MACORETTA CL, MIRANDA LA. Development of cooling protocols for fish embryos and evaluation of larval quality.

MIRANDA LA; MACORETTA CL

Congreso; 58th Annual Meeting of the Society for Cryobiology; 2021

The cooling technique has emerged as a promisingalternative to transport and synchronize embryos from different spawning eventsin fish like pejerrey (Odontesthes bonariensis) and the Siamese fightingfish (Betta splendens), with high aquaculture and scientific interestworldwide. Early- and middle-stage embryos were microinjected and/or immersedin different cryoprotectants solutions (CSs), and cooled for 1- to 24-hours atlow (14 and 5 °C) or sub-zero (-14 and -20 °C) temperatures. After cooling, embryonicand larval parameters were evaluated. Larvae were stained with Alcian Blue orO-Red-Oil, and the cranial cartilages, and swim bladder area were measured. Furthermore,a comparative study using medaka (Oryzias latipes) embryos from atransgenic line (TG955) with GFP and mCherry on the skeletal and cardiacmuscle, respectively, was done. The most suitable protocol forcooling middle-stage O. bonariensis embryos at -14 °C for 1-hour was theslow temperature decrease in combination with a CS composed of 2.5 M methanol,1.4 M Me2SO, 0.3 M sucrose, and 0.08 M NaCl, obtaining 87% hatchingand 30% normal-looking larvae. If cooling was performed until -20 °C, hatchingdecreased to 60% and only 4.5% normal-looking larvae were obtained when a CSwas microinjected in the perivitelline space. In the case of B. splendensembryos, the selected CSs for its storage for 9-hours at 5 °C or 24-hours at 14°C, obtaining 77% hatching and 52% normal larvae or 88% hatching and 81% larvaewith mild abnormalities, were those composed of 0.25 M sucrose and 0.75 M methanol;and 0.125 M sucrose and 0.375 M methanol, respectively. Larval malformationsincluded craniofacial, tail and muscle-formation abnormalities, cardiac edema,and deflated swim bladder. Remarkably, a partial recovery was noticed in somelarvae. These results establishthe conditions for further studies on the preservation of O. bonariensisand B. splendens embryos.(295 words)