Quality cloned embryos derived by oocyte bisection technique for the establishment of porcine embryonic stem cell lines

本試驗目的為改善複製胚之品質以提高複製豬與建立胚胎幹細胞之效率。試驗一測定抗壞血酸 (ascorbic acid, AA) 與曲古菌素A (Trichostatin A: TSA) 以及兩者共同 (TA) 添加對複製豬胚再程式化與發育之影響。各處理對囊胚率以AA處理組 (50 and 100 μg/mL) 之囊胚率高於對照組 (49.6% and 44.0% vs. 30.7%, P < 0.05)，TSA處理組 (30 and 40 nM) 兩者皆為60%，並顯著高於對照組 (29.4, P < 0.05)，而TA處理組之囊胚率則較AA組高 (58.9% vs. 43.5%, P < 0.05)。TSA與TA處理之組蛋白 (acH3K9 and K14)乙醯化程度高於AA與對照組。經由TUNEL分析，AA與TA皆可降低囊胚之細胞凋亡率。試驗二則以胚聚合改善複製胚之品質，將兩個(2×)或三個胚(3×)聚合可提高囊胚率 (63.0% 與 73.6%)，而未經胚聚合之對照組僅35.1%。重組囊胚之總細胞數隨聚合之胚數量而不同 (P < 0.05)，最高為3×之126而最低為對照組之55。比較重組囊胚之內細胞團 (inner cell mass) 與總細胞數發現，2× (25.1%) 與3× (26.1%) 之重組胚總細胞數皆顯著高於對照組者(15.3%)。此外，細胞凋亡之比例在2× (2.7%) 與3× (2.2%) 皆低於對照組 (4.7%)。最後將3×之重組囊胚轉殖入代理孕母後產下七頭仔豬。試驗三之目的在於以重組囊胚建立胚幹細胞株。將2×與3×之重組囊胚培養於飼養層細胞上後，貼盤率、擴增與初級細胞群落皆優於未經胚聚合之對照組 (P < 0.05)。飼養層細胞 (STO與 MEF) 與血清種類 (FBS 與 KSR) 對胚幹細胞之建立並無顯著影響。然而，將胚幹細胞培養於含KSR之培養液，則細胞增殖率下降、外觀異常甚至發生分化或死亡。胚幹細胞培養於α-MEM中(於前第三代)較於DMEM與DMEM/F12中之存活率高 (16.7%, 4.3% 及 6.8%)。此外，初級細胞群落與第三代後仍存活之細胞量，以TSA處理組 (36.7%與26.7%) 顯著高於對照組(23.1%與11.5%)。綜合上述，添加ascorbic acid與TSA並配合不同處理可改善複製胚之發育潛能。胚聚合可提高複製胚之品質，經由細胞數量的增加、多能性基因表現與降低細胞凋亡基因之表現；將3×重組囊胚轉殖入母體亦可產下仔豬。雖然TSA與胚聚合可顯著改善複製豬胚品質與其胚幹細胞建立，但仍需更進一步的研究。The objectives of this study were to improve the quality of cloned embryos for efficient production of cloned pigs and for the establishment of embryonic stem (pES) cell lines derived from cloned embryos. In Study 1, we first investigated the effect of ascorbic acid (AA), Trichostatin A (TSA), and their combined treatment (TA) on the reprogramming and development of cloned pig embryos. The AA-treated embryos (50 and 100 μg/mL) had higher blastocyst rates than controls (49.6% and 44.0% vs. 30.7%, P < 0.05). TSA-treated embryos had a blastocyst rate of 60%, which was significantly higher (P < 0.05) than that of control embryos (29.4%). Higher levels of histone acetylation (acH3K9 and K14) were observed in the TSA- and TA-treated embryos than in the AA-treated and untreated embryos. By TUNEL assay, both AA and TA treatments reduced apoptotic rates of blastocysts. In Study 2, we refined the quality of cloned embryos by embryo aggregation. Aggregation with two (2×) and three (3×) embryos had improved blastocyst rates (63.0% and 73.6%) compared to the non-aggregated group (35.1%). Total cell numbers differed among treatments (P < 0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control. The ICM/total cell number ratio in the 2× group (25.1%) was comparable to that in the 3× group (26.1%), both of which were all significantly better than that in the control group (15.3%). A lower apoptotic cell number was found in the 2× (2.7%) and 3× (2.2%) groups than in the control (4.7%). Seven piglets were born to two surrogates after embryo transfer of 3× aggregated blastocysts. In Study 3, we employed embryo aggregation to produce good quality cloned embryos for ES cell establishment. After culture of aggregated (2× and 3×) blastocysts on feeder cells, rates of embryo attachment, outgrowth formation, and primary colonies were greater (P < 0.05) than those of non-aggregated group. Types of feeder cells (STO and MEF) and serum sources (FBS and KSR) showed no effects on ES cell derivation efficiency. However, we found that ES cells cultured in KSR-containing medium had a low proliferation rate, defected morphology, and eventually differentiated or died in subsequent passages. When DMEM, DMEM/F12, and α-MEM media were compared, apparently more ES cells cultured in α-MEM media (16.7%) survived beyond passage 3 compared to those in DMEM and DMEM/F12 (4.3% and 6.8%). As expected, primary colonies and the amount of ES cells survived more than passage 3 were significantly (P < 0.05) higher in the TSA-treated blastocyst group (36.7% and 26.7%) compared to the control group (23.1% and 11.5%). In conclusion, ascorbic acid and TSA treatments improve development of cloned embryos via differential mechanisms. Cloned embryos have achieved better development and quality via an aggregation procedure, which renders a better survival and full-term development. Although the combined treatment of TSA with embryo aggregation apparently promotes establishment of pig ES cell lines derived from cloned embryos, more studies are required to improve the pluripotency of cloned embryo-derived ES cell lines in pigs.TABLE OF CONTENTS Page ACKNOWLEDGMENTS------------------------i CHINESE ABSTRACT------------------------------------ii ENGLISH ABSTRACT ---------------------------- iii TABLE OF CONTENTS--------------------------- v LIST OF TABLES------------------------------- viii LIST OF FIGURES-----------------------x CHAPTER 1: Literature review-----------------------------1 1. Introduction-----------------------2 Part I: Somatic cell nuclear transfer (SCNT)----------5 2. Handmade cloning (HMC) or oocyte bisection cloning technology (OBCT)--7 3. Oocyte bisection cloning procedure ------- 8 3.1. Zona pellucida removal--------10 3.2. Enucleation--------------------10 3.3. Fusion and activation----------12 3.4. Embryo culture-------------13 4. Factors effecting SCNT-----------------13 4.1. Quality of recipient oocytes---13 4.2. Enucleation methods----------15 4.3. Fusion and activation methods--17 4.4. Nuclear reprogramming and genomic activation-----------21 4.5. Epigenetic effects------------25 4.6. Strategies for improving cloned embryo development and quality----------28 Part II: Embryonic stem (ES) cells-------------------39 5. Establishment of ES cell lines from porcine embryos (pES cells)---39 5.1. The age and the source of embryos-40 5.2. Inner cell mass (ICM) isolation---41 5.3. Culture conditions------------ 42 5.4. Feeder cell layers---------------44 5.5. ES cell characterization----------------------45 CHAPTER 2 Study 1: The effect of Trichostatin A and ascorbic acid treatments on nuclear reprogramming and development of cloned porcine embryos--------47 1. Abstract----------48 2. Introduction-----------------------49 3. Materials and Methods-----------------51 4. Results--------------------60 5. Discussion---------------------76 CHAPTER 3 Study 2: Birth of cloned miniature pigs by aggregation of embryos cloned by oocyte bisection cloning technique at the 4-cell stage--------------------80 1. Abstract--------------------81 2. Introduction-------------------------82 3. Materials and Methods------------------84 4. Results-------------------------92 5. Discussion--------------------106 CHAPTER 4 Study 3: Cloned porcine ES-like cell lines derived from the aggregation of 4-cell embryos cloned by oocyte bisection cloning technique-------------111 1. Abstract--------------------------112 2. Introduction------------------------113 3. Materials and Methods---------------115 4. Results----------------------124 5. Discussion---------------------140 CONCLUSIONS------------------------------------144 APPENDICES------------------------145 COMET assay--------------------146 TUNEL assay----------------------147 In vitro fertilization----------------------148 Fusion and activation medium------------154 TCM199 medium for porcine oocyte maturation---156 Porcine zygote medium (PZM-3)---------------157 Preparation of feeder cells------------------159 Cryopreservation of MEF and STO cells--------161 Preparation pES cell culture medium---------163 Alkaline phosphatase (AP) staining-----------166 REFERENCES-------------------------------------------16

Improving feed efficiency and antioxidant properties in Thai native chickens by supplementing Thai rice paddy herb (Limnophila aromatic (Lam.) Merr.) powder

This study aimed to investigate the benefits of adding Thai rice paddy herb (Limnophila aromatic (Lam.) Merr.) powder to the diet of Thai native chickens. Using a randomized completely block design (RCBD), 200 mixed-gender chickens were assigned to groups that received varying amounts of rice paddy herb powder (RPHP): 0% (Control), 1% (RPHP1), 2% (RPHP2), and 3% (RPHP3), along with oxytetracycline (OTC) at 0.25% in the diet. The addition of RPHP to the diet did not result in significant changes in body weight, body weight gain, or average daily gain (P>0.05). When compared to the control group, the RPHP-supplemented group had significantly lower feed intake and feed conversion ratio (P0.05). In contrast, the inclusion of OTC increased total feed costs, resulting in a lower benefit-cost ratio (P0.05). Additionally, supplementation of RPHP, particularly RPHP3, increased SOD and T-AOC levels in meat while decreasing O2- and MDA levels (P<0.05). Taken together, these findings indicated that Thai native chickens fed Thai rice paddy herb powder increased feed efficiency without changing meat quality or carcass characteristics. The inclusion of RPHP at a concentration of 3% in the diet showed potential in enhancing the antioxidant properties of meat

Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos

Novel histone deacetylase inhibitors and embryo aggregation enhance cloned embryo development and ES cell derivation in pigs.

The histone deacetylase inhibitor (HDACi) has been investigated for treating cancers and many other diseases as well as enhancing the reprogramming efficiency in cloned embryos for decades. In the present study, we investigated the effects of two novel HDAC inhibitors, i.e., HDACi-14 and -79, at the concentrations of 0, 1, 2, or 4 μM on the development of embryos cloned by the oocyte bisection cloning technique (OBCT). Blastocyst rates for the reconstructed embryos reached 60% in the 2 μM HDACi-14-treated groups, which was higher (P 0.05). Histone acetylation profile by both HDACi-14 (2 μM) and -79 (2 μM) treatments demonstrated a drastic increase (P < 0.05) mainly in two-cell stage embryos when compared to the control group. After seeding on the feeder cells, the aggregated cloned blastocysts produced by the HDACi-79 treatment showed a significant increase of primary outgrowths compared to the control group (60.0% vs. 42.9%; P < 0.05). Finally, the cloned embryo-derived ES cell lines from aggregated cloned embryos produced from the HDACi-79-treated, HDACi-14-treated and control groups were established (5, 3, and 2 lines, respectively). In conclusion, the novel histone deacetylation inhibitors improve blastocyst formation and potentially increase the derivation efficiency of ES cell lines from the cloned porcine embryos produced in vitro. Depending on the purposes, some fine-tuning may be required to maximize its beneficial effects of these newly synthesized chemicals

Trichostatin A and Ascorbic Acid Assist in the Development of Porcine Handmade Cloned Embryos via Different Physiologic Pathways

The objective was to determine the effects of ascorbic acid (AA), trichostatin A (TSA), and their combined treatment (TA) on reprogramming and development of cloned porcine embryos. Embryos treated with AA (50 and 100 mg/mL) had a higher blastocyst rate than controls (49.6% and 44.0% vs 30.7%, P < .05). Blastocyst rates of handmade cloned (HMC) embryos were nearly 60% in both the 30 and 40 nmol/L TSA treatment groups, which were higher (P < .05) than the control (29.4%). The TA treatment groups had a higher blastocyst rate compared with the AA treatment alone (58.9% vs 43.5%, P < .05). Histone acetylation was much higher in the TSA and TA treatments (primarily in 2- and 4-celled embryos) but was not significantly different between AA-treated and untreated embryos. Both AA and TA treatments reduced apoptotic rates of blastocysts. In conclusion, AA supplementation improved blastocyst development in porcine HMC embryos mainly by a traditional antioxidant pathway rather than by cellular reprogramming

Apoptotic cells of blastocysts from different HDAC trreatments were detected by TUNEL assay.

Hoechst staining for the total cell counts; dUTP: Green color represents apoptotic cells. No significant difference was observed among groups (P > 0.05).</p