Abstract
The study was carried out to produce improved broodstocks of Rohu (Labeo rohita) using cryopreserved
sperm and to assess their quality through DNA microsatellite markers. In order to achieve the goals several experiments like, standardization of the cryopreservation protocol, production of fry using cryopreserved
and fresh sperm at different experimental sites, rearing the produced fry and comparing their growth performances, quality assessment through DNA microsatellite markers etc. were conducted. In 0.4% NaCl
solution, highest motility of 95.2±2.5% and swimming duration of 47.2±0.84 min of sperm were observed. In
most cases 5% and 10% concentrations of cryoprotectant produced good results at 5 to 10 min incubation,
but 15% concentration produced worst results, and seemed toxic to sperm. Alsever's solution with 10%
DMSO produced best equilibration (91.6±3.20%) and post-thaw motility (85.2±1.78%). Along with Alsever's
solution and egg-yolk citrate with 10% DMSO at the dilution ratio of 1:9 and 1:4, sperm produced
48.33±2.51% and 33.66±3.51% fertilization, and 43.81±4.63% and 20.57±6.33% hatching respectively. At three public hatcheries 67.5±7.23%, 78.33±4.04% and 59.66±4.5% fertilization, and 43.32±5.74%, 37.79±2.8% and 44.55±3.38% hatching; and 80.5±4.43%, 86.66±3.51% and 74.0±2.64% fertilization, and 56.63±4.35%,
49.36±2.51% and 61.14±1.4% hatching were obtained by cryopreserved sperm of the Halda River origin and
fresh sperm of hatchery-reared male origin respectively. Similarly, 69.00±3.05%, 55.33±7.02% and 62.33±6.0%
fertilization and 48.30±5.91%, 30.01±4.49% and 35.9±5.94% hatching; were obtained from cryopreserved
sperm of the Halda River male; and 74.33±2.51%, 85.66±2.51% and 79.66±3.51% fertilization, and
64.15±4.51%, 52.87±4.02% and 53.05±7.54% hatching were obtained from fresh sperm of hatchery-reared
male at three private hatcheries. The average final length and weight were 18.06±0.36 cm and 19.33±0.23 cm,
and 64.04±0.56 g and 83.18±0.48 g; average length and weight gain were 12.75±0.39 cm and 13.08±0.21 cm,
and 61.88±0.49 g and 80.20±0.44 g; SGRs of fry ranged from 0.23% to 6.24%/day; survival rate were
85.0±2.45% and 86.0±5.35% after 64 days of rearing under the same physico-chemical conditions of ponds of
the fry of fresh sperm of hatchery-reared male and cryopreserved sperm of the Halda River male,
respectively. Highest motility of sperm was recorded as 93.03±1.28% and motility remained for 1.73±0.06
min. Average fertilization rates of cryopreserved sperm of cryopreserved sperm-originated (F1) male and fresh sperm of hatchery-reared male were 78.5±2.12%, 82.5±4.94% and 94.5±2.12%, and 83.5±4.94%,
86.5±4.94% and 95.5±3.53% respectively; whereas the average hatching rates of cryopreserved sperm of
cryopreserved sperm-originated (F₁) male and fresh sperm of hatchery-reared male were 69.49±6.01%,
76.56±0.48% and 89.95±3.91%, and 72.69±7.81%, 83.63±1.99% and 91.74+4.14% respectively at two public and
one private hatchery. The average final length and weight were 16.89±0.47 cm and 17.87±0.36 cm, and
69.56±4.50 g and 91.02±3.54 g; average length and weight gain were 12.64±0.06 cm and 13.32±0.04 cm, and
65.93±1.81 g and 86.72±0.16 g; SGRs were 3.84±0.01%/day and 3.96±0.06%/day; survival rate were
85.72±4.04% and 91.43±4.04% after 78 days of rearing under the same physico-chemical conditions of ponds
of the fry of fresh sperm of hatchery-originated male and cryopreserved sperm of cryopreserved sperm- originated (F₁)male respectively. When fry were produced using fresh sperm of Halda river origin male and
fresh sperm of cryopreserved sperm-originated (F₁) male the average final length and weight were 18.57±0.16 cm and 19.79±0.14 cm, and 99.40±2.93 g and 115.42±2.53 g; average length and weight gain were
13.13±0.02 cm and 13.63±0.11 cm, and 94.43±2.61 g and 109.62±2.55 g; SGRs were 3.95±0.04% and
3.69±0.04%; and survival rate were 81.43±6.06% and 84.29±6.06% after 78 days of rearing under the same
physico-chemical conditions of ponds respectively. Seven DNA microsatellite markers Lr3, Lr12, Lr14b, Lr21,
Lr22, Lr24 and Lr27 were used to determine the parental genetic inheritance to the fry produced with
cryopreserved sperm. The fry were produced by fertilizing eggs collected from the six selected hatcheryreared females with cryopreserved sperm of Labeo rohita of the Halda River origin. Among the seven
primers, stable and repeated relevant alleles were obtained from all the primers in the fry produced at FSMF, Shombhuganj; stable and repeated relevant alleles were obtained from primers Lr14b, Lr22, Lr24 and
Lr27 in the offspring at FSMF, Maskanda; and stable and repeated relevant alleles were obtained from
primer Lr24 in the offspring at BAU hatchery. Similarly, stable and repeated relevant alleles were obtained
from primers Lr3, Lr12 and Lr14b in the offspring at Sotota Matshya Prozonon Kendra and Fishery; stable
and repeated relevant alleles were obtained from primer Lr12 in the offspring at Biswas Agro Fisheries and
Hatchery; and stable and repeated relevant alleles were obtained from primer Lr21 in the offspring at
Deshbandhu Matshya Hatchery and Nursery. All the above primers showed that cryopreserved sperm
originated fry shared alleles with their parents, i e. parental genetic inheritance was maintained to all the
cryopreserved sperm-originated fry following Mendelian rules.