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

Accepted Manuscript Title: Retrieval and cryopreservation of sperm in spermatophores from cadaveric Indian white shrimp, Fenneropenaeus indicus (H. Milne Edwards, 1837) Authors: Narasimman Selvakumar, Krishnamoorthy Dhanasekar, Buelah Carmel D., Natesan Munuswamy PII: DOI: Reference:

S0378-4320(17)31053-9 https://doi.org/10.1016/j.anireprosci.2018.03.008 ANIREP 5786

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

16-12-2017 12-2-2018 6-3-2018

Please cite this article as: Narasimman S, Krishnamoorthy D, Buelah CD, Natesan M, Retrieval and cryopreservation of sperm in spermatophores from cadaveric Indian white shrimp, Fenneropenaeus indicus (H. Milne Edwards, 1837), Animal Reproduction Science (2010), https://doi.org/10.1016/j.anireprosci.2018.03.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Retrieval and cryopreservation of sperm in spermatophores from cadaveric Indian white shrimp, Fenneropenaeus indicus (H. Milne Edwards, 1837) Narasimman Selvakumar, Krishnamoorthy Dhanasekar, Buelah Carmel. D &

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Natesan Munuswamy*

*Unit of Aquaculture & Cryobiology, Department of Zoology, University of Madras,

Guindy Campus, Chennai, Tamil Nadu, India

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[email protected], [email protected], [email protected], [email protected]

*Correspondence: Prof.N. Munuswamy; Unit of Aquaculture & Cryobiology, Department of

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Zoology, University of Madras, Guindy Campus, Chennai - 600 025, Tamil Nadu, India; Tel.: +91 44 22202841; fax: +91 44 22300899; E-mail address: [email protected] (N. Munuswamy)

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Highlights Effect of cryoprotectants on sperm quality of F. Indicus on cryopreservation.

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One-step freezing at cooling rate of -0.5 °C/min between 4°C and -80°C before LN2.

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Development of freezing protocol with Ca-F saline and 10% egg yolk containing DMSO

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5% + MeOH 5%. 

Comparison of sperm quality such as HOST and DNA integrity test in fresh and cryopreserved spermatophores.

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Abstract

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This study focused on the quality of sperm obtained from spermatophores of cadaveric

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shrimp after long-term storage. Spermatophores were collected using the stripping method,

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which has resulted in maximum sperm viability when this approach was previously used. Cryoprotectants toxicity assessment of samples was conducted using dimethyl sulfoxide

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(DMSO), methanol (MeOH), ethylene glycol (EG), glycerol (Gly), dimethyl acetamide (DMA)

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and propylene glycol (PG) at different concentrations (5%, 10% and 30% v/v), prepared in Ca-F

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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

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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

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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 ± 3.6% and 53.9 ± 4.7%, respectively. Further observations with HOST and DNA integrity analyses of the cryopreserved sperm, resulted in percentages of 45.6 ± 4.2%; 58.1 ± 1.7% compared to control values of 82.3 ± 4.8%; 94.3 ± 1.9%, respectively. Use of the one-step slow freezing protocol at 2

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

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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.

Keywords: Fenneropenaeus indicus; Cadaveric; Spermatophores; Dimethyl sulfoxide +

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Methanol; Hypo osmotic swelling test; DNA integrity.

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1. Introduction

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Cadaveric fish spermatozoa are potential means to recover the male gametes from

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organisms that die unexpectedly due to environmental disasters (Koteeswaran and Pandian, 2002). In the event of such emergencies, there is no method available for the restoration of

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shrimp species in a short time. Death of genetically improved brood fish in culture ponds causes

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considerable economic loss to the farmers worldwide (Routray et al., 2006). Sustainable

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aquaculture practices by judicious use of available aquatic resources are desirable, where endangered species have a competitive niche (Costa-Pierce 1996; Costa-Pierce and Bridger,

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2002).

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Another possible solution that can help to mitigate the limited availability of improved

broodstock is the use of cryopreservation of high quality gametes from selected individuals (Frankel, 2013). Thus, cryopreservation has a significant role in addressing concerns such as aquatic biodiversity and environmental conservation (Chao and Liao, 2001). The number of threatened and endangered species is increasing (Knapp, 2000), hence, cryopreservation is 3

considered an effective strategy to save the endangered species by facilitating the storage of gametes in a gene bank (Gausen, 1993). Spermatozoa cryopreservation, therefore, has proved an invaluable technique for sustaining endangered species (Polge et al., 1949; Polge and Rowson,

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1952). Sperm in decapod crustaceans have unique shapes and are non-motile, although, some decapods contain microtubules that may be responsible for the immobility of the sperm in all decapods. The immobility of the sperm in vivo make viability assessment difficult (Moses, 1961; Clark et al., 1981; Niksirat et al., 2013). The non-motile sperm of decapod crustaceans consist of

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two groups, the multistellate reptantian and unistellate natantian sperm (Talbot and Summers,

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1978).

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Fenneropenaeus indicus, being an endangered species, similar to other shrimp species have been added to the seafood red list by Greenpeace International (FAO, 2010). The main

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threats to this species that result in it being endangered are habitat degradation and the

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introduction of exotic species. Besides, over exploitation and over harvesting of wild larvae and brooders, destruction of mangroves, misuse of wetlands, pollution of waterways and

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ground water due to pond effluents are the main causes of loss of biodiversity (Jelks et al., 2008;

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Frankel, 2013; FAO, 2014). Among the penaeid shrimp species identified as important for culture practices, F. indicus is considered as the most ideal candidate species due to its short

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lifespan, desirable growth characteristics and excellent survival rates in culture. This is one of the major commercial penaeid species in the world that contribute to about 2.4% of global fisheries production (FAO, 2008). Furthermore, the culturing of this species allows for collection of viable spermatophore from wild caught penaeid shrimp for artificial insemination or aquaculture practices. Retrieval of spermatophores from cadaveric shrimp, thus, is a potential option when 4

optimal time intervals and storage conditions are understood for the dead animals. Previous studies are limited to the shrimp species Litopenaeus schmitti and Litopenaeus vannamei (Fernandes et al., 2014; Castelo-Branco et al., 2015).

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The present study, therefore, was conducted to assess whether the practice of retrieving spermatophores from the cadaveric shrimp, F. indicus, to augment the quality of sperm and the utilization of spermatophores for long-term storage by means of cryopreservation. 2. Material and methods

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2.1. Experimental animals

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Sexually mature males of F. indicus, that were alive and dead (cadaveric) were obtained

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from professional fishermen at Ennore and Pazhaverkadu estuaries near Chennai, Tamilnadu,

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India. The shrimps (cadaveric) were collected within 1 h of capture before freezing. The samples

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were immediately transferred to the laboratory in insulated boxes covered with ice and stored at

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4°C for further processing. Specimens without any injury/damage and integrity of the antennae, rostrum, pleopods, pereopods and spermatophores were evaluated for selection of individuals.

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Male shrimp of an average weight of 34.1 ± 2.5 g and length of 16.1 ± 1.1cm (n=50) were used

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for the study.

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2.2. Collection of spermatophores Specimens that were alive and dead (cadaveric) were cleaned with sterile sea water and

70% ethanol sprayed around the gonophores prior to collection of spermatophores. Manual ejaculation of spermatophore was performed on all males by stripping the area around the base of the coxa of the fifth pair of walking legs (pereiopods), just below the opening of the

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gonopore within 1 h after death and subsequent storage at 4°C. Spermatophores obtained from each male weighed about 0.059 ± 0.08g. 2.3. Assessment of sperm viability

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Sperm viability of fresh and cryopreserved spermatophores during storage was determined using a modified eosin-nigrosin protocol (Jeyalectumie and Subramonium, 1989; Nimrat et al., 2005). The sperm suspension (50 µL) was transferred onto a slide, mixed with 50 µL of 0.5 % eosin and 10 % nigrosin, air dried and examined using bright field microscopy

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(40 X magnification, Leica microscope DM 2500, Germany). Live sperm were unstained and

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were very visible on the red background of nigrosin staining, while dead sperm stained with pink

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color. Percentage of viable sperm was calculated in triplicate by counting a minimum number of

2.4. Toxicity assay of cryoprotectants

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250 cells.

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Cryoprotectants such as dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol

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(EG), glycerol (Gly), dimethyl acetamide (DMA) and propylene glycol (PG) were used

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(Sigma-Aldrich, St. Louis, MO, USA). Each cryoprotectant solution was prepared to a final concentration of 5%, 10% and 30% (v/v) using sterile calcium free saline (Ca-F saline) as an

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extender. The Ca-F saline (Leung-Trujillo and Lawrence, 1987) contained 21.63g NaCl, 1.12g KCl, 0.53g H3BO3, 0.19g NaOH and 4.93g MgSO4.7H2O in 1 liter of double distilled water.

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Each spermatophore was directly suspended in 0.5 mL of a cryoprotectant solution in vials. For each control, the freshly collected spermatophore was immersed in Ca-F saline without

cryoprotectant. The apparent sperm viability was assessed after 30 and 60 min of exposure time at room temperature, by removing the cryoprotectants from the vials. Spermatophores were

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rinsed twice with Ca-F saline at a similar temperature. A random sample was taken and stained with eosin-nigrosin stain and as many as 250 viable sperm were counted using bright field (40 X magnification, Leica microscope DM 2500, Germany). Each treatment had three replicates

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of observation. 2.5. Hypo osmotic swelling test

Sperm functional integrity was evaluated using the hypo osmotic swelling test (HOST) as described by Lomeo and Giambersio, (1991); Chan et al. (1992) with some modifications. Sperm were subjected to hypo-osmotic media (2:1 mixtures of Ca++FASW andDDH2O) and the

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osmotic response, as indicated by volume changes, was determined as an index for membrane

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integrity (Bhavanishankar and Subramonium, 1997). Viable sperm become swollen (or) everted

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upon exposure to hypo-osmotic medium and this variable was used as an indication of membrane

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2.6. DNA integrity test

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integrity.

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The DNA integrity of sperm was evaluated using acridine orange as described by Varela

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junior et al. (2012). The metachromatic colorant acridine orange emits green fluorescence on reaction with double-stranded DNA and an orange or red fluorescence on reaction with single

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stranded DNA, therefore, identifying breaks in the DNA. For this assessment, 45µL of the sperm sample was diluted with 50 µL of TNE (0.01 M Tris HCl, 0.15 M NaCl, 0.001 M EDTA;

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pH 7.2) and incubated for 30 seconds, followed by addition of 200 µL triton 1x solution. At 30 seconds subsequent to the addition of the triton solution, samples were treated with 50 µL

of acridine orange (2 mg/mL in deionized water) and observed under a fluorescence microscope (Nikon E 200, Japan).

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2.7. Cryopreservation by rate controlled freezing and vitrification Toxicity of cryoprotectants (i.e.) DMSO (5%), MeOH (5%), and DMSO (5%) + MeOH (5%) was assessed to develop a freezing protocol including egg yolk (10%) as

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co-cryoprotectant and trehalose (0.25M) with the extender Ca-F saline. The spermatophores collected from the cadaveric shrimp served as the control (day 0). The spermatophore was stored in cryoprotectant solution at room temperature for 30 min. Slow and fast freezing rates were imposed from the initial temperature of 4°C, at freezing rates of 0.5°C/min and-10°C/min to the final temperature of -80°C. These freezing protocols were achieved using a programmable

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freezer (Cryoplanner, Kryo 360 - 1.7 planer plc, Sunbury, Middlesex, UK). Two freezing rates

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were attempted; (-0.5ºC Protocol A, -10ºC Protocol B) spermatophores were cooled to the final

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temperature of -80°C, and held for 5 min before storing in LN2. Similarly, vitrification

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(Protocol C) procedures were performed with samples in vials being positioned for 10 min at 5

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cm above the liquid surface of LN2 before plunging the samples into LN2 for storage. The

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samples were directly plunged into LN2 (-196°C) and assessed for sperm quality at intervals of 15, 30 and 45 days after initiation of storage. Thawing was performed in a water bath at 30°C for

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1 min for all samples.

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2.8. Statistical analysis

The data were assessed using a two-way analysis of variance (ANOVA) followed by

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post-hoc test (Duncan’s multiple range test) to determine the level of significance. Percentages of viable sperm considering cryoprotectant toxicity, as assessed for HOST and DNA integrity were compared after arcsine transformation. Difference from the control specimens was considered significant at P<0.05. The SPSS version 21.0 was used for the analysis.

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3. Results 3.1. Sperm quality assessment Sperm obtained from the spermatophore of fresh (live) and cadaveric (dead) shrimp had

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viabilities of 96.4 ± 2.7% and 92.8 ± 2.9%, respectively. Similarly, HOST and DNA integrity tests of sperm samples resulted in values of 91.6 ± 2.4, 87.9 ± 1.5% and 94.3 ± 2.1%, 93.2 ± 1.9%, respectively. These observations on sperm quality served as the control values for the studies performed with different cryoprotectants.

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3.2. Effect of cryoprotectants on sperm viability

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Values relative to cryoprotectant toxicity on sperm when DMSO (5%) and MeOH (5%)

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were used were less compared with control values. The use of other cryoprotectants (EG, Gly,

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DMA and PG) resulted in values that were less as compared with the control values for viability of sperm (P< 0.05). The use of the combination of DMSO (5%) and MeOH (5%) resulted in

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greater sperm viability compared to samples treated with either DMSO or MeOH alone. The

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amount of time sperm were stored in different concentrations of cryoprotectants influenced

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sperm viability. Storage for 30 min in DMSO (5%), MeOH(5%) and DMSO (5%) + MeOH(5%) resulted in values for sperm viability of 82.8 ± 3.8%, 81.8 ± 2.9% and 83.7 ± 2.5% and with 60

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min of storage the viability was less (74.1 ± 1.5%, 73.2 ± 4.1% and 76.5 ± 2.3% respectively;Table.1). When the other cryoprotectants were used at different concentrations,

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there was a lesser sperm viability at 30 and 60 min of storage. The cryoprotectants EG, Gly, DMA and PG were moderately toxic at minimum concentrations and highly toxic at greater concentration when stored for 30 and 60 min. When the concentration of cryoprotectants and storage time was increased, there was a reduction in sperm viability (P< 0.05).

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3.3. Freezing protocols for long-term storage The spermatophores cryopreserved using Protocol A had sperm that were of greater quality compared with use of the freezing Protocol B and C (Figs.1-3). Sperm quality was

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greater when using the slow freezing Protocol A (-0.5°C/min) with sperm viability, HOST and DNA integrity being 53.9 ± 4.9%, 45.6 ± 4.2% and 58.1 ± 1.7%, respectively, with DMSO (5%) + MeOH (5%) after 45 days of storage. The sperm cells of spermatophores subjected to freezing Protocol B (-10°C/min) had a viability, HOST and DNA integrity of 38.8 ± 4.3%, 25.2 ± 2.5% and 44.9 ± 2.5%, respectively, after 45 days of storage with use of the DMSO (5%) +

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MeOH (5%) extender. Sperm cells in spermatophores stored using the vitrification method,

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Protocol C, had a viability, HOST and DNA integrity of 31.7 ± 4.1%, 16.5 ± 1.9% and

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34.0 ± 2.5%, respectively, when using the combination of DMSO (5%) + MeOH (5%) for

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45 days of storage. Sperm mortality was great in the spermatophores preserved using vitrification

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due to the rapid freezing that results with use of this procedure. Use of vitrification resulted in a

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lesser sperm quality compared with use of the other freezing protocols. When using the other cryoprotectants, there was a minimum sperm quality after different durations of storage when all

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freezing protocols were used, and there was no differences when the different protocols were used (P> 0.05). The sperm in spermatophores preserved using Protocol A with DMSO (5%) +

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MeOH (5%) had greater viability after 15, 30 and 45days of storage as compared with use of

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other cryoprotectants. Variations of sperm quality parameters on different storage days, however, were significant (P< 0.05). 4. Discussion

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Cryopreservation techniques applied to aquatic organisms have direct application to aquaculture and conservation of threatened species (Subramonium, 1994). There are reports indicating that appreciable numbers of viable gametes from both live and dead fish for

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preservation purposes. Gametes can be successfully stored using proper cryopreservation protocols for propagation of progeny of several species. The possibility of using a simple and widely practicable method of post-mortem preservation of sperm of teleostean fish has been attempted (Koteeswaran and Pandian, 2002). In addition, the time interval between death and sperm collection is associated with cell viability (Kroon et al., 2012). Earlier investigations in

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this field clearly indicate that the successful use of cadaveric sperm was accomplished for fin

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fish and shellfish. The study of Fernandes et al. (2014) on sperm viability (L. schmitti) indicated

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that there was a greater mean survival (50.96 ± 19.5%) with use of the procedure of reducing

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temperature by -0.5°C/min to -32°C. A study performed by Castelo-Branco et al. (2015) with cadaveric shrimp L.vannamei indicated the maximum sperm viability of 69 ± 2% occurred with

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storage at 4°C. In the present study with F. Indicus, a sperm viability of 53.9 ± 1.8% registered

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with cryopreserved spermatophores using the slow freezing protocol with DMSO (5%) +

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MeOH (5%) as cryoprotectants for 45 days of storage in. With reference to the cryoprotectant toxicity, it may not affect the viability, but could

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have a negative effect on fertilization capacity of sperm (Jeyalectumie and Subramoniam, 1989).

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In the present study, both DMSO and MeOH were less toxic to F. indicus sperm at the relatively lesser concentrations whereas, Gly and EG were moderately toxic, PG and DMA highly toxic. Different concentrations of DMSO from 5% to 20% have been found to be suitable in various experiments for sperm cryopreservation; the concentrations of 5% and 10% of EG, PG, MeOH and Gly are also effective from a post-thaw fertilization perspective (Memon et al., 2012). 11

Similarly, in Penaeus monodon, sperm viability gradually decreased over time when stored in DMSO (5%) and use of the greater concentrations (10%, 15% and 20%) had a greater effect on loss of sperm viability (Bart et al., 2006; Vuthiphandchai et al., 2007). Permeation of embryos by

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MeOH occurs within a shorter duration than with DMSO (Zhang and Rawson, 1995). Permeation of P. monodon in the larval stages by occurred more efficiently (Arun and Subramoniam, 1997). Simon et al. (1994) reported the more rapid permeation by MeOH through the hatching envelope of F. indicus embryos. In the present study, toxicity assessment clearly indicated that an increase in the concentration of DMSO and MeOH for 30 and 60 min of storage

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was toxic to the sperm.

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Earlier studies indicated the use of a combination of a variety of cryoprotectants resulted

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in a reduction the toxicity on sperm, whereas, there are several in consistencies regarding the

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toxicity neutralization while using multiple cryoprotectants (Fahy et al., 1984; Fahy et al., 1990).

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In the present study, the use of a combination of DMSO (5%) and MeOH (5%) resulted in

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greater sperm viability of sperm contained in spermatophores and the effects were more pronounced when used at -196ºC. Diwan and Joseph (1999) reported a 61% to 86% survivability

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rate of the F. indicus sperm using DMSO and Gly as cryoprotectants. A markedly greater viability of 95% was reported for Scylla serrata sperm after 30 days of storage in Gly and

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DMSO with the addition of trehalose (Jeyalectumie and Subramoniam, 1989). There was not any

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significant variation in the toxicity assay data for the viability of sperm when the combination of Gly and DMSO were used. Simon et al. (1994) reported that there was greater viability of F. indicus embryos when using cryoprotectant mixtures containing MeOH in combination with other low penetrating cryoprotectants such as DMSO, EG and PG. Consistent with the finding in these experiments, in the present study there were lesser toxic effects of cryoprotectants with use 12

of DMSO (5%), MeOH (5%) and DMSO (5%) + MeOH (5%). Use of the other cryoprotectants, EG, PG, Gly and DMA, at the greater concentrations resulted in a maximum sperm mortality. Slow freezing protocols are usually suitable for sperm cryopreservation of decapods

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(Anchordoguy et al., 1988; Vuthiphandchai et al., 2007; Salazar et al., 2008). With slow freezing rates, ice formation would begin to occur in the extracellular regions, by dehydrating water molecules from inside to outside the cell due to the greater solute concentrations (Mori et al., 2012). Thus, the present study clearly documented that a greater percentage of sperm survived slow freezing (Protocol A), with combination of DMSO (5%) +MeOH (5%). The slow freezing

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procedure used in the present study was effective in protection of F. indicus sperm quality with

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cryopreservation at a temperature of -196°C. An earlier study on P. monodon confirmed that the

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viability of the sperm was retained with the usage of 10% DMSO at a freezing rate of -6ºC/min

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to -80ºC. Arun and Subramoniam (1997), however, have reported desirable survival rates of

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penaeid shrimp larvae at greater freezing rates. In the present study, however, the sperm

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mortality was greater in cells from the spermatophores preserved with Protocol B for which the freezing rate (-10ºC/min to-80°C) was quite rapid which probably damaging the cells. There was

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a significant decrease in the viability of sperm, hence a slow freezing protocol was utilized.

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Vitrification is a typical process that when used requires greater precautions for membrane protection, thus DMSO (5%), MeOH (5%) and DMSO (5%) + MeOH (5%) was

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selected as the preferred intracellular cryoprotectant based on the toxicity assessment when this procedure is used. Castelo-Branco et al., (2015) reported the greatest viability of sperm of 91.3 ± 2.3% when using the vitrification method in L. vannamei. In the present study, there was a decrease in sperm viability during vitrification with the use of DMSO (5%) + MeOH (5%) being less effective with spermatophores of F. indicus for all storage durations. Thus, the findings of 13

the present study clearly reveal that the rapid freezing rates affect sperm viability, whereas slower freezing rate appear to be preferable for the preservation of sperm contained in spermatophores.

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Sperm survival can generally be assessed based on sperm motility, but this method of evaluation is not applicable for sperm of decapod crustaceans (Akarasonan et al., 2004). The sperm of F. indicus are atypical consisting of a head with an elongated spike. The posterior main body is an elongated sphere containing an uncondensed nucleus followed by a central cap region, which contains acrosomal vesicles (Diwan and Joseph, 1999).The sperm capacitation in

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decapods and its possible relationship with the quality of cryopreserved sperm should be

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considered in future studies of sperm cryopreservation in decapods.

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The viability and membrane integrity of sperm can be evaluated using eosin and nigrosin staining (Curry and Watson, 1994), whereas, the integrity and functional activity of the sperm

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membrane are of fundamental importance in the fertilization process (Sliwa, 1993) and

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assessment of plasma membrane function can be evaluated by using the HOST technique (Jeyendran et al., 1984). The evaluation of sperm membranes can provide important information

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regarding the effects of cryopreservation because the membranes are extremely susceptible to

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cryoinjury. In the present study, the HOST assessment of cryopreserved sperm revealed swelling of the sperm head when slow freezing rates were used. The sperm cells showed a partial swelling

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in the HOST with the sperm cells processed by fast freezing and vitrification protocols. This may be due to the sudden osmotic change during rapid freezing that causes damage to the cell membrane integrity. Sperm DNA integrity is associated with male infertility (Bilodeau et al., 2000). Sperm with bent or absent spike and questionable morphology stained light green rather than yellow, 14

orange or red (Wang et al., 1995). The use of DNA integrity test with the cryopreserved sperm of F. indicus in the present study resulted in a green fluorescence in a majority of the cells indicating less DNA damage during storage when the slow freezing rate was used. The sperm,

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however, stained orange and red fluorescence when the fast freezing and vitrification methods were used indicating DNA damage due to rapid freezing.

In conclusion, the fertility potential of both fresh and cryopreserved sperm was assessed based on the use of hypo-osmotic swelling test which revealed the maximum activity of the sperm after cryopreservation when the slow freezing rate was used for processing. Compilation

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of data clearly documents the sperm viability of cells was greater in spermatophores that were

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retrieved from cadaveric shrimp when the extender was composed of DMSO (5%) + MeOH(5%)

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and the freezing rate was -0.5°C/min to -80°C for long-term storage of F. Indicus sperm. Conflict of Interest

Conflict of Interest

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None

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The authors declare there is no conflict of interest.

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Acknowledgement Financial assistance from the Department of Science and Technology (DST) Government

of India, New Delhi (SR/SO/AS-59/2012) is gratefully acknowledged.

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Figure legends Fig.1: Post-thaw viability of sperm subjected to long-term storage with individual and combination of cryoprotectants using various freezing Protocols (A, B and C) for different

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storage periods. Bars with different letters indicate differences (P< 0.05).

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Fig.2: Hypo-osmotic swelling test for the functional integrity of post-thaw sperm with use of individual and combination of cryoprotectants with various freezing Protocols (A, B and C) for

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different storage periods. Values depicted with bars associated with different letters are different (P< 0.05).

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Fig.3: DNA integrity of post-thaw sperm with use of individual and combination of

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cryoprotectants with various freezing Protocols (A, B and C) for different storage periods.

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Values depicted by bars associated with different letters are different (P< 0.05).

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Table legend Table 1 Percentage of viable sperm obtained from spermatophores equilibrated with cryoprotectants at various concentrations and various exposure durations.

91.64 ± 1.76a

90.62 ± 2.30a

DMSO (5%) DMSO (10%) DMSO (30%)

82.89 ± 3.80b 78.13 ± 3.20c 62.44 ± 1.90d

74.13 ±1.58b 58.58 ±3.91c 45.87 ±6.61d

EG (5%) EG (10%) EG (30%)

78.22 ± 1.88b 70.62 ±2.74c 58.44 ± 5.16d

69.38 ±2.70b 61.20 ±3.35c 52.80 ±1.73d

81.87 ± 2.92b 75.87 ±1.58c 58.22 ± 2.55d

73.20 ±4.15b 68.18 ±1.51c 42.13 ±2.12d

71.33 ± 4.21b 62.93±2.87c 46.04 ±3.06d

62.22 ±4.53b 49.11 ±3.80c 37.91 ±1.99d

Gly (5%) Gly (10%) Gly (30%)

77.60 ±1.73b 65.64 ± 2.89c 53.29 ±5.49d

62.36 ±2.37b 46.40 ±4.15c 37.29 ±2.22d

PG (5%) PG (10%) PG (30%)

69.07 ±2.35b 53.29 ±4.68c 45.11 ±2.99d

58.49 ±1.72b 48.62 ±2.07c 35.38 ±3.75d

83.78 ± 2.51b

76.53 ±2.35b

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Cryoprotectants (%) *Control

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Viability (%) Equilibration time (min) 30 60

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DMA (5%) DMA (10%) DMA (30%)

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MeOH (5%) MeOH (10%) MeOH (30%)

DMSO (5 %) + MeOH (5%) x̅ ± SD of three observations a-d

Values with different letters in the same column are different (P<0.05) among cryoprotectants

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*Control represents freshly collected spermatophores that were suspended in Ca-F saline without

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cryoprotectants

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