Effect of Sperm-Egg Ratio and Thawing Protocol on Hatching Success of Cryopreserved Rohu (Labeo rohita) Milt

Sexually mature male and female rohu broodstock were selected based on external morphological characteristics during the breeding season. The male broodfish were held separately in shadowed tanks and supplied with well aerated water. In accordance with carp cultivation protocol, tanks were prepared and managed. A commercial diet was given to broodstocks at a rate of 2% body weight per day until they were satiated.

The brood stocks were collected from ponds 12-24 h prior to planned stripping and transported to the hatchery for acclimatization. Male brood fishes were conditioned in a 5000-liter tank under continuous showering condition for 6 h before hormonal induction.

The male broodfish were induced with WOVA-FH (Biostadt India Limited, Mumbai) at a dose of 0.2 mg/kg body weight. Prior to the collection of milt, the male brood fish were anesthetized using phenoxyethanol (Bernath et al., 2016) at 0.4 ml/L. The abdomen was wiped with tissue paper to remove moisture and water if any, to avoid unexpected activation of the spermatozoa. The milt was collected 4 h after hormonal administration by gentle hand stripping into dry plastic sperm collection box.

Selected extender and cryoprotectant combinations were used for cryopreservation. Milt was diluted at a ratio of 1:5.3:0.7 (milt: extender: cryoprotectant). Extender was first mixed with milt, followed by the gradual addition of cryoprotectant. The diluted milt was loaded into 2 ml, 5 ml, and 12ml cryovials using stepper (NICHIRYO, F 19500031, Japan) and equilibrated for 12 min before vapour-phase freezing. Cryovials were placed on a steel stand positioned above the liquid nitrogen surface during vapour-phase freezing. The distance from the liquid nitrogen surface was 2.5 cm for 2 ml cryovials, 2.0 cm for 5 ml cryovials, and 1.5 cm for 12 ml cryovials. Samples were exposed to nitrogen vapour for 12 min, achieving an approximate cooling rate of −10 °C min⁻¹ before plunging into liquid nitrogen (−196 °C) for storage.

Cryovials were removed from liquid nitrogen and immediately subjected to thawing in water bath (REMI, RWB 6, India). Cryovials were thawed in an ice–water mixture (10:90, ice: water, v/v) until complete melting of the contents. Subsequently, samples were exposed to the designated thawing temperatures (40–55°C) according to the experimental treatments before fertilization.

Female broodfish were induced with WOVA-FH at a dose of 0.5 mg kg⁻¹ body weight via intraperitoneal injection. Eggs were collected by hand stripping 6 h after hormonal induction following anesthesia with phenoxyethanol. Eggs from a single female were pooled and divided into treatment batches in triplicate.

For fertilization trials, one spoonful of eggs (approximately 16,000 eggs) was fertilized with varying volumes of cryopreserved milt according to treatment. Thawed milt was mixed thoroughly with eggs, and spermatozoa were activated by adding 5 ml of tap water. For control treatments, 300 µl of fresh milt collected 4 h after hormonal induction was used. Fertilized eggs were incubated under standard hatchery conditions, and hatching percentage was recorded.

All experiments were conducted in triplicate. Data were expressed as mean±standard deviation (SD). Statistical differences among treatments were analyzed using one-way ANOVA followed by post hoc multiple comparison tests. Differences were considered significant at p < 0.05.

Hatching success varied significantly among different sperm–egg ratios (Table I). Based on sperm concentration analysis and the volume of milt used for fertilization, the optimal treatment corresponded to approximately 1.88 × 10⁶ spermatozoa per egg. The treatment using one spoonful of eggs fertilized with one vial (2 ml) of cryopreserved milt resulted in the highest hatching rate (51%), followed by one spoon of egg with two vials (25%). Lower hatching rates were observed in treatments using one spoonful of eggs with 1.5 vials (22.91%) and 1.5 spoonful of eggs with one vial (21.94%). The control group fertilized with fresh milt exhibited the highest hatching rate (79%).

Values with different superscripts differ significantly (p < 0.05).

Thawing temperature significantly affected hatching success across different cryovial sizes (Table II). The 2 ml and 5 ml cryovials thawed at 40°C yielded hatching rates of 51% and 48%, respectively. For 12 ml cryovials, the highest hatching rate (47.9%) was obtained at 45°C, while higher thawing temperatures of 50°C and 55°C resulted in reduced hatching rates (45.3% and 40.82%, respectively). The control group, using fresh sperm, had the best hatching rate (79%).

Hatching success obtained in the present study is comparable with earlier reports on cryopreserved carp sperm. Cejko et al. (2022) reported hatching rates ranging from 36% to 48%, depending on the type of extender and sperm-to-egg ratio. Kumar et al. (2023) reported the use of approximately 1.5 × 10⁶ sperm cells per egg for fertilization using cryopreserved milt in common carp. It is important to observe that comparing the hatching % without comparing the sperm: Egg ratio will have a major effect on the results (Di Lorio et al., 2019). Previous studies have reported wide variation in optimal sperm–egg ratios among species, including 8 × 10⁵ sperm per egg in silver barb (Vuthiphandchai et al., 2015), 4.6 × 10⁶ sperm per egg in Brycon orbignyanus (Maria et al., 2006), 1 × 10⁵ sperm per egg in common carp (Warnecke and Pluta, 2003), and 5 × 10⁵ sperm per egg in rainbow trout (Sahin et al., 2013). The optimal sperm–egg ratio determined in this study (1.88 × 10⁶ spermatozoa per egg) was higher than values reported for several freshwater species. The relatively higher sperm requirement may be attributed to the use of cryopreserved milt and the reduced egg quality associated with late breeding season conditions.

Previous reports indicate that larger cryovials can be effectively used for fish sperm cryopreservation when appropriate thawing protocols are applied. Kumar et al. (2023) reported the hatching rate of using 2 ml and 5 ml cryovials 55.6±11.2 and 53.4±7.6 in common carp. Varkonyi et al. (2019) reported higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryovial than with the pike extender in common carp. Beirao et al. (2011) reported the hatching rate of 34.5±19.3% in sea bream. Ji et al. (2004) reported the use of 1.8 ml cryovial in sea perch with fertilization and hatching rate of 84.8% and 70.1%, respectively. In the present study best hatching rate of using 12 ml cryovial is 47.9% demonstrating the feasibility of large-volume cryopreservation. Through this research, it will help the seed producer to store more sperm per each drop of liquid nitrogen. Further studies required to develop the suitable sperm egg ratio and freezing of fish sperm for large quantity for commercial seed production. Rapid thawing at 40–45°C likely minimized ice recrystallization damage, while higher temperatures (50–55°C) may have caused thermal stress to sperm membranes. The higher sperm requirement observed for cryopreserved milt may be due to reduced post-thaw motility and membrane integrity.

The study demonstrated that hatching success of cryopreserved rohu (Labeo rohita) milt is significantly influenced by sperm–egg ratio and thawing protocol. Optimal hatching was achieved when one spoonful of eggs was fertilized with one 2 ml vial of cryopreserved milt, while thawing at 40°C for 2 ml and 5 ml cryovials and 45°C for 12 ml cryovials yielded the best results. Although fresh milt produced higher hatching rates, optimized cryopreserved milt showed satisfactory performance. These findings confirm the feasibility of using large-volume cryovials for upscaled milt cryopreservation and highlight their potential application in commercial carp seed production.

The authors sincerely thank ICAR–NBFGR, Lucknow, for providing facilities and support to carry out this research.

The study received no externa funding.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors have declared no conflict of interest.