Kajian Molekuler dan Fisiologis Pematangan Gonad dan Pemijahan Ikan Sepatung (Pristolepis grootii, Blekeer 1852)

Abstract

Ikan sepatung (Pristolepis grootii) salah satu jenis ikan endemik Indonesia yang potensial dibudidayakan. Ikan ini disukai masyarakat dan bernilai ekonomi. Ikan ini mampu bertahan hidup di perairan dengan keasaman dan oksigen terlarut rendah, mudah beradaptasi dalam lingkungan budidaya serta dapat menerima pakan buatan. Ikan ini dimanfaatkan sebagai lauk pauk, dan juga ikan hias. Produksi ikan sepatung masih mengandalkan hasil tangkapan dari alam liar. Peningkatan aktivitas penangkapan dan kerusakan habitat menyebabkan penurunan populasi ikan sepatung di alam. Indikasinya adalah sulitnya nelayan mendapatkan ikan sepatung dalam jumlah banyak. Jika penurunan populasi ikan sepatung berlangsung terus-menerus, maka dapat menyebabkan punahnya spesies ini. Oleh karena itu, sebelum ikan ini punah, perlu dilakukan usaha pembudidayaannya. Sebagai langkah awal dalam usaha budidaya ikan liar seperti spesies ini, perlu dilakukan domestikasi/penjinakan di dalam wadah budidaya. Ikan yang baru didomestikasi di lingkungan budidaya biasanya mengalami gangguan fisiologi reproduksi. Hal ini disebabkan perbedaan kondisi lingkungan budidaya dengan alam liar habitatnya. Tujuan umum penelitian ini adalah mendomestikasi ikan sepatung, sedangkan tujuan khusus adalah (1) menganalisis karakter gen penyandi hormon gonadotropin ikan sepatung, (2) mengaji pematangan gonad ikan sepatung yang diinjeksi hormon luteinizing hormone-releasing hormone analogue (LH-RHa), dan (3) mengaji pemijahan ikan sepatung yang diinduksi dengan salmon gonadotropin releasing hormone analogue dan domperidone (sGnRHa+D). Penelitian ini terdiri atas tiga topik. Penelitian pertama dilakukan untuk mengkarakterisasi gen gonadotropin ikan sepatung. Kelenjar hipofisa ikan sepatung diambil, lalu dilakukan ekstraksi total RNA menggunakan GENEzolTM Reagent, cDNA disintesis dan diamplifikasi dengan metode RT-PCR selanjutnya dikloning, dan disekuensing. Penelitian kedua dilakukan untuk memacu pematangan gonad dan menginduksi ekspresi gen gonadotropin. Ikan betina diinjeksi secara intramuskular dengan LH-RHa dosis berbeda (0, 1, 10, dan 50 µg/kg bobot ikan), Penelitian ketiga, ikan betina dan jantan diinjeksi sGnRHa+D dosis 0,5 ml/kg bobot ikan. Telur dan sperma dikeluarkan dengan metode stripping. Fertilisasi dilakukan secara eksternal. Setelah fertilisasi, telur diinkubasi dalam akuarium sampai menetas, dan larva dipelihara sampai umur 3 hari setelah menetas. Hasil penelitian pertama berupa informasi karakteristik gen gonadotropin; follicle-stimulating hormone subunit-β (FSH-β) dan luteinizing hormone subunit-β (LH-β) ikan sepatung. Panjang sekuens parsial gen FSH-β ikan sepatung yang berhasil diisolasi adalah 279 bp, menyandikan 93 residu asam amino termasuk 2 residu prolin dan 8 sistein, serta 2 situs N-glikosilasi. Situs N-glikosilasi teridentifikasi pada urutan asam amino ke 25-27 (NIS: asparagin, isoleusin, serin), dan situs kedua pada urutan asam amino ke 77-79 (YTT: tirosin, treonin, treonin). Panjang sekuens parsial gen LH-β ikan sepatung yang berhasil diisolasi adalah 300 bp, menyandikan 100 residu asam amino termasuk 7 residu sistein, dan 1 situs N-glikosilasi. Situs N-glikosilasi pada LH-β teridentifikasi pada urutan asam amino ke 44-46 (NCT: asparagine, sistein, treonin). FSH-β ikan sepatung menunjukkan kemiripan tertinggi (91%) dengan ikan betok (Anabas testudineus), sedangkan LH-β dengan ikan cupang (Betta splendens) (90%). N-glikosilasi bersifat konservatif bagi FSH-β dan LH-β. Analisis filogenetik FSH-β ikan sepatung menunjukkan kesamaan tertinggi dengan ikan betok (A. testudineus) (nilai bootstrap 81%), sedangkan LH-β dengan ikan cupang (B. splendens) (nilai bootstrap 91%). Pada penelitian kedua, hasil yang diperoleh menunjukkan bahwa injeksi LH-RHa mampu memacu perkembangan dan pematangan gonad, serta menginduksi ekspresi gen FSH-β dan LH-β ikan sepatung, dengan dosis LH-RHa terbaik yakni 50 µg/kg bobot ikan. Peningkatan dosis LH-RHa dari 0, 1, 10, dan 50 µg/kg bobot ikan serta waktu pengamatan hari ke-0, 7, 14, dan 21 setelah injeksi kedua, ekspresi gen FSH-β dan LH-β cenderung mengalami peningkatan. Ekspresi gen LH-β lebih tinggi dari gen FSH-β pada setiap perlakuan dan waktu pengamatan. Gen FSH-β dan LH-β pada ikan sepatung tereskpresi secara bersamaan. Pada akhir pengamatan berdasarkan hasil histologi, ikan yang diinjeksi LH-RHa dosis 10 µg/kg bobot ikan, memasuki fase vitellogenesis akhir dan dosis 50 µg/kg bobot ikan, memasuki fase pematangan. Dengan demikian hasil histologi gonad sejalan dengan pola ekspresi gen FSH-β dan LH-β di kelenjar hipofisa. Pada penelitian ketiga, ikan sepatung yang diinjeksi dengan sGnRHa+D berhasil ovulasi dan spermiasi. Rata-rata jumlah telur ikan sepatung yang diovulasikan adalah 3.272±428 butir per induk, dengan diameter telur 748,58±79,36 µm. Tingkat pembuahan telur sebesar 85,74±4,27%, tingkat penetasan 49,18±5,70 %, rata-rata kelangsungan hidup larva sampai umur tiga hari (D0-D3) 42,24±14,70% dan rata-rata panjang larva umur tiga hari 7,7±0,8 mm. Kesimpulan umum penelitian ini adalah ikan mampu bertahan hidup, dapat tumbuh dan berkembangbiak dalam lingkungan budidaya. Kesimpulan khusus penelitian ini adalah (1) Karakterisasi gen gonadotropin telah berhasil dilakukan. Gen gonadotropin (FSH-β dan LH-β) ikan sepatung masing-masing mirip dengan ikan betok (A. testudineus) dan ikan cupang (B. splendens). Gen gonadotropin ikan sepatung telah dipublikasikan di bank gen (NCBI), merupakan pendaftar pertama. (2) Hasil penelitian menunjukkan bahwa LH-RHa menginduksi ekspresi gen gonadotropin dan memacu perkembangan dan kematangan gonad. (3) Hormon sGnRHa+D mampu menginduksi ovulasi dan spermiasi serta pembuahan buatan berhasil dilakukan pada ikan sepatung.

The Indonesian leaffish (Pristolepis grootii) is one of Indonesia's endemic fish species that has the potential to be cultured. This fish is preferred by consumers and has economic value. These fish are able to survive in waters with low acidity and dissolved oxygen, are easily adaptable in aquaculture environments and can accept artificial feeds. This fish is used as a side dish, as well as ornamental fish. The production of Indonesian leaffish relies on catches from wild nature. The increase of fishing activities and habitat destruction has led to a decline in the population of it in nature. The indication is the difficulty of fishermen acquiring fish in large quantities. If the decline in the Indonesian leaffish population continues, can lead to the extinction of this species. Therefore, before this species becomes extinct, it is necessary to cultivate it. The first step in the culture of wild fish such as this species, it is necessary to do domestication in the aquaculture environment. Newly domesticated fish in aquaculture usually experience reproductive physiological disorders. This is due to differences in the conditions of the cultivated environment and its wild habitat. The general objective of this study were to domesticate the Indonesian leaffish, while the specific aims were (1) to analyze the character of the gene coding for the gonadotropin hormone in Indonesian leaffish (2) to study the gonadal maturation and gonadotropin gene expression of Indonesian leaffish that was injected with the luteinizing hormone-releasing hormone analogue (LH-RHa) hormone. (3) to study the spawning of Indonesian leaffish that was injected with the salmon gonadotropin releasing hormone analogue (sGnRHa+D). This study consists of three topics. The first study was carried out to characterize the gonadotropin genes of Indonesian leaffish. Pituitary gland samples were taken from each fish. Total RNA was extracted using GENEzolTM Reagent. The cDNA was synthesized and amplified by the RT-PCR method then cloned, and sequenced. The second study was conducted to stimulate gonadal maturation and induce gonadotropin genes expression. The female fish were injected intramuscularly with different doses of LH-RHa (0, 1, 10, and 50 g/kg body weight). In the third study, the spawning of Indonesian leaffish, the females and males were injected with sGnRHa+D at a dose of 0.5 mL/kg of body weight. Eggs and sperm were collected by stripping method. Fertilization was carried out externally. After fertilization, the eggs were incubated in the aquaria until they hatched, and the larvae were reared for up to 3 days after hatching. The results of the first study were information on the characteristics of the gonadotropin genes; follicle-stimulating hormone subunit-β (FSH-β) and luteinizing hormone subunit-β (LH-β) of Indonesian leaffish. The partial sequence length of the FSH-β gene isolated from the Indonesian leaffish was 279 bp, encoding 93 amino acid residues including 2 proline and 8 cysteine residues, and 2 N-glycosylation sites. The N-glycosylation site was identified on the 25-27th amino acid sequence (NIS: asparagine, isoleucine, serine), and the second site on the 77-79th amino acid sequence (YTT: tyrosine, threonine, threonine). The partial sequence length of the LH-β gene isolated from the Indonesian leaffish was 300 bp, encoding 100 amino acid residues including 7 cysteine residues, and 1 N-glycosylation site. The N-glycosylation site on LH-β was identified at amino acid sequence 44-46 (NCT: asparagine, cysteine, threonine). FSH-β of Indonesian leaffish showed the highest identity (91%) with climbing perch (Anabas testudineus), while LH-β with betta fish (Betta splendens) (90%). The N-glycosylation is conservative for FSH-β and LH-β. The phylogenetic analysis of FSH-β showed the highest similarity with A. testudineus (81% bootstrap value), while LH-β with B. splendens (91% bootstrap value). In the second study, the results showed that LH-RHa injection was able to stimulate gonadal development and maturation and induce the expression of the FSH-β and LH-β genes in Indonesian leaffish, with the best LH-RHa dose of 50 g.kg-1 body weight. The increasing of LH-RHa doses from 0, 1, 10, and 50 g.kg-1 body weight and observation time on days 0, 7, 14, and 21 after the second injection, the expression of FSH-β and LH-β genes tended to increase. The expression of the LH-β gene was higher than the FSH-β gene in each treatment and observation time. The FSH-β and LH-β genes in Indonesian leaffish are expressed simultaneously. At the end of the observation base on histological results, the fish injected with LH-RHa at a dose of 10 g.kg-1 body weight, entered the late vitellogenesis phase and at a dose of 50 g.kg-1 body weight, entered the maturation phase. Thus, the histological results were in line with the expression patterns of the FSH-β and LH-β genes in pituitary gland. In the third study, the Indonesian leaffish injected with sGnRHa+D successfully ovulated and spermiated. The average number of ovulated Indonesian leaffish eggs was 3,272±428 eggs per brooder, with an egg diamter of 748.58±79.36 µm. The egg fertilization rate was 85.74±4.27%, the hatching rate was 49.18±5.70%, the average survival of larvae to three days post-hatched (D0-D3) was 42.24±14.70%, and the average length of larvae at three days of age was 7.7 ±0.8 mm. The general conclusion of this study I s that fish are able to survive, grow and reproduce in a cultured environment. The specific conclusions of this study are (1) The characterization of the gonadotropin gene has been successfully carried out. The gonadotropin genes (FSH-β and LH-β) of Indonesian leaffish are similar to those of climbing perch (A. testudineus) and betta fish (B. splendens). The gonadotropin gene of P Indonesian leaffish has been published in the gene bank (NCBI), which is the first registrar. (2) The results showed that LH-RHa induces gonadotropin gene expression and stimulates gonadal development and maturation. (3) The sGnRHa+D was able to induce ovulation and spermiation, as well as artificial fertilization was successfully carried out on Indonesian leaffish.