Retrieval and cryopreservation of sperm in spermatophores from cadaveric Indian white shrimp, Fenneropenaeus indicus (H. Milne Edwards, 1837).

This study focused on the quality of sperm obtained from spermatophores of cadaveric shrimp after long-term storage. Spermatophores were collected using the stripping method, which has resulted in maximum sperm viability when this approach was previously used. Cryoprotectants toxicity assessment of samples was conducted using dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG), glycerol (Gly), dimethyl acetamide (DMA) and propylene glycol (PG) at different concentrations (5%, 10% and 30% v/v), prepared in Ca-F saline. Based on the results from the cryoprotectant toxicity assay, DMSO and MeOH were used individually as well as in combination for the subsequent study. Samples along with cryoprotectants were subjected to slow and fast freezing protocols (i.e. -0.5, and -10 °C/min to a final temperature of -80 °C) and were subsequently stored in LN2 (196 °C). Similarly, vitrification was performed by plunging the samples directly in to LN2. Samples of control and cryopreserved spermatophores that were stored for 45 days had sperm viabilities of 91.4 ±â€¯3.6% and 53.9 ±â€¯4.7%, respectively. Further observations with HOST and DNA integrity analyses of the cryopreserved sperm, resulted in percentages of 45.6 ±â€¯4.2%; 58.1 ±â€¯1.7% compared to control values of 82.3 ±â€¯4.8%; 94.3 ±â€¯1.9%, respectively. Use of the one-step slow freezing protocol at the rate of -0.5 °C/min between 4 °C and -80 °C in LN2 with DMSO (5%) + MeOH (5%) was a desirable preservation strategy of spermatophores, compared to other freezing protocols. Unlike sperm viability, the HOST results affirm the fertility potential of the sperm that have the capacity to participate in the fertilization process. Thus, the results of this study demonstrate that long term storage of sperm in spermatophores of Fenneropenaeus indicus collected from cadaveric specimens can result in viable sperm after cryopreservation if extender (Ca-F saline) containing DMSO and MeOH are used.

Effect of cryoprotectants and cooling rates on fertility potential of sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man).

This study evaluates freezing protocol with suitable cryoprotectants and their effects on the fertility potential of sperm in the cryopreserved spermatophores of Macrobrachium rosenbergii. Spermatophores, collected using electroejaculation, were suspended in dimethyl sulfoxide (DMSO), propylene glycol (PG), methanol, glycerol and ethylene glycol (EG) at different concentrations (10, 15 & 20% v/v), prepared in sterile-filtered pond water. Based on the cryoprotectant toxicity assay, DMSO and PG were used individually as well as in combination with three freezing protocols (i.e. -1.5, -3 and -5°C/min and to final temperature of -39°C) and plunged into liquid nitrogen at -196°C. After 90 days of storage (-196°C) thawing was done at 35°C in a water bath for 1min. Results showed that fresh and cryopreserved spermatophores held for 90 days registered sperm viability of 91.4±2.9% and 50.4±1.9% respectively. Further, fertility potential of sperm was assessed based on acrosome reactivity using calcium ionophore (A23187). Observations indicated that cryopreserved sperm registered 28.3±2.2% of acrosome reactivity compared to freshly collected spermatophores (85.3±2.5%). Thus, one-step slow cooling rate of -1.5°C/min between 27°C and -39°C stored in liquid nitrogen at -196°C with DMSO (10%)+PG (10%) seems to be amenable for cryopreservation of spermatophores, compared to other cooling rates.

TBT effects on the development of intersex (ovotestis) in female fresh water prawn Macrobrachium rosenbergii.

The impact of tributyltin (TBT) on the female gonad and the endocrine system in Macrobrachium rosenbergii was studied. Prawns were exposed to environmentally realistic concentrations of 10, 100, and 1000 ng/L of TBT for 6 months. Dose dependent effects were noticed in TBT exposed prawns. At 1000 ng/L TBT caused ovotestis formation (formation of male germ cells in ovary). Presence immature oocytes, fusion of developing oocytes, increase in interstitial connective tissues, and its modification into tubular like structure and abundance of spermatogonia in the ovary of TBT treated prawns. The control prawn ovary showed normal architecture of cellular organelles such as mature oocytes with type 2 yolk globules, lipid droplets, normal appearance of yolk envelop, and uniformly arranged microvilli. On the other hand, type 1 yolk globules, reduced size of microvilli, spermatogonial cells in ovary, spermatogonia with centrally located nucleus, and chromatin distribution throughout the nucleoplasm were present in the TBT treated group. Immunofluorescence staining indicated a reduction in vitellin content in ovary of TBT treated prawn. Moreover, TBT had inhibited the vitellogenesis by causing hormonal imbalance in M. rosenbergii. Thus, the present investigation demonstrates that TBT substantially affects sexual differentiation and gonadal development in M. rosenbergii.