nan Alimuddin

Enhancement of EPA and DHA biosynthesis by over-expression of masu salmon Δ6-desaturase-like gene in zebrafish

The nxa0−xa03 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have important nutritional benefits in humans. Farmed fish could serve as promising sources of EPA/DHA, but they need these fatty acids or their precursors in their diets. Here we transferred masu salmon Δ6-desaturase-like gene in zebrafish to increase its ability for synthesizing EPA and DHA. Expression of this gene in transgenic fish elevated their EPA content by 1.4-fold and DHA by 2.1-fold. On the other hand, the α-linolenic acid (ALA) content decreased, it being a substrate of Δ6-desaturase, while the total lipid remained constant. This achievement demonstrates that fatty acid metabolic pathway in fish can be modified by the transgenic technique, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.

Expression of Masu Salmon Δ5-Desaturase-Like Gene Elevated EPA and DHA Biosynthesis in Zebrafish

Farmed fish could substitute for marine capture fish as a source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) beneficial for human health; however, they require these compounds in their diets. In the present study on a model fish species, we modified the EPA/DHA biosynthesis pathway by overexpression of masu salmon Δ5-desaturase-like gene in zebrafish to increase its ability to synthesize EPA and DHA. Expression of this gene in transgenic fish fed a commercial diet and Artemia helped to improve their EPA content by 1.21-fold and DHA by 1.24-fold. In similar fish that were fed only Artemia the increments were 1.14-fold for EPA and 1.13-fold for DHA, compared with nontransgenic fish. In contrast, eicosatetraenoic acid content decreased, as it is a substrate of Δ5-desaturase, while the total lipid remained constant. The results demonstrated that masu salmon Δ5-desaturase is functional in zebrafish and can modify its fatty acid metabolic pathway. The technique could be applied to farmed fish to generate a nutritionally richer product for human consumption.

Cloning and nutritional regulation of polyunsaturated fatty acid desaturase and elongase of a marine teleost, the nibe croaker Nibea mitsukurii

We identified fatty acid desaturase (fads)-like and elongase (elovl)-like genes from nibe croaker to better understand the molecular basis of n-3 highly unsaturated fatty acid metabolism in marine fish. Phylogenetic analysis revealed that the fads-like and elovl-like genes were classified into the fads6 and elovl5 groups, respectively. We investigated the effects of various levels of docosahexaenoic acid (DHA)-enriched live feed, Artemia nauplii, on larval growth, survival, and fads-like and elovl-like gene expression. After a 15-day feed trial, total length, body weight, and survival were all significantly improved by the supplementation of Artemia with DHA. This result indicates that nibe croaker cannot endogenously produce enough DHA. Furthermore, the fads-like gene transcripts in larvae fed on oleic acid-enriched Artemia were significantly higher than those in larvae on 100% DHA-enriched Artemia. In contrast, no significant differences were observed in the transcript levels of the elovl-like gene. These data indicate that the fads6-like gene was controlled by negative feedback from the quantity of DHA stored in the larval body. These results have implications for the functionality of the fads-like gene in nibe croaker.

Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22xa0days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.

Aromatase gene expression and masculinization of Nile tilapia immersed in water 36 °C containing 17α-methyltestosterone

ABSTRACT Immersion of undifferentiated larval tilapia in high temperature and 17α-methyltestosterone (MT) cab increase the male ratio. However, the effectiveness of immersion in high temperature of water containing MT remains to be evaluated. The purposes of this study were: 1) evaluate the male ratio, growth, and survival of tilapia, and 2) analyze the aromatase brain-type gene expression level in tilapia after immersing in high temperature (36 °C) containing MT at 2 mg/L for four hour with single and double immersion. Aromatase gene expression was analyzed by semi-quantitative RT-PCR (sqRT-PCR) method. The result showed that higher monosex male ratio was obtained by single immersion of MT at 36 °C at room temperature. Gene expression level of aromatase brain-type was lower on single immersion and increased significantly at second immersion compared to control (immersion at room temperature without MT). Immersion using MT and high temperature had no significant effect on fish survival. However the specific growth rate and fish biomass were higher than control. Thus, monosex male tilapia can be produced by single immersion of undifferentiated larvae at 36 °C temperature containing MT. Keywords: male ratio, aromatase, Oreochromis niloticus , temperature, 17α-methyltestosterone ABSTRAK Perendaman larva ikan nila yang belum terdeferensiasi kelaminnya dengan suhu tinggi dan hormon 17α-metiltestosteron (MT) dapat meningkatkan nisbah kelamin jantan. Tetapi, efektivitas perendaman menggunakan MT pada suhu tinggi belum diteliti. Tujuan dari penelitian ini adalah 1) mengevaluasi nisbah kelamin jantan, pertumbuhan, dan kelangsungan hidup ikan nila, dan 2) menganalisis ekspresi gen aromatase tipe-otak pada ikan direndam menggunakan MT dengan dosis 2 mg/L selama empat jam sebanyak satu dan dua kali perendaman pada suhu 36 °C. Ekspresi gen aromatase dianalisis menggunakan metode RT-PCR semi-kuantitatif (sqRT-PCR). Hasil penelitian menunjukkan bahwa kombinasi perendaman MT satu kali pada suhu 36 °C lebih tinggi menghasilkan ikan nila jantan monoseks dibandingkan perendaman MT satu kali pada suhu ruang. Tingkat ekspresi gen aromatase tipe otak pada perendaman satu kali lebih rendah, dan meningkat secara signifikan pada perendaman kedua dibandingkan dengan kontrol (perendaman pada suhu ruang tanpa MT). Perendaman larva menggunakan MT dan suhu 36 °C tidak berpengaruh nyata terhadap kelangsungan hidup, tetapi laju pertumbuhan spesifik dan biomassa ikan perlakuan tersebut lebih tinggi daripada kontrol. Dengan demikian, ikan nila jantan monoseks dapat diproduksi dengan perendaman satu kali pada larva yang belum terdeferensiasi jenis kelaminnya menggunakan MT pada suhu 36 °C. Kata kunci: rasio jantan, aromatase, Oreochromis niloticus , suhu, 17α-metiltestosteron

GROWTH IN NURSERY AND GROW-OUT PHASES OF WHITE SHRIMP AFTER IMMERSED IN RECOMBINANT GIANT GROUPER GROWTH HORMONE

The growth of white shrimp ( Litopenaeus vannamei ) can be improved by using recombinant fish growth hormone through immersion. This research was performed to evaluate the white shrimp growth at nursery and grow-out phases after recombinant giant grouper growth hormone (rElGH) immersion. Shrimp were immersed at different stages in one liter seawater containing 15 mg rElGH for two hours. At the nursery stage 30,000 PL4 that previously immersed at nauplius stage (treatment A1), at PL4 (treatment B1) and control without rElGH immersion (K1) were reared in fiber tanks containing 750 L seawater for 8 days. At the grow-out phase, 1 00 PL11 that have been immersed in rElGH solution at nauplius stage (treatment A2), at PL4 (B2), PL11 (C2) and control without rElGH immersion (K2) were separately reared in fiber tanks containing 750 L seawater for 55 days. Each treatment consisted of three replications. The results showed that at the end of the nursery phase, B1 treatment increased 19% of body length, 30.2% of the body weight and decreased the coefficient of length variation 34.9% compared to control K1 (P<0.05). At the grow-out phase, C2 treatment enhanced 38.2% of body weight and 32% of biomass compared with control K2 (P<0.05). Thus, hatchery is better to immerse PL4, and the farmer should used rElGH-immersed PL11 for growing-out. Keywords : growth promoting, nursery, grow out , Litopenaeus vannamei , rElGH

IMPROVEMENT METHOD OF GENE TRANSFER IN Kappaphycus alvarezii

Method of foreign gene transfer in red seaweed Kappaphycus alvarezii has been reported, however, li-mited number of transgenic F 0 (broodstock) was obtained. This study was conducted to improve the method of gene transfer mediated by Agrobacterium tumefaciens in order to obtain high percentage of K. alvarezii transgenic. Superoxide dismutase gene from Melastoma malabatrichum (MmCu/Zn-SOD) was used as model towards increasing adaptability of K. alvarezii to environmental stress. The treat-ments were the culture media and recovery duration, and each treatment consisted of three replica-tions. The best method was co-cultivation using liquid media, then recovery was conducted in liquid media for 10 days. That treatment allowed higher transformation percentage ( 90% ) , regeneration effi-ciency ( 90% ) , putative bud efficiency (100%) , number of buds and explants sprouted (100%) and transgenic explants ( 100% ) . The transgenic explants showed an amplification PCR product of Mm-Cu/Zn-SOD gene fragment , whereas the non-transgenic explants showed no amplification product.xa0 All results revealed that suitable method of transgenesis for K. alvarezii has been developed. Keywords :xa0xa0xa0xa0xa0xa0 Agrobacterium tumefaciens , culture media, Kappaphycus alvarezii , recovery duration, transformation

Performance of broodstock and hybrid juvenile of Egyptian and sangkuriang Clarias gariepinus strains

ABSTRACT This study was conducted to evaluate reproduction of broodstock and intraspecific hybrid juvenile performance of Egyptian (M) and sangkuriang (S) strain African catfish Clarias gariepinus at nursery phase. Intraspecific hybridization of African catfish was carried out reciprocally (SM and MS) and purebreed (SS and MM), each was with three replicates. Fish spawning was conducted by artificial fertilization, and larvae were reared at 1 st , 2 nd and 3 rd nursery phases, subsequently. The results showed that broodstock performance (male and female) of both strains were not significantly different (P>0.05) on all reproduction traits, except female’s gonadosomatic index. Fertilization and hatching rates of all hybrids were not significantly different (P>0.05). MM juvenile had higher growth performances than other juvenile hybrids. Heterosis of total length, standard length, and body weight were varied, whereas the survival showed positive heterosis. SM juvenile showed positive growth heterosis in 3 rd nursery phase (total length, standard length, and body weight were 2.61%; 2.16%, and 4.79% respectively). Survival heterosis of MS juvenile (24.20% for total length; 103.13% for standard length and 11.62% body weight) was higher than SM juvenile (6.86%; 48.57%, and 3.09%) on all nursery phases Keywords: African catfish, intraspecific hybridization, growth, survival, heterosis ABSTRAK Penelitian ini dilakukan untuk menguji performa reproduksi induk dan benih hasil hibridisasi intraspesifik ikan lele Afrika Clarias gariepinus strain Sangkuriang (S) dan Mesir (M) pada fase pendederan. Hibridisasi intraspesifik ikan lele Afrika dilakukan secara resiprokal (SM dan MS) dan galur murni (SS dan MM) masing-masing dengan tiga ulangan. Pemijahan dilakukan secara buatan dan larva yang dihasilkan dipelihara pada fase pendederan pertama, kedua, dan ketiga. Hasil penelitian menunjukkan bahwa performa induk ikan lele jantan dan betina pada kedua strain tidak berbeda nyata (P>0,05) pada seluruh parameter reproduksi, kecuali indeks gonadosomatik (GSI) pada induk betina. Derajat pembuahan dan penetasan telur pada seluruh persilangan tidak berbeda nyata (P>0,05). Pertumbuhan benih persilangan MM lebih tinggi, dibandingkan benih hasil persilangan lain. Nilai heterosis panjang total, panjang standar, dan bobot tubuh yang dihasilkan pada setiap stadia pendederan bervariasi, sedangkan nilai heterosis kelangsungan hidup menunjukkan hasil seluruhnya positif dibandingkan rataan galur murni. Heterosis pertumbuhan benih persilangan SM pada pendederan ketiga menunjukkan hasil yang positif (2,61% untuk panjang total; 2,16% untuk panjang standar dan 4,79% untuk bobot tubuh). Nilai heterosis kelangsungan hidup benih persilangan MS (24,20% untuk panjang total; 103,13% untuk panjang standar dan 11,62% untuk bobot tubuh) lebih tinggi dibandingkan benih persilangan SM (6,86%, 48,57% dan 3,09%) pada setiap stadia pendederan. Kata kunci: ikan lele Afrika, hibridisasi intraspesifik, pertumbuhan, kelangsungan hidup, heterosis

A rapid chromosome preparation technique without metaphase arrest for ploidy determination in Nile tilapia, Oreochromis niloticus

Abstract Most chromosome preparation techniques use colchicine or colcemid in order to arrest cell division during metaphase, which usually involves the use of high cost materials. This research was aimed to simplify fish chromosome preparation technique without using colchicine or colcemid. Ten fish of different ages, namely one-, two- and three-month-old Nile tilapias, were used in this research. Tissues were collected on the caudal fin of the fish. Chromosome spreads was easily observed, using Giemsa or silver staining, five hours after the whole preparation process. The results showed that the one- and two-month-old fish had better mitotic index (0.008 and 0.01, respectively) compared to those of the three-month-old fish (0.005). This method is rapid, cost-efficient and applicable for ploidy level determination, and also keeps fish alive for subsequent evaluations.

Testicular cell transplantation of neon tetra Paracheirodon innesi into common carp fry

ABSTRACT Neon tetra Paracheirodon innesi is an ornamental fish that have high export value. However, production is still relatively low due to low fecundity (approximately 180 eggs/female). Technology of testicular cell transplantation of neon tetra as donor to common carp as recipient fish which have high fecundity provides a promising way to overcome problem of neon tetra production. This research was performed to determine the optimum age of common carp fry that is able to receive donor cells and allow high success of transplantation. In this research, the testes of neon tetra fish were dissociated by 0.5% trypsin solution. The testicular cells were labeled with PKH-26 fluorescent dye, and then transplanted into the peritoneal cavity of seven, ten, and 14 days post hatching common carp fry. The results showed that the survival of seven day-old transplanted fry (31.25%) was lower than that of ten day-old (37.75%) and 14 day-old transplanted fry (56.25%). Percentage of fish colonized testicular cells donor at 21 days post-transplantation on seven days old and ten days old fry were similar (80%), while on 14 day-old fry was 60%. Based on the cumulative transplantation success rate (survival and colonization rates), transplantation on 14 days old fry (33.75%) showed higher result compared to transplantation on seven days old fry (25.00%) and ten day-old fry (30.00%). It can be concluded that transplantation of neon tetra testicular cells to common carp fry have been successfully carried out, and the optimum age of common carp fry to transplantation was 14 days after hatching. Keywords: transplantation, colonization, testicular cells, common carp, neon tetra ABSTRAK Ikan neon tetra Paracheirodon innesi merupakan ikan hias yang memiliki nilai ekspor yang tinggi. Namun demikian, tingkat produksinya masih relatif rendah karena fekunditas ikan neon tetra yang sedikit (sekitar 180 telur/induk). Teknologi transplantasi sel testikular ikan neon tetra (ikan donor) ke ikan mas yang memiliki fekunditas telur yang banyak dan diharapkan mampu mengatasi ketersediaan benih ikan neon tetra. Penelitian ini bertujuan untuk menentukan umur optimum benih ikan mas (calon ikan semang) yang mampu menerima sel donor dengan baik dan memiliki keberhasilan kolonisasi yang tinggi. Testis ikan neon tetra didisosiasi menggunakan larutan tripsin 0,5%. Sel testikular diwarnai dengan PKH-26, kemudian ditransplantasikan ke rongga peritoneal benih ikan mas umur tujuh, sepuluh, dan 14 hari setelah menetas. Hasil penelitian menunjukkan bahwa tingkat kelangsungan hidup ikan mas perlakuan transplantasi umur tujuh hari (31,25%) lebih rendah dibandingkan dengan perlakuan transplantasi umur sepuluh hari (37,50%) dan 14 hari (56,25%). Persentase ikan terkolonisasi sel donor pada hari ke-21 pascatransplantasi pada benih umur tujuh dan sepuluh hari adalah sama (80%), sedangkan transplantasi benih umur 14 hari sebesar 60%. Berdasarkan keberhasilan transplantasi secara kumulatif (tingkat kelangsungan hidup dan kolonisasi), transplantasi pada benih umur 14 hari (33,75%) menunjukkan hasil lebih tinggi dibandingkan dengan perlakuan transplantasi pada benih umur tujuh hari (25,00%) dan benih umur sepuluh hari (30,00%). Transplantasi sel testikular ikan neon tetra pada benih ikan mas telah berhasil dilakukan, dan umur optimum benih ikan mas adalah 14 hari setelah menetas. Kata kunci: transplantasi, kolonisasi, sel testikular, ikan mas, ikan neon tetra