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Optimizing Germ Cell Culture and Cryopreservation in Endangered Beluga Sturgeon: A Strategy for Accelerated Reproductive Maturity

Hediye Fadakar 1, Tooba Mirzapour 1, *, Shirin Jamshidi 2, Tooraj Sohrabi 2 

1 Department of Aquaculture and Animal Production, College of Agriculture and food Sciences, King Faisal University, Al Hofuf, Kingdom of Saudi Arabia

2 Animal and Fish Production Department, Faculty of Agriculture, Alexandria University, Saba Basha, P.O. Box 21531, Alexandria, Egypt
  1. Department of biology, Faculty of Science, University of Guilan, Rasht, Gilan, Iran
  2. International Caspian Sturgeon Research Institute, Iranian Fisheries Science Research institute, Agricultural Research Education and Extension Organization (AREEO), Rasht, Gilan, Iran

* Corresponding author: Mirzapour, T. : dr.tooba72@gmail.com; Tel: +981333333647

 

Article info:

 Submitted: 5/5/2025

Revised: 23/5/2025

Accepted: 12/6/2025

Online: 14/6/2025

Abstract: The formation and growth of gonads, essential for proper gametogenesis and sexual maturity, rely heavily on germline stem cells. In this study germ cells were isolated from the gonads of six-month-old beluga sturgeons (Huso huso) using various concentrations of trypsin and collagenase enzymes. The isolated cells were cultured for three weeks in two different culture mediums: Leibovitz 15 (L15) and Dulbecco's Modified Eagle Medium (DMEM). The formation of germline colonies was compared between the two culture media. The cells' identity was confirmed by immunocytochemistry utilizing a VASA antibody. The expression of specific genes (Vasa, Nanos, Bax and p53-like) was investigated using qRT-PCR. Cryopreservation of germ cells was performed with two different freezing solutions: dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Based on results isolating germ cells from gonads with 0.5% trypsin- EDTA and 1 mg/ml collagenase caused minimal damage to the cells.  In this manner the digestion time decreased to half an hour and the percentage of cell viability also increased to 95%. L15 medium supported the growth of larger colonies due to better pH stability.
(101.87±8.40). However, prolonged culture beyond 20 days led to a reduction in both the number and diameter of colonies. The expression of Nanos and Vasa genes was highest during mid-growth in both culture media (1.38±0.04, 1.27±0.04 respectively), while the expression of Bax and P53-like genes increased at the end of the culture period (after 33 days, 1.82±0.09, 0.48±0.04 respectively). Cell viability after thawing was significantly improved with DMSO compared to ethylene glycol (78±3.00). Given that beluga sturgeons have a prolonged reproductive cycle and reach sexual maturity late, successful isolation, proliferation, and cryopreservation of their germ cells could enable future transplantation into the peritoneal cavity of newly hatched larvae from female fish species with shorter reproductive cycles. This approach offers a promising strategy by reduction long maturation time for conserving these endangered species.

Keywords: Huso huso, Germ cells culture, Assisted reproduction, Cryopreservation, Cell viability, Beluga Sturgeons.

Cite as: Fadakar, H., Mirzapour, T., Jamshidi, S., Sohrabi, T. (2015) Optimizing Germ Cell Culture and Cryopreservation in Endangered Beluga Sturgeon: A Strategy for Accelerated Reproductive Maturity. Animal Reports, 1 , 1:19. In press

Formation of germ cells colony in culture medium and Vasa immunoreactivity

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