Xenotransplantation of giant gourami testicular germ cells into larvae of nile tilapia

Abstract

We were attempting to develop testicular germ cell (TGC) transplantation as a tool to produce surrogate broodstock of commercially valuable fish and long generation time species. Giant gourami testis had been used as a model for donor and Nile tilapia larvae as recipient. The research was conducted to study the competency of giant gourami TGC as donor and Nile tilapia larvae as recipient for xenotransplantation of giant gouramy TGC into larvae of Nile tilapia. We developed TGC xenotransplantation system by some steps as follow : 1) The characterization of spermatogonia to identify optimal donor of giant gourami based on body weight using histological approach, 2) The determination of dissociation method for giant gourami testicular tissue by comparing two different composition of dissociation medium with 5 hours incubation time, 3) Optimizing the timing of intraperitoneal TGC transplantation into peritoneal cavity of 1, 3, 5 and 7 days post hatching (dph) recipient by investigating the colonization efficiency of donor cell labelled PKH 26 fluorescent membrane dye under fluorescent microscope and of molecular technique using growth hormone of giant gourami specific primer, 4 ) Analyzing the proliferation of spermatogonia colonized in recipient gonad using molecular approach, and 5) The evaluation of TGC isolated from testis of giant gourami which preserved at 4 oC using NaCl 0.7% for 6, 12, 24 and 48 hours and then transplanted into peritoneal cavity of recipient. The result showed that the donor with abundant spermatogonia stem cell and A type spermatogonia (cell diameter = 14.43–20.53 μm) were found in giant gourami with body weight ranged from 500–1000 g. The dissociation method produced high number of spermatogonia with high viability was one used medium PBS containing trypsin, DNase, CaCl2, HEPES, FBS and incubated for 3 hours. The highest colonization efficiency was observed at 3 dph recipient (61.1±34.71%) suggesting that 3 dph Nile tilapia larvae was the optimum recipient for transplantation. Intraperitoneally transplanted xenogenic spermatogonia efficiently colonized the ovary as well with sex ratio male out of female was 1:1, and possibly proliferated indicated by cell cluster forming and the increase of DNA concentration of donor in recipient testis during time interval 1 month to 2 or 3 month pt. The successful colonization of spermatogonia isolated from preserved testis were also observed with colonization efficiency not differed significantly as from non preserved testis. In conclusion : the testis of giant gouramy was composed of cells that had competency as donor for xenotransplantation using Nile tilapia larvae as recipient.

Lambatnya pertumbuhan ikan gurami (Osphronemus goramy) tidak hanya berdampak pada lamanya ikan gurami mencapai ukuran konsumsi tetapi juga pada lamanya ikan gurami mencapai ukuran induk (matang kelamin) sehingga ketersediaan induk untuk menghasilkan benih ikan gurami menjadi terbatas. Keterbatasan induk dan benih tentu saja menjadi kendala bagi kegiatan peningkatan produksi ikan gurami. Saat ini berkembang satu sistem pembenihan untuk produksi ikan yang lama matang kelamin yaitu benih diproduksi oleh induk lain atau induk pengganti (surrogate broodstock). Untuk aplikasi sistem pembenihan ini dibutuhkan suatu teknologi yang disebut xenotransplantasi sel testikular yaitu transplantasi sel testikular yang berasal dari jenis ikan (donor) yang ingin diproduksi ke jenis ikan (resipien) berbeda yang memiliki pertumbuhan cepat. Jika teknologi ini diterapkan pada budidaya ikan gurami diharapkan sel spermatogonia dari suspensi sel testikular ikan gurami yang ditransplantasikan akan tumbuh dan berkembang bersama-sama dengan sel testikular resipien hingga dikeluarkan sebagai sel gamet ikan gurami dalam waktu yang lebih cepat. Pada penelitian ini resipien yang digunakan adalah ikan nila karena cepat matang kelamin, masa rematurasi 2 bulan, viabilitas larva tinggi dan mudah beradaptasi.