Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 153-161
Author(s):  
Jacek A. Modliński
Keyword(s):  
Survival Rate ◽  
Cytochalasin B ◽  
Single Case ◽  
Cell Stage ◽  
Morula Stage ◽  
Homozygous Diploid ◽  
Haploid Embryos ◽  
Early Developmental

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos, which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72–80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.

68 TRANSCRIPTIONAL GENOME ACTIVATION IN CANINE EMBRYOS COLLECTED IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 146 ◽  
Author(s):  
S. Chastant-Maillard ◽  
C. Viaris de Lesegno ◽  
S. Thoumire ◽  
M. Chebrout ◽  
K. Reynaud
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Staining Intensity ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Stage ◽  
Morula Stage

Early embryonic stages are supported by maternal transcripts from the oocyte cytoplasm. Progressive transcription of embryonic genome is a key step for further embryonic development, especially during in vitro culture. To date, in vitro culture from fertilization to the blastocyst stage is inefficient in the canine species. The objective of this work was to identify minor and major activation in in vivo-produced dog embryos. Ovariectomies were performed in 31 Beagle bitches from 102 to 266 h after ovulation (post-ov), precisely timed by transabdominal ultrasonography. Embryos were collected by tubal flushing with M199-Hepes and immediately transferred into transcription buffer. Transcriptional activity was evaluated through 5-bromouridine 5′-triphosphate (BrUTP) incorporation in nascent RNA, without microinjection (Aoki et al. 1997). Oocytes from anoestrus ovaries were used as positive controls. 5-Bromouridine 5′-triphosphate incorporation was revealed by immunocytochemistry (anti-bromodeoxyuridine primary antibody) and embryonic DNA was stained by ethidium homodimer-2. Staining was quantified under laser scanning confocal microscopy. Transcriptional activity was calculated as (mean nuclear intensity – cytoplasmic mean intensity) × nuclear area and expressed in arbitrary units (AU). It was compared to 1 (similar intensity in nucleus and cytoplasm; i.e. no transcriptional activity) by t-test; levels of transcriptional activity were compared between stages by variance analysis. Seventy embryos (from 7 to 21 per stage) from 31 bitches were analysed, from 2 pronuclei to morula stage. Between 28 and 125 nuclei were quantified per stage. At each stage, transcriptional activity was calculated per embryo and per nucleus. A significant transcriptional activity was detected as early as the 2 pronuclei stage (102–132 h post-ov; 1.15 ± 0.05 AU). Transcriptional activity per embryo significantly increased between the 2- and the 4-cell stage and between the 8-cell and the morula stage. In early 8-cell embryos, staining intensity of the various nuclei was markedly heterogeneous within the same embryo, all nuclei being intensively stained from the late 8-cell stage onwards. Transcriptional activity per nucleus increased also from the 2- to the 4-cell stage (respectively, 120–161 h post-ov, 1.15 ± 0.02 AU and 133–154 h post-ov, 1.35 ± 0.04 AU) until the 8-cell stage (153–225 h post-ov, 5.12 ± 0.55 AU). Transcriptional levels at these 3 stages differed significantly. It decreased between the 8-cell and the morula stage (230–266 h post-ov, 3.06 ± 0.13 AU), probably reflecting the acquisition of a selectivity in gene expression at major activation, as in other species; Nothias et al. 1995). Addition of the transcriptional inhibitor α-amanitin during BrUTP incubation decreased the transcriptional activity by 60% (P < 0.05). Embryonic gene expression (minor activation) thus begins in the canine embryo as early as the 2 pronuclei stage, with major activation taking place during the 8-cell stage.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

2008 ◽  
Vol 56 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Chang-Liang Yan ◽  
Qi-En Yang ◽  
Guang-Bin Zhou ◽  
Yun-Peng Hou ◽  
Xue-Ming Zhao ◽  
...  
Keyword(s):  
Survival Rate ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Significant Loss ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Fresh Embryos

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3–100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8–89.5%) and hatched blastocyst rates (61.1–69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3–30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

The developmental potential of mouse somatic cell nuclear-transferred oocytes treated with trichostatin A and 5-aza-2′-deoxycytidine

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Yuta Tsuji ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Treatment Duration ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

SummaryTo facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

25 THE EFFECTS OF OOCYTE SOURCES AND ACTIVATION PROTOCOLS ON THE DEVELOPMENT OF CLONED GOAT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 131
Author(s):  
M. Apimeteeumrong ◽  
A. Thuangsanthia ◽  
N. Leingchaloen ◽  
V. Yiengvisavakul ◽  
A. Harintharanon ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Cytochalasin B ◽  
Vero Cells ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates ◽  
Matured Oocyte

The objective of this study was to compare the development to the morula and blastocyst stages, after either cycloheximide (CHX) or ethanol (ETOH) activation, in somatic nuclear transfer (NT) goat embryos derived from 2 sources of oocytes. In vivo- and in vitro-matured oocytes were obtained from FSH-stimulated goats (Native, Saanen, and Native-Saanen crossbred goats). Gonadotropin treatment was performed with a modified program of a previous report (Reggio et al. 2001 Biol. Reprod. 64, 849-856). In vivo-matured oocytes were flushed from the oviduct of donor goats by exposing the reproductive tract via a small ventral laparotomy incision. In vitro-matured oocytes were aspirated and cultured in maturation medium (M199 + 10% FCS, 10 �g mL-1 FSH, 10 �g mL-1 LH, and 1 �g mL-1 17�-estradiol) for 22 h, at 38.5�C in 5% CO2 and air. Donor cells were prepared from ear skin fibroblasts of a female goat (Native breed). Cells, at passage 3-9, starved by culturing in 0.5% FCS for 4-8 days, were used for NT. Matured oocytes were enucleated, and cell-cytoplast couplets (n = 162 in vivo-, and n = 190 in vitro-matured oocyte groups, respectively) were fused by applying 2 DC pulses of 2.2 kV cm-1 for 30 �s. One to 2 h after fusion, fused embryos were either incubated in 10 �g mL-1 cycloheximide plus 5 �g mL-1 cytochalasin B for 5 h (CHX treatment) or in 7% ethanol for 5 min followed by a 4-h incubation in 2 mM 6-dimethylaminopurine plus 5 �g mL-1 cytochalasin B (ETOH treatment). NT embryos were then cultured in B2 medium supplemented with 5% FCS and Vero cells for 9 days. At the end of the culture period, the NT embryos were fixed and stained with Hoechst 33342 (Begin et al. 2003 Theriogenology 59, 1839-1850). The numbers of nuclei were counted under ultraviolet light. Fusion, cleavage, and development rates were compared using chi-square test or Fisher&apos;s exact test. For the in vivo-matured oocyte group, there were no significant differences in fusion rates (78.1% vs. 68.7%), cleavage rates (87.7% vs. 87.0%, based on the numbers of embryos fused) between the CHX and ETOH treatment groups, respectively (P &gt; 0.05). However, the development rates to morula and blastocyst stages of NT embryos derived from either in vivo- or in vitro-matured oocytes were significantly higher in the ETOH group than in the CHX group (in vivo: 15.2% vs. 0%, and in vitro: 7.1% vs. 0%, for ETOH and CHX groups, respectively; P &lt; 0.05). For the in vitro-matured oocyte group, no significant differences were found between the CHX and ETOH groups in fusion rates (78.6% vs. 83.6%; P &gt; 0.05), cleavage rates (80.5% vs. 83.9%: P &gt; 0.05, based on the numbers of embryos fused). NT embryos from the CHX treatment group derived from in vivo- or in vitro-matured oocytes did not develop beyond the 16-cell stage. These results demonstrate that activation with CHX plus cytochalasin B treatment affects the development to the blastocyst stage of cloned goat embryos whether derived from in vivo- or in vitro-matured oocytes. This work was supported by the RGJ PhD program, Thailand Research Fund, and the Bureau of Biotechnology in Animal Production, Department of Livestock Development.

scholarly journals 194 BIRTH OF KITS AFTER STORAGE IN CULTURE AND TRANSFER OF IN VIVO EMBRYOS IN THE FARMED EUROPEAN POLECAT MUSTELA PUTORIUS)

2005 ◽  
Vol 17 (2) ◽  
pp. 247 ◽  
Author(s):  
H. Lindeberg ◽  
K. Kananen-Anttila ◽  
M. Eronen ◽  
E. Reinikainen ◽  
A. Helin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mustela Putorius ◽  
Cell Stage ◽  
Cell Numbers ◽  
Embryo Recovery ◽  
Morula Stage

The effect of in vitro culture on viability of pre-implantation stage embryos in the farmed European polecat was studied, aimed at developing assisted reproductive technology for conservation of endangered mustelids, particularly the European mink (Mustela lutreola). Embryo storage in culture would enable embryo recovery and transfer in different locations. Ferret (Mustela putorius furo) kits have been produced from embryos that were cultured for 3 days in serum-containing medium (Li et al. 2001 Reproduction 122, 611–618). In our earlier studies, polecat embryos were maintained for 24 h in culture conditions (Lindeberg et al. 2003 Theriogenology 60, 965–970). Fourteen estrous donors were kept in the same cage with a fertile male overnight and sacrificed 3 days after the start of mating for recovery of embryos from the oviducts. Embryos were flushed with Emcare™ Complete ultra flushing medium (ICPBio, Auckland, New Zealand), washed twice in it, washed once in Emcare™ embryo holding solution and transported in the holding solution at room temperature for 1 h to the laboratory. Embryos of seven donors were pooled and cultured in 30-μL drops of TCM199 + glutamax I (GIBCO™) supplemented with fatty acid-free albumin (FAFBSA, Sigma-Aldrech, Helsinki, Finland) under a cover of paraffin oil (Medicult) for 3 days in a humidified atmosphere (39°C) and in 5% of O2. At the end of the culture, the embryos were evaluated and the ones that had developed at least to morula stage were chosen for transfers. The selected embryos were transported at room temperature in Emcare™ embryo holding solution for 1 h to the farm where they were surgically transferred under general anesthesia into seven recipients. The recipients had been mated the same way as the donors but with vasectomized males either on the same day as the donors (the first set: 7 donors, 3 recipients) or one day later than the donors (the second set: 7 donors, 4 recipients). Five embryos were cultured a total of 6 days to the blastocyst stage and stained for a count of cell numbers. A total number of 169 one- to 16-cell-stage embryos were recovered. At the end of the 3-day culture period, a total of 139 (139/169, 82%) had developed to morula (56.6%), compact morula (9.8%), early blastocyst (30.3%), or blastocyst stage (3.3%). Of these 139 embryos, a total of 102 were surgically transferred. Five of the 7 recipients delivered one to 5 kits each 43 to 45 days after the mating. Altogether 21 kits were born and the success rate was 21% (21 kits/102 transferred embryos). Cell numbers of the five Day 6 blastocysts varied from 130 to 430. In conclusion, this preliminary trial confirms that polecat embryos can be stored in culture for 3 days. In this study polecat embryos were cultured in 5% oxygen and without addition of serum which resulted in considerably better cell numbers for Day 6 blastocysts than in our earlier studies (90 to 165 cells; Lindeberg et al. 2003 Theriogenology 60, 965–970).

scholarly journals Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

2015 ◽  
Vol 35 (7) ◽  
pp. 605-612
Author(s):  
Daniel R. Arnold ◽  
Carolina A.P. Corrêa ◽  
Laura L.G. Lorena ◽  
Roberta C. Gaspar ◽  
Guilherme F. Rossi ◽  
...  
Keyword(s):  
Histone Modification ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Cytochalasin B treatment of mouse oocytes during intracytoplasmic sperm injection (ICSI) increases embryo survival without impairment of development

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 361-369 ◽  
Author(s):  
Li-li Hu ◽  
Xing-hui Shen ◽  
Zhong Zheng ◽  
Zhen-dong Wang ◽  
Zhong-hua Liu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Intracytoplasmic Sperm Injection ◽  
Cytochalasin B ◽  
Polar Body ◽  
Term Treatment ◽  
Short Term Treatment ◽  
Embryo Survival ◽  
Mouse Oocytes

SummaryIntracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.

Expression of pregnancy-associated glycoprotein 1 and 2 genes in in vivo, in vitro and parthenogenetically derived preimplantation pig embryos

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Hyun-Jin Do ◽  
Jae-Hwan Kim ◽  
Lalantha R. Abeydeera ◽  
Yong-Mahn Han ◽  
Robert L. Matteri ◽  
...  
Keyword(s):  
Southern Blotting ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Specific Primers ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Reverse Transcription Pcr ◽  
Morula Stage

The objective of this study was to determine whether porcine PAG (poPAG) genes are expressed in embryos as they develop from the 1-cell stage to expanded blastocysts, and whether expression differed according to how embryos had been derived. Embryos at various preimplantation stages were assayed after in vivo fertilisation, after in vitro fertilisation of in vitro-matured oocytes, or following parthenogenetic activation of in vitro-matured oocytes. The presence of PAG transcripts was determined at the1-, 2-, and 4-cell, compact morula and blastocyst stages by reverse transcription-PCR procedures with PAG 1- and PAG 2-specific primers, followed by Southern blotting. The mRNAs for poPAG 1 and 2 were detected in in vitro-derived, in vivo-derived and parthenogenetically derived blastocyst stage embryos. In some replications poPAG 1 could be detected as early as the compact morula stage and poPAG 2 could be detected as early as the 4-cell stage. Our study revealed that poPAG 1 and 2 genes are expressed as early as the compact morula stage and 4-cell stage, respectively, in normal embryos and in parthenogenetically derived blastocysts. Thus it appears that the poPAGs are not maternally imprinted and they may be useful as potential candidates for markers of developmental competence.

scholarly journals The Effectiveness of Candidate Probiotic Bacteria to Control Vibriosis Disease

2017 ◽  
Vol 5 (2) ◽  
pp. 1
Author(s):  
Mulyati Mulyati ◽  
Suryati Suryati ◽  
Irfani Baga
Keyword(s):  
Survival Rate ◽  
Sea Water ◽  
Challenge Test ◽  
Probiotic Bacteria ◽  
Pathogenicity Test ◽  
Inhibitory Ability ◽  
Significant Difference ◽  
Portunid Crab

The study aims to isolate, characterize, and examine probiotic bacteria's inhibitory ability against Vibrio harveyi bacteria, both in-vitro and in vivo. Methods used in the study consist of 1) An Isolation of Candidate Probiotic Bacteria, 2) An Antagonistic Test of Candidate Probiotic Bacteria in vitro, 3) An Identification of Bacteria, 4) A Pathogenicity Test of Candidate Probiotic Bacteria, 5) An Antagonistic Test of Candidate Probiotic Bacteria against V. harveyi in vivo. According to the isolation of candidate probiotic bacteria, there are 18 isolated candidate probiotic. After being tested for its inhibitory ability in vitro, there are 8 isolates with zone of inhibition as follows: isolate MM 7 from intestine (22 mm), isolate MM 6 from intestine (12 mm), isolate MM 10 from sea water (10 mm), isolate MM 5 from intestine (9 mm), isolate MM 4 from intestine (8 mm), isolate MM 3 from intestine (7 mm), isolate MM 2.2 from intestine (7 mm), isolate MM 2.1 from intestine (7 mm). Eight genera of the candidate probiotic bacteria is derived from Portunid crab, they are Staphylococcus, Streptococcus, bacillus, vibrio, Alcaligenes, Lactobacillus, micrococcus. Before proceeding the V. harveyi bacterial challenge test in vivo, three potential isolates consisting of MM6, MM7 and MM10 as the probiotic bacteria are pathogenicity-tested against V. harveyi. The survival rate of Portunid crab on pathogenicity test using MM6, MM7 and MM10 generates 91.11-100%, while the control generates 100% survival rate. Variance analysis result through post-hoc Tukey's Honest Significant Difference (HSD) test at 95% confidence interval indicates that isolate MM7 and MM10 are significantly able to increase hatchling Portunid crab's survival rate.

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