oocyte and cell is 70% and embryotic follicle rate is 12%.
5 CHAPTER 2- RESEARCH METHODOLOGY 2.2.1. Method of classifying months through the season 2.2.2. Method of collecting and preserving the ovary 2.2.3. Method of measuring dimension and weight of ovary 2.2.4 Method of cleaved follicle group 2.2.5. Collecting oocyte method 2.2.6. Classifying oocyte quality method 2.2.7. Measuring dimension method 2.2.8. Method of transplanting oocyte 2.2.9. Method of apprising matureness after cultivating 2.2.10. Method of freezing sperm from the epididymis 2.2.11. Method of testing sperm quality before and after freezing 2.2.12. In vitro fertilization method 2.2.13. Fertilization state appraising method 2.2.14. Frozen embryo method 2.2.15. Selection and transplantation Pig’s somatic cell method 2.2.16. Method of selection and nourishment Pig’s somatic cell 2.2.16. Cell cultivation method 2.2.18. Cloning method 2.2.19. Embryos nourishment method 2.2.20. Hoechst dyeing method 2.2.21. Orcein dyeing method 2.2.22. Analyze and handle data method
6 METHOD DIAGRAM Surface The potential Normal follicle's Post-harvest of oocyte dimension and dimension oocyte quality exploitation weight survey survey survey through season CONTENT 1 Oocyte mature Select oocyte rate by season mature medium CONTENT 2 Fertilization Select optimum Optimum medium (TALP, medium (TCM- cultivate Pig), sperm 199, POM) oocyte, concentration IVF and 5 6 (1x10 , 1x10 embryo % follicle process TT/ml) embryo, oocyte after optimum cell/embryo IVF fertilization maturity Cumulus status regime Embryos Ban Pig's medium Cafeine Freezing sperm collect (NCSU-37, concentration(2 and PZM-3) mM, 5 mM) and preservation fertilization time Embryo (3 hours, 6 bank hours) CONTENT 3 3 Kind of donor cell Ban Pig cells bank Ban Pig's cloning embryo CLONING producing system Cultivate Embryo Landrace medium Pig's oocyte (NCSU-37, mature PZM-3) CONTENT 4
7 CHAPTER 3 RESULT AND DISCUSSION 3.1. Morphological characteristics of the ovary and the potential exploitation of Ban Pig's oocyte in each season This content was performed by 3 basic research: 1. Survey of the effect of Spring, Summer, Fall, Winter on the size and the volume of Ban Pig's ovaries. 2. Survey of the effect of season on the potential exploitation of the ovary according to the proportion of oocytes types A, B, C/ovary. 3. Survey of the effect of season on the size of oocytes (types A, B) The experiments were conducted on Ban Pigs of the same ages, caring regulation, have a volume of about 20-25kg. 3.3.1. Morphological characteristics of the size of Ban Pig's ovary in each season Table 3.1. Size and weight of Ban pig ovaries according to the seasons Total Spring Summer Fall Winter ovary means means means means SEM (n) SEM SEM SEM Length 10 (17±0.8)a (15±1)b (15.2±0.6)b (17.4±0.8)a (mm) Width 10 (12.2±0.3)a (10.9±0.7)b (11.3±0.4)b (11.9±0.5)a (mm) Thickness 10 (8.1±0.4)a (6.3±0.3)b (7±0.3)b (8±0.5)a (mm) Volume 10 (2.4±0.1)a (2.0±0.2)b (2.1±0.2)b (2.3±0.2)ab (g) Data are presented as means SEM. a.b Differ significantly (p < 0.05). (Aspin-Welch T-test) Our research results on the effects of season on ovarian size and weight in our Ban pigs also different from those of Landrace-Yorkshire pigs by Kyzeková and assistant. These results show that the characteristics of different breeds. weather. food. and feeding regime have a certain influence on the size and volume of ovaries of each breed of pig in each locality. 3.1.2. Distribution of surface cysts of Ban pig ovaries The experiments were conducted with 10 ovaries collected from 5 pigs/season. The results are presented in Table 3.2 shows that the average number of cysts/ovary with sizes of 3-5 mm is similar in size of spring and winter
8 (0.7±0.2; 0.6±0.2). significantly higher than summer and autumn (0.1±0.1; 0.1±0.1) (Table 3.2). Table 3.2. The average number of follicles by size Total follicles (n. follicles/ovaries) Total of follicles Season (n. < 2 mm 2-3 mm 3-5 mm >5mm follicles/ovaries) 102 56 7 165 Spring 0 (0) (10.2±0.7) (5.6±0.3)a (0.7±0.2)a (16.5±0.8)a 93 39 1 133 Summer 0 (0) (9.3±0.6) (3.9±0.4)b (0.1±0.1)b (13.3±0.7)b 105 59 1 165 Fall 0 (0) (10.5±0.5) (5.9±0.3)a (0.1±0.1)b (16.5±0.6)a 94 53 6 153 Winter 0 (0) (9.4±0.4) (5.3±0.4)a (0.6±0.2)a (15.3±0.7)a Data are presented as means SEM. a.b Differ significantly (p
9 of type A oocytes/ovary in spring and winter is higher than in summer and autumn (7.6±0.4 and 8.3±0.8 compared to 6.2±0.4 and 6.8±0.9). 3.1.3.2. The size of Ban Pig's oocyte Survey results for a total of 30 oocytes in each season show that the season does not affect the oocyte size of A+B quality oocytes. In which the oocyte size does not have light membrane verb 110.1±2.4 to 113.6±2.1. Synthesizing the research in this section we find out the effects of season on the harvesting potential of Ban pig's ovaries is shown through the regular changes in the ovarian size correlation. the cyst development. and oocyte quality. Ovaries are collected in spring and winter have larger sizes than those in autumn and summer. the ratio of A+B oocytes obtained/ovary in spring and winter is also higher than in summer and autumn. Similarly. the average number of large follicles (> 2 mm) per ovary was positively correlated with the number of A+B oocytes/ovary and was seasonally dependent. The average number of A+B oocytes obtained/ovary in the spring also highest (18.1). and the number of A+B oocytes collected in winter (17.5). followed by the rate in autumn (16.3) and in summer reached the lowest rate (13.4). The results of our study show that the season does not affect the oocyte size type A+B. The results were similar for the change in the size of the seasonal foreign pig's oocyte have been announced by some authors on foreign pigs. The research by Kyzekova and cs. 2019 showed that oocytes obtained from follicles ≤ 3 mm had the smallest average size in spring and biggest in winter. 3.2. Results of research on maturation of Ban pig oocytes This part of the research was conducted to investigate the maturation ability of Ban pig oocytes according to each season. thereby determining the optimal sampling time. and at the same time selecting a suitable maturation medium with the development of Ban Pig oocytes. The research was arranged with 2 basic experiments: 1) Survey the development status of oocytes in 4 seasons after 44-46 hours of culture in NCSU-37 environment with the following criteria: Oocytes rate in the stage of GV. GVDB. MI. MII. rate of oocytes in the stage other. 2) Research of culturing oocyte matures in the five basic types of medium used today are: NCSU 37+pFF. NCSU- 37+FBS. TCM-199+pFF. TCM-199+FBS. POM and choose the culture medium suitable for the development of Ban pig oocytes. 3.2.1. Effects of season on results of maturity Ban pig oocytes
10 Table 3.5. Maturity of Ban Pig oocytes Season Total GV GVBD MI oocytes MII Other stage oocytes(n oocytes oocytes (n.%) oocytes oocytes ) (n.%) (n.%) (n.%) (n.%) Spring 638 42 90 19 433 54 a (6.6±0.7) (14.1±0.7) (2.9±0.4) (67.8±0.7) (8.4±1.1) Summ 547 52 79 40 322 54 b er (9.5±1.1) (14.5±1.0) (7.3±0.3) (58.8±1.0) (9.9±1.5) Fall 611 47 65 42 375 82 b (7.7±0.9) (10.7±1.3) (6.8±0.7) (61.2±1.8) (13.4±1.7) Winter 644 29 89 25 437 64 a (4.4±0.9) (13.8±0.9) (3.8±0.7) (67.9±0.5) (9.9±1.3) Data are presented as means SEM. a.b Differ significantly (p < 0.05). (Aspin-Welch T- test) The results showed that in the same cultural environment as NCSU- 37+10% pFF. the maturation rate of Ban pig oocytes varies from season to season. specifically in the spring and winter is higher than summer and autumn (67.8±0.7% and 67.9±0.5% compared to 58.8±1.0% and 61.2±1.8%) ( the difference was statistically significant. P
11 rate of oocyte matures in the POM medium and the TCM-199+pFF medium is similar ( 74.5±2.7% and 73.4±2.5%) and higher in NCSU-37+pFF .NCSU- 37+FBS. TCM-199+FBS. (64.1±0.6%; 61.6±2.4%; 55.8±2.0%) (Table 3.6). Thus. the medium TCM-199+pFF and POM is more suitable for the development of Ban pig oocytes. Table 3.6. Results of maturity Ban pig oocytes Medium Total GV oocytes MI oocytes MII Other stage oocytes (n) (n.%) (n.%) oocytes oocytes (n.%) (n.%) NCSU- 131 12 23 84 12 b b 37+pFF (9.0±1.9) (17.9±3.7) (64.1±0.6) (9.0±2.1) NCSU- 130 12 31 80 7 a b 37+FBS (9.3±1.5) (23.8±0.5) (61.6±2.4) (5.2±3.5) TCM- 135 13 8 99 15 c a 199+pFF (9.6±1.3) (5.9±1.2) (73.4±2.5) (11.1±3.0) TCM- 136 15 21 91 24 b c 199+FBS (11.2±2.4) (15.6±1.8) (55.8±2.0) (17.4±2.5) POM 145 11 9 108 17 c a (7.6±1.3) (6.2±0.6) (74.5±2.7) (11.7±3.5) Four replications were performed. each time collecting oocytes from 5 female pigs. Data are presented as means SEM. a.b Differ significantly (p < 0.05). (Aspin-Welch T- test) 3.3. Results of creating Ban pig embryos by in vitro fertilization 3.3.1. Research on freezing of Ban pig sperm 3.3.1.1. Surveying the quality of Ban sperm collected from the epididymis Sperm motility. sperm concentration. survival and abnormal rate pre-and post-cryogenic were examined by the method of sperm quality test presented in the method section. in which each indicator is checked 4 times to calculate the average data. Research results on sperm volume. sperm motility. sperm concentration rate of live sperm. abnormal rate of sperm right after obtained are presented in Table 3.7. From the results of Table 3.7 we see that the survival rate and motility of sperm in all 4 groups of sperm in our study are >50%. in which the potency of TLB 001 and TLB 004 is lower than TLB 002 and TLB 003 (72±1.5 and 76±1.1 versus 50±1.1 and 61.6±1.6%). The results of our study showed that the survival and potency in the group TLB 002 and TLB 003 can be used to freeze and create in vitro embryos. Thus. based on the criteria of sperm after collection. we found that TLB 002 and TLB 003 have the best and the most suitable criteria for IVF research and creation of Ban pig embryos.
12 3.3.1.2. The quality of frozen sperm Frozen sperm were thawed and examined for potency. survival. and abnormal rate. The results showed the potency of TLB 002 and TLB 003 were similar. but the survival rate of sperm after freezing of TLB 003 was higher than TLB 002 (45.8±0.6 vs. 38.6±2.3) (Table 3.8). The survival rate of sperm after freezing in our study was higher than that of the same published in Ban pigs by Nguyen and cs. 2015 Table 3.8. The quality of Ban Pig sperm after freezing Symbol Sperm potency Rate of live Abnormal rate (%) sperm (%) (%) a b TLB 002 26.6±0.8 38.6±2.3 13±0.5 a a TLB 003 25.7±1.4 45.8±0.6 12±1.5 Data are presented as means SEM. a.b Differ significantly (p < 0.05). (Aspin-Welch T-test) 3.3.1.3. Check sperm quality through in vitro fertilization results Oocytes after cultured in TCM-199 were divided into 2 experimental groups: Group fertilized with TLB 002. group 2 fertilized with TLB 003 in Pig FM with a concentration of 1x106 sperm/ml. embryos were cultured in PZM-3 and the division rate and formation blastocysts at day 5. 6. and 7 after fertilization. Table 3.9. Testing the ability of sperm to create embryos after freezing Blastocyst Blastocyst Blastocyst Hatching Cleaved Symbol s day 5 s day 6 s day 7 embryos day (n.%) (n.%) (n.%) (n.%) 7 (n.%) 47 8 12 16 1 TLB 002 (52.2±1.1) (8.9±2.9) (13.3±1.9) (17.7±2.9) (1.1±1.1) 49 9 13 17 4 TLB 003 (54.4±4.4) (10±1.9) (14.4±1.1) (18.8±1.1) (2.2±1.1) Data are presented as means SEM. a.b Differ significantly (p < 0.05). (Aspin-Welch T-test) The results presented in Table 3.9 showed the survival rate and sperm potency after thawing of TLB 003 was higher than that of TLB 002 (Table 3.8). the rate of the blastocyst at days 5.6.7 after fertilization had no statistically significant difference between the 2 groups of spermatozoa. Although the embryogenesis rate in our study is still lower than that of exotic pig breeds. it is relatively high compared to other breeds of pigs with similar studies in domestic pigs that have been previously published. Thus. we have selected and
13 successfully frozen 2 groups of sperm. namely TLB 002 and TLB 003 as raw material for IVF in the next research steps. 3.3.2. Research the optimal fertilization regime 3.3.2.1. Influence of the fertilization medium and the concentration of sperm on the results of in vitro fertilization Table 3.10. Evaluate the fertilization status of the oocytes Fertilizatio Sperm Total Penetrated Normally Monosperm n concentrati oocytes (n.% total) fertilized fertilized culture on (n) (n.% a total) (n.% (sperms/ml Penetrated) ) Pig-FM 1x105 138 19 11 16 b (11.9±3.7) (6.0±3.2) (74.7±18.8) 6 Pig FM 1x10 181 47 21 28 a (25.8±4.4) (11.5±3.3) (61.4±9.9) 5 TALP- 1x10 177 13 7 10 b PVA (7.2±2.1) (3.8±1.3) (78.3±15.7) 6 TALP- 1x10 159 17 4 10 b PVA (10.5±1.8) (2.3±1.1) (63.3±11.0) IVF was performed for 3 hr using oocytes with partially denuded cumulus cells. Five replications were performed. Data are presented as means SEM. a.b Differ significantly (p
14 under a microscope to check the rate of oocytes with sperm penetration. normal fertilization rate and the rate of monosperm fertilization. The results of our research show that it is possible to exploit the procedure of removing most cumulus cells around the oocytes and leaving only a portion of the cells before fertilization to establish an effective IVF Ban pig embryo- forming system. Table 3.11. Effect of cumulus cells layer on fertilization status in Ban pig Cumulus status Total Penetrated Normal Monospermy oocytes (n.% total) fertilization (% (n) (n.% a total) penetrated) 4 3 3 Intact 96 b b (4.2±1.2) (3.1±0.1) (83.3±16.6)b 13 13 13 Partial denuded 87 a a (15.2±1.9) (15.2±1.9) (100±0)a Completely 84 0 (0)c 0 (0) c 0 (NA) denuded IVF was performed in Pig-FM medium. within 3 hours. the concentration was 1x106 sperm/ml. Three replications were performed. Data are presented as means SEM. a.b.c Differ significantly (p
15 The study was performed in Pig-FM medium. partially isolated cumulus cells. at a concentration of 1×106 sperm/ml for 3 hours or 6 hours. Pig-FM fertilization medium supplemented with 2 mmol/l or 5 mmol/l caffeine. The fertilization state was checked by staining 10 hours after fertilization. The results of our research show that when combining increasing caffeine concentration to 5mM with 6 hours IVF interval can increase the penetration rate of oocytes by more than 50%. The penetration rate of sperm into oocytes in the experimental group using the medium containing 5mM/l caffeine. insemination in 6 hours was significantly higher than the group using the medium containing 2mM/l caffeine. in 3 hours (58.8±10.9% compared with 15.2±1.9%) (Table 3.12). Besides. the fertilization rate of single spermatozoa has also increased by over 50%. The results showed that the penetration rate of sperm into oocytes in the experimental group using the medium containing 5mM/l of caffeine. insemination in 6 hours was significantly higher than that of the group using the medium containing 2mM/l caffeine. fertilized in 3 hours (58.8±10.9% compared with 15.2±1.9%). 3.3.3. The effects of maturing culture on fertilization and embryo development 3.3.3.1. Effects of oocytes culture on results of fertilization and embryo culture Table 3.13. Maturity and fertilization of Ban pig oocytes after culturing Culture Total GVBD* Maturity Penetrated Normal Male Monosperm medium oocyte (n.% a total) (n.% a (n.% fertilization pronucleus ic (% s (n) total) GVBD) (n.% (n.% penetrated GVBD) penetrated) TCM- 133 116 101 35 87 40 b b 199+10 134 (99.1±0.8) (85.7±4.4) (75.7±1.3) (26.6±3.5) (86.5±2.1) (40.6±4.0) % pFF POM 142 124 124 45 122 46 a a 145 (97.8±0.9) (85.5±4.5) (87.5±2.9) (32.1±7.1) (98.3±1.1) (38.2±7.2) Five replications were performed. Data are presented as means SEM. a.b.c Differ significantly (p
16 cells layer before fertilization. Oocytes after 10 hours of fertilization are fixed in a culture of 1 acetic acid: 3 ethanol and stained in orcein 1% to evaluate the fertilization state. including the following criteria: the rate of oocytes with spermatozoa. the rate of fertilization. Usually. male pronucleus rate and monosperm fertilization rates. The results showed that there was no difference between the two experimental groups in the maturation rate of oocytes (85.7±4.4% and 85.5±4.5%). The percentage of oocytes with sperm infiltration after IVF in TCM-199+10% pFF medium was lower than in POM medium (75.7±1.3 vs. 87.5±2.9) (Table 3.13. p.89). The fertilized oocytes were cultured in TCM-199 medium and POM medium and monitored for embryo development for 7 days after culturing. In this study. we use POM defined medium supplemented with 10 ng/ml EGF and gonadotrophin as oocytes culture medium. The production of pig embryos using the unidentified medium as TCM-199+10% pFF and NCSU- 37+10% pFF has been previously announced with maturation rates of 87% and 93% and blastocysts formation rates are 19% and 30%. The addition of gonadotrophins to the indeterminate medium (TCM-199+10% pFF) during the first 22 hours has also been used to provide good oocytes development efficiency. Table 3.14. Effects of oocytes culture on the development of Ban pig embryos after fertilization Oocytes Total Mature Cleaved Blastocyst Blastocyst Blastocysts Cells/ Hatching culture oocyt oocyte (n.% a day 5 (n.% day 6 (n.% day 7 blastocy day 7 medium es (n) (n.%) total) total) total) (n.% total) sts (n.% total) Day 7 TCM- 132 111 14 25 32 3 52.1 199+10 195 (67.6±2.5)a (57.4±3.4) (7.5±3.2) (13.3±4.3) (16.8±4.3)a (1.5±1.0) ±1.2b % pFF 147 106 23 34 41 11 75.6 199 a a POM (73.8±3.0) (53.7±3.9) (12.1±3.6) (17.8±5.2) (21.4±5.0) (5.7±1.1) ±3.4a Experiment repeated 5 times. data showing mean±SEM. data with different annotations (a. b) in the same column were statistically significant differences (P 0.05 (Table 3.14).
17 3.3.3.2. Effects of culture medium on the development of in vitro fertilized Ban pig embryos Oocytes were cultured in POM for 44-46 hours. Pig-FM supplemented with 5 mmol/l of caffeine is used as the fertilization medium. remove a part of cumulus cells. the sperm concentration is 1x106 sperm/ml. the time of insemination is 6 hours. The oocytes after IVF were separated from spermatozoa and were divided into 2 experimental groups as follows: Group 1: Embryo culture in NCSU-37; Group 2: Embryo culture in PZM-3. Checking embryos cleaved on day 2. blastocyst rate at day 5.6.7 and the number of embryos on day 7 by staining. Table 3.15. Effects of culture medium on the development of Ban pig embryos after fertilization Grou Total Cleaved Blastocy Hatching p oocyte (n.% a Blastocyst Blastocysts Cells/b stday 5 day 7 s (n) total) day 6 (n.% day 7 (n.% lastocy (n.% (n.% total) total) sts total) total) Day 7 101 16 25 35 5 53.7± 1 195 (51.5±3.2) (8.2±1.2) (12.8±0.4) (17.7±1.4)b b (2.5±0.3)b 6.2b 109 20 34 43 12 72.3± 2 208 (52.3±0.8) (9.5±1.1) (16.3±1.0) (20.6±0.8)a a (5.8±1.7)a 3.9a Three replications were performed. Data are presented as means SEM. a.b.c Differ significantly (p