Successful cryopreservation of spermatogonia in critically endangered Manchurian trout (Brachymystax lenok)

Because of the lack of cryopreservation techniques for fish eggs and embryos, 22 cryopreservation of fish spermatogonia and subsequent generation of eggs and sperm would be 23 an exit strategy for the long-term preservation of genetic resources. This study aimed to optimize 24 cryoprotectants, cooling rates, and thawing temperatures for slow freezing of spermatogonia from 25 endangered Manchurian trout (Brachymystax lenok). Whole testes were frozen with a 26 cryomedium containing 1.3 M methanol, 0.2 M trehalose, and 10% egg yolk at a cooling rate of 27 −1°C/min and then stored in liquid nitrogen for 2 days. After thawing at 30°C in a water bath, 28 testicular cells from thawed testes were intraperitoneally transplanted into allogeneic triploid 29 hatchlings. Transplanted spermatogonia migrated toward and were incorporated into recipient 30 gonads, where they underwent gametogenesis. Transplantation efficiency did not significantly 31 differ between frozen and fresh testes, demonstrating that Manchurian trout spermatogonia can be 32 successfully cryopreserved in liquid nitrogen

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1,185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish

Long-term (5 years) cryopreserved spermatogonia have high capacity to generate functional gametes via interspecies transplantation in salmonids

Although sperm cryopreservation is a powerful tool widely applicable in biodiversity conservation and broodstock management, cryopreservation of teleost eggs and embryos remains challenging. In the present study, we demonstrated that spermatogonia of rainbow trout (Oncorhynchus mykiss) cryopreserved for 5 years possessed the ability to differentiate into functional eggs or sperm in the gonads of triploid recipient masu salmon (O. masou). After cryopreservation for 5 years in liquid nitrogen, intraperitoneally transplanted spermatogonia migrated toward, and incorporated into, the gonads of xenogeneic recipients. The transplanted spermatogonia resumed spermatogenesis and oogenesis in male and female recipients, respectively, and differentiated into sperm or eggs within the gonads of male and female recipients at 2 years posttransplantation. The differentiated sperm and eggs generated normal rainbow trout representative of donor phenotypes. Thus, cryopreservation of spermatogonia is a powerful and reliable method for long-term preservation of fish genetic resources

OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH

Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3) rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD), Δ5-desaturase-like (OmΔ5FAD) and elongase-like (MELO) encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou) were individually transferred into zebrafish (Danio rerio) as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05) than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05) than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05) than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over-expressing gene encoding enzymes involved in fatty acid biosynthesis, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food

Expression patterns of gdnf and gfrα1 in rainbow trout testis.

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Setoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes

Enrichment of Spermatogonial Stem Cells using Side Population in Teleost

Spermatogenesis originates from a small population of spermatogonial stem cells; this population can maintain continuous sperm production throughout the life of fish via self-renewal and differentiation. Despite their biological importance, spermatogonial stem cells are not thoroughly characterized because they are difficult to distinguish from their progeny 5 cells that become committed to differentiation. We previously established a novel technique for germ cell transplantation to identify spermatogonial stem cells based on their colonizing activity and their ability to initiate donor-derived gametogenesis in the rainbow trout (Oncorhynchus mykiss). Although spermatogonial stem cells can be retrospectively identified after transplantation, there is currently no technique to prospectively enrich for or purify spermatogonial stem cells. Here, we describe a method for spermatogonial stem-cell enrichment using side-population. With optimized Hoechst 33342 staining conditions, we successfully identified side-population cells among type A spermatogonia. Side-population cells were transcriptomically and morphologically distinct from non-side-population cells. To functionally determine whether the transplantable spermatogonial stem cells were enriched in the side-population fraction, we compared the colonization activity of side-population cells with that of non-side-population cells. Colonization efficiency was significantly higher with side-population cells than with non-side-population cells or with total type A spermatogonia. In addition, side-population cells could produce billions of sperm in recipient. These results indicated that transplantable spermatogonial stem cells were enriched in the side-population fraction. This method will provide biological information that may advance our understanding of spermatogonial stem 20 cells in teleosts. Additionally, this technique will increase the efficiency of germ-cell transplantation used in surrogate broodstock technology

OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH

Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3) rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD), Δ5-desaturase-like (OmΔ5FAD) and elongase-like (MELO) encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou) were individually transferred into zebrafish (Danio rerio) as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05) than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05) than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05) than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over-expressing gene encoding enzymes involved in fatty acid biosynthesis, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food

Growth, Survival, and Body Composition of Transgenic Common Carp Cyprinus carpio 3rd Generation Expressing Tilapia Growth Hormone cDNA

Transgenic has been known as one of the applicable methods to improve growth performance of cultured fish. This study was performed to evaluate the growth performance, survival, and body composition of the 3rd generation of growth hormone (GH) transgenic common carp (TG). Juveniles (BW: 1.53 ± 0.03 g) were reared for 60 days in 250-L glass aquarium with stocking density of 25 fishes/aquarium. Fishes were fed with commercial feed (protein content 36%), three times a day to satiation. Growth and survival were measured every 20 days. Our results showed that TG fish has 1.49 times higher in average weight growth (p &lt; 0.05) compared with the non-transgenic common carp (NT). Higher total feed consumption, survival, body protein content, protein and lipid retention, hepatosomatic index, and lower feed conversion ratio were also shown on TG fish compared with NT fish (p &lt; 0.05). However, body lipid content and blood glucose level of TG fish were lower (p &lt; 0.05) compared with the NT fish. Total ammonium nitrogen level in rearing media of TG fish was 51.78% lower (p &lt; 0.05) than that of the NT fish. In conclusion, culturing of GH-TG common carp showed potential to achieve high productivity, efficient, and environmental-friendly aquaculture

ISOLASI DAN KARAKTERISASI PROMOTER β-ACTIN DARI IKAN KERAPU BEBEK (Cromileptes altivelis)

Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasilan transgenesis.  Penelitian isolasi dan karakterisasi promoter β-actin (ktBA) dari ikan kerapu bebek (Cromileptes altivelis) dalam rangka pembuatan ikan kerapu autotransgenik telah dilakukan. Promoter β-actin memiliki aktivitas tinggi pada jaringan otot. Sekuens promoter ktBA diisolasi menggunakan metode degenerate PCR. Sekuensing dilakukan menggunakan mesin ABI PRISM 3100. Analisis sekuens menggunakan software BLAST, GENETYX versi 7 dan TFBind. Fragment DNA hasil amplifikasi PCR yang dipotong dari vektor kloning selanjutnya diligasi dengan pEGFPN1 untuk membuat konstruksi pktBA-EGFP. Konstruksi pktBA-EGFP dimikroinjeksi ke embrio ikan zebra (Danio rerio) fase 1 sel untuk menguji aktivitas promoter ktBA. Ekspresi gen EGFP diamati menggunakan mikroskop fluoresens. Analisis sekuens menunjukkan bahwa panjang fragmen DNA hasil amplifikasi PCR sekitar 1,6 kb dan memiliki faktor transkripsi yang conserved pada promoter β-actin, yaitu CCAAT, CArG dan boks TATA. Selanjutnya, sekuens ktBA dalam konstruksi pktBA-EGFP mampu mengendalikan ekspresi gen EGFP pada jaringan otot embrio ikan zebra yang dimikroinjeksi dengan konstruksi tersebut. Dengan demikian dapat disimpulkan bahwa fragmen DNA hasil amplifikasi PCR tersebut merupakan sekuens promoter β-actin ikan kerapu bebek. Pembuatan ikan kerapu autotransgenik selanjutnya dapat dilakukan dengan mengganti gen EGFP pada pktBA-EGFP dengan gen-gen asal ikan kerapu yang mengkodekan karakter penting dalam budi daya ikan.Promoter as gene expression regulator is one of the factors affecting the successful of transgenesis. Isolation and characterization of β -actin promoter (ktBA) from humpback grouper (Cromileptes altivelis) towards generation of autotransgenic grouper have been conducted.  β -actin promoter has high activity in muscle. Sequence of ktBA promoter was isolated by using degenerate PCR method. Sequencing was performed using ABI PRISM 3100 machine. Analysis of sequences was conducted using BLAST, GENETYX version 7 and TFBind softwares. DNA fragment of PCR amplification product digested from the vector cloning was then ligated with pEGFPN1 to generate pktBA-GFP construct. The construct was microinjected into one-cell stage of zebrafish (Danio rerio) embryos to test the ktBA promoter activity. EGFP gene expression was observed by fluorescence microscope. The result of sequence analysis showed that the length of DNA fragment obtained is about 1.6 kb and containing the evolutionary conserved sequences of transcription factor for β -actin promoter including CCAAT, CArG and TATA boxes. Furthermore, ktBA sequence in pktBA-EGFP construct could drove GFP expression in muscle of zebrafish embryos injected with the construct. The results suggested that PCR amplification product is the regulator sequence of humpback grouper β -actin gene. Autotransgenic grouper can be then produced by changing GFP gene fragment of pktBA-EGFP construct with genes from grouper encoding important traits in aquaculture